Int J Mol Med. 2010 Aug;26(2):249-56.
Takata A, Kuromatsu R, Ando E, Iwamoto H, Fukushima N, Sumie S, Torimura T, Sata M.
Division of Gastro-enterology, Department of Medicine, Kurume University School of Medicine, Fukuoka 830-0011, Japan. oika@med.kurume-u.ac.jp.
Abstract
In recent years, the number of elderly patients with hepatocellular carcinoma (HCC) has been increasing. The aim of this study was to compare the liver function and the background factors of HCC patients with hepatitis C virus (HCV) infection by generation and to examine the characteristics of this disease in the elderly. A total of 1096 patients (776 men and 320 women) diagnosed with HCV-related HCC at our institution from 1995 to 2006 were divided into 4 groups as follows: D group, 75 years of age or older; C group, 65-74 years of age; B group, 55-64 years of age; A group, 54 years of age or younger, and the liver function and other clinical characteristics were compared among these 4 groups. The average age at initial diagnosis of HCV-related HCC was 66.9 years of age. The A, B, C and D groups were comprised of 87, 363, 514 and 132 patients, respectively. The rate of Child-Pugh class A patients in the D group was significantly higher than that of the other groups (P<0.05). The average levels of ALT, TB and PT-INR in the D group were significantly lower than the levels in the other groups (P<0.05). The average Alb level in the D group was significantly higher than that in the other groups (P<0.05). In conclusion, we found that HCV-related HCC in the elderly occurred against a background of chronic liver disease with mild inflammation and fibrosis.
PMID: 20596605 [PubMed - in process]
Source
July 6, 2010
Insult to Injury: VA Hospital Forgot to Tell Patients About HIV Scare
Getty Images
The Miami Veteran Administration Hospital has added insult to injury for dozens of patients who underwent a colonoscopy at the hospital.
79 patients who may have been exposed to HIV a year ago are just now being notified.
By TODD WRIGHT
Updated 6:00 PM EDT, Tue, Jul 6, 2010
Hospital officials admitted Tuesday that it failed to notify 79 patients who may have been exposed to deadly diseases as a result of dirty and contaminated equipment used in the procedure.
Dr. Robert Jesse, the VA principal deputy undersecretary for health, said in a telephone conference call Tuesday the 74 patients should have been included when about 2,400 former Miami VA patients were notified in March 2009 to get tests for HIV, hepatitis and other infections.
Jesse called it an "inexcusable situation."
It's unclear how the 79 patients were left out of the loop, but the delay could be potentially fatal. Hospital director Mary Berrocal, who said she was heartbroken when the news broke last year, has been reassigned as a result of the recent mistake.
At least two Miami patients have already been reported to have contracted HIV after having a colonoscopy at the Miami VA hospital.
The VA sent letters to more than 3,000 veterans who had colonoscopies at the Miami VA hospital informing them that improperly cleaned equipment might have exposed them to hepatitis B, hepatitis C and HIV. Similar problems arose at VA hospitals in Tennessee and Georgia with more than 11,000 veterans potentially exposed to the unsanitary equipment.
Records show that among the patients at the three hospitals who heeded VA warnings to get follow-up blood checks, eight tested positive for HIV. Twelve former patients have tested positive for hepatitis B and 37 have tested positive for hepatitis C.
Juan Rivera of North Miami was the first to file a lawsuit against the government after he claims he contracted HIV after a colonoscopy. The VA claims there is no way to know if patients actually received diseases from the contaminated equipment.
Copyright Associated Press / NBC Miami First Published: Jul 6, 2010 4:34 PM EDT
Source
New Website Evaluates Stem Cell Claims
By Todd Neale, Staff Writer, MedPage Today
Published: July 01, 2010
Patients seeking stem cell treatments for various conditions now have an online resource to help them evaluate the claims of clinics and companies around the world.
The recently launched website -- http://www.closerlookatstemcells.org/ -- set up by the International Society for Stem Cell Research (ISSCR), provides information about stem cell biology as well as questions to ask clinics offering these often-experimental treatments.
The site will eventually contain a list of clinics that did -- or did not -- provide information to the ISSCR on issues such as whether a medical ethics committee is involved in a treatment to protect patients' rights, or whether there is oversight from a regulatory body like the FDA or the European Medicines Agency.
The website is the brainchild of a task force convened by Irving Weissman, MD, director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine in California, during his tenure as president of the ISSCR, which ended earlier this month.
Reporting in the July 2 issue of Cell Stem Cell, members of the task force -- researchers, clinicians, ethicists, jurists, and patient advocates -- said they have become increasingly aware of misleading direct-to-consumer advertising touting expensive stem cell therapies.
Exploiting patients' hopes and fears, these ads often make exaggerated claims of efficacy, underestimate the potential risks, fail to disclose methods, and leave out information on whether the treatment is conducted with regulatory oversight, according to the authors.
"These so-called therapies therefore fail to meet minimum ethical, scientific, and medical standards that such oversight entails, including appropriate support through preclinical data; commensurability of risks and benefits; phased, structured assessment of safety, efficacy, dosing, or appropriate administration; and independently assessed and approved informed consent," Weissman and his colleagues wrote.
They said such unethical marketing "could place individual patients at risk and also jeopardize the progress of legitimate stem cell clinical translation."
The website set up by the ISSCR aims to give patients and their physicians the information needed to assess clinics' claims and decide whether to proceed with treatment.
Using a brief form, patients are able to submit websites for investigation by the ISSCR. The results of the inquiries will be posted on the ISSCR website.
According to the task force, any of the clinics investigated by the group should be able to provide evidence of adherence to a translational pathway from basic science to clinical applications.
For treatments deemed experimental or innovative -- and thus not needing a history of clinical testing in humans -- there will be the following note on the ISSCR website accompanying a clinic listing: "The position of the ISSCR Task Force is that the provision of an untested experimental or innovative therapy to more than two participants is a departure from recommended practice and should be tested in a regulated and authorized clinical trial prior to being offered for sale."
Weismann is a co-founder and director of StemCells Inc. and owns more than $10,000 stock in Amgen for services on their scientific advisory board from 1981 to 1988. His co-authors reported relationships with iPierian, Cellerant Therapeutics, and Tacere Therapeutics.
Primary source: Cell Stem Cell
Source reference:
Taylor P, et al "Patients beware: commercialized stem cell treatments on the web" Cell Stem Cell 2010; 7: 43-49.
Source
Published: July 01, 2010
Patients seeking stem cell treatments for various conditions now have an online resource to help them evaluate the claims of clinics and companies around the world.
The recently launched website -- http://www.closerlookatstemcells.org/ -- set up by the International Society for Stem Cell Research (ISSCR), provides information about stem cell biology as well as questions to ask clinics offering these often-experimental treatments.
The site will eventually contain a list of clinics that did -- or did not -- provide information to the ISSCR on issues such as whether a medical ethics committee is involved in a treatment to protect patients' rights, or whether there is oversight from a regulatory body like the FDA or the European Medicines Agency.
The website is the brainchild of a task force convened by Irving Weissman, MD, director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine in California, during his tenure as president of the ISSCR, which ended earlier this month.
Reporting in the July 2 issue of Cell Stem Cell, members of the task force -- researchers, clinicians, ethicists, jurists, and patient advocates -- said they have become increasingly aware of misleading direct-to-consumer advertising touting expensive stem cell therapies.
Exploiting patients' hopes and fears, these ads often make exaggerated claims of efficacy, underestimate the potential risks, fail to disclose methods, and leave out information on whether the treatment is conducted with regulatory oversight, according to the authors.
"These so-called therapies therefore fail to meet minimum ethical, scientific, and medical standards that such oversight entails, including appropriate support through preclinical data; commensurability of risks and benefits; phased, structured assessment of safety, efficacy, dosing, or appropriate administration; and independently assessed and approved informed consent," Weissman and his colleagues wrote.
They said such unethical marketing "could place individual patients at risk and also jeopardize the progress of legitimate stem cell clinical translation."
The website set up by the ISSCR aims to give patients and their physicians the information needed to assess clinics' claims and decide whether to proceed with treatment.
Using a brief form, patients are able to submit websites for investigation by the ISSCR. The results of the inquiries will be posted on the ISSCR website.
According to the task force, any of the clinics investigated by the group should be able to provide evidence of adherence to a translational pathway from basic science to clinical applications.
For treatments deemed experimental or innovative -- and thus not needing a history of clinical testing in humans -- there will be the following note on the ISSCR website accompanying a clinic listing: "The position of the ISSCR Task Force is that the provision of an untested experimental or innovative therapy to more than two participants is a departure from recommended practice and should be tested in a regulated and authorized clinical trial prior to being offered for sale."
Weismann is a co-founder and director of StemCells Inc. and owns more than $10,000 stock in Amgen for services on their scientific advisory board from 1981 to 1988. His co-authors reported relationships with iPierian, Cellerant Therapeutics, and Tacere Therapeutics.
Primary source: Cell Stem Cell
Source reference:
Taylor P, et al "Patients beware: commercialized stem cell treatments on the web" Cell Stem Cell 2010; 7: 43-49.
Source
Progress Toward an Artificial Liver Transplant
The decellularized livers retain their networks of blood vessels.
Image courtesy of B.E. Uygun.
June 28, 2010
Liver transplantation is currently the only available treatment for severe liver failure, but there aren’t enough donors to fill the need. Researchers have now made transplantable liver grafts for rats that may point the way toward a successful liver transplant substitute for humans.
The decellularized livers retain their networks of blood vessels. Image courtesy of B.E. Uygun. Your liver's job is to help fight infections and clean your blood. It also helps digest food and stores energy for when you need it. People needing a liver transplant are placed on a national waiting list kept at the United Network for Organ Sharing. Their blood type, body size and severity of sickness all play a role in when they’ll receive a liver. Whole livers can come only from people who have just died. Currently, there’s an estimated shortfall of about 4,000 livers per year.
Liver cell transplantation has shown some promise, but has limited uses. To be successful, an artificial transplant must be sufficiently large to provide enough liver function. That requires a network of small blood vessels—called a microvascular network—to transport oxygen and nutrients throughout the structure.
Decellularization—the process of removing cells from a structure but leaving a scaffold with the architecture of the original tissue—has shown some success in other organs. One group of scientists reported the decellularization of an entire heart that preserved the original architecture and microvascular network. A research team led by Dr. Korkut Uygun at Massachusetts General Hospital tried a similar approach for the liver. Their work was supported by NIH’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and others.
In the June 13, 2010, advance online edition of Nature Medicine, the team explained the process they developed. They used a gentle detergent over 3 days to decellularize the liver while preserving its structure. A matrix of proteins remained behind to hold the liver’s shape. Using a dye, the researchers showed that the microvascular network in each eerily translucent liver was intact.
The researchers were then able to successfully introduce functional hepatocytes—a type of liver cell—back into the matrix. When they tested the recellularized matrix, they found that it carried out liver-specific functions at levels comparable to a normal liver.
Grafts transplanted into rats maintained their functional hepatocytes as well, for a few hours. The researchers note, however, that successful engineering of an entire functional liver will require other types of cells.
"As far as we know, a transplantable liver graft has never been constructed in a laboratory setting before," Uygun says. "Even though this is very exciting and promising, it is a proof-of-concept study only. Much more work will be required to make long-term functional liver grafts that can actually be transplanted into humans."
That said, the study highlights the feasibility of using this approach. There is currently a large pool of livers that are unsuitable for transplantation, including livers from people who have died of heart attacks. These could potentially be used to make decellularized liver matrices.
—by Harrison Wein, Ph.D.
Related Links:
Also See: Functional, Transplantable Rat Liver Grafts: Discarded Livers Have Potential to Be Reengineered Into Usable Replacement Organs
Hepatitis C virus infection: A "liaison a trois" amongst the virus, the host, and chronic low-level inflammation for human survival
Download the pdf here
Articles in Press
Jnl of Hepatology
Vincenzo Barnaba
Departimento of Medicina Interna, Sapienza Universita di Roma, Fondazione Andrea Cesalpino, Fondazione Cenci Bolognetti, Italy
Received 28 February 2010; received in revised form 20 May 2010; accepted 9 June 2010. published online 02 July 2010.
Uncorrected Proof
ABSTRACT: Herein, various ambiguous aspects of the immune system that render this complex biological network so highly flexible and able to defend the host from persisting infections such as that induced by the hepatitis C virus (HCV) are reviewed. This ambiguity stems mainly from the property of the immune system to be both protective and harmful. Immunity cannot be fully protective without producing a certain degree of damage (acute hepatitis resulting in resolving HCV infection). In addition, the balance between protection and tissue damage is critical for the development of chronic HCV infection. The establishment of a state of chronic low-level inflammation is instrumental to limit liver immunopathology, to limit viral spread, and ultimately to ensure a long-lasting survival of the host. It is dictated by a fine equilibrium maintained by multiple immunologic mechanisms, including: sensory perception of innate immunity, virus-specific T and B cell functions, control of immune responses, and finally the balance between immunity and immunopathology that has principally evolved to favor the species survival.
Abbreviations: HCV, hepatitis C virus, pDCs, plasmocytoid dendritic cells, cDCs, conventional dendritic cells, PRRs, pattern-recognition receptors, TLRs, toll-like receptors, NOD, intracellular nuclear oligomerisation domain, PAMPs, pathogen-associated molecular patterns, LPS, lipopolysaccharide, IL, interleukin, TRIF, toll-IL-1 receptor domain-containing adaptor inducing IFN-ß, IFN, interferon, DAMPs, damage-associated molecular patterns, UTR, untraslated region, RIG-I, retinoid acid-inducible gene I, IPS-1, adapter molecule IFN-ß promoter stimulator protein 1, p, plasmocytoid, JAK, Janus kinases, E, envelope, NS, non-structural, c, conventional, CCR7, Cys-Cys chemokine receptor 7, TGF, transforming growth factor, Th, T helper, NK, natural killer, KIR, NK cell inhibitory receptor, PD-1, programmed death-1 receptor, TCR, T cell receptor, L, ligand, Treg, T regulatory, Foxp, forkhead box P, IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked, SHPs, Src homology 2-containing tyrosine phosphatases, pSTAT-5, STAT-5 phosphorylation
Conclusions
Through the different (non-mutually exclusive) mechanisms illustrated above, the host survives for a long time in parallel with both the persistent HCV infection and a low-grade liver inflammation that can degenerate into liver failure after several decades. The ambiguous co-existence of virus and inflammation results in an advantage for the evolutionary process and hence for human species survival. If immune responses were invariantly strong and aggressive during a persistent infection such as HCV, they would be unable to eliminate that infection, because of its acquired capacity to escape or to subvert them. In such a situation, exuberant (but non-protective) responses would produce prompt irreversible tissue (hepatic failure) damage, leading to catastrophic epidemic infections. Considering this point of view, chronic (low-level) inflammatory diseases seem to represent a sort of safeguard for the human survival. We can assume that chronic inflammation may be defined as the "Yin and Yang" of the immune system. On the one hand, it guarantees the long-term survival of human hosts despite pathogen persistence. On the other hand, the imbalance of the homeostatic mechanisms maintaining chronic inflammation may degenerate into severe "side-effects" (i.e., the development of either autoimmune diseases or tumours) in a minority of infected individuals. From an evolutionary point of view, the onset of autoimmune diseases or the development of some tumours might be the price to pay following the establishment of chronic inflammation. Indeed, a status of pre-existing chronic inflammation can contribute to the development of cancer, by the production of growth and angiogenic factors eventually promoting cancer-cell survival, implantation, and growth. In addition, chronic inflammation can affect the immune-surveillance directly via its own intrinsic mechanisms (i.e., expansion of Treg cells, T cell exhaustion, etc.), and indirectly by the incapacity to limit the immunosuppressive effects of tumours. The production of soluble factors (i.e., pro-inflammatory or cell growth cytokines) that favor cell proliferation, generally needed for the immune system to defend the host efficaciously, can also facilitate the mitotic cycle of non-lymphoid cells. In the long run, this prolonged stimulation can induce, as in the case of liver cirrhosis by both HBV and HCV, necrosis, cell renewal, and even neoplastic transformation [84]. A further example in HCV infection, is the chronic stimulation of B lymphocytes that can induce the monoclonal expansion of anti-IgG antibodies, which are responsible for the formation of cryoglobulins, autoantibodies, or even the establishment of follicular B cell lymphomas [85].
The immune system simultaneously expresses different strategies that are seemingly opposite but eventually result in an evolutionary advantage. On the one hand, the immune response contributes to species survival; on the other hand it can lead to the sacrifice of single individuals. During the evolutionary process, selective pressure has led to the generation of multiple ambiguous mechanisms to help counteract aggressive infectious agents. Although this is obtained at the cost of severe side-effects (tumour development, autoimmune diseases) in some individuals, these side-effects are considered irrelevant in terms of the survival of the species.
The challenge for scientists is to eliminate the side-effects that emerge in the chronic HCV-host relationship (i.e., cirrhosis, liver failure, HCC, autoimmunity, etc.), possibly via ad hoc modeling and production of new antiviral drugs, immuno-modulatory molecules, therapeutic antiviral antibodies, antiviral small interference (si)RNAs, systems restoring T cell exhaustion (by inhibiting PD-1, Treg cell function, IL-10, or TGF-ß, etc.), and new vaccination strategies.
Article Outline
Introduction
Hepatitis C virus (HCV) is a positive-stranded RNA virus belonging to the Flaviviridae family (reviewed in [1]). HCV eludes host defenses in a considerable portion of infected individuals, developing a status of viral persistence, representing the major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (reviewed in [1], [2], [3]). This review considers the various mechanisms of HCV persistence, and mainly concentrates on those by which T cell responses have been evolved to favor long-term host survival, in spite of chronic HCV-dependent liver disease.
Innate immunity and HCV infection
Resolution of acute infections is dependent on a complex interplay between innate and adaptive immunity. Innate immune cells and molecules play a central role in promptly controlling infections in the early phases and providing the environment required for priming efficient adaptive immune responses. Innate immune cells (mainly monocytes, neutrophiles, dendritic cells [DCs]) are promptly activated upon the recognition of infecting agents by a wide array of pattern-recognition receptors (PRRs), such as the toll-like receptors (TLRs), or the intracellular nuclear oligomerisation domain (NOD)-like receptors [4], [5], [6], [7], [8]. TLRs identify infectious signals derived by molecular patterns common to different pathogens (pathogen-associated molecular patterns [PAMPs], such as lipopolysaccharide [LPS], bacterial DNA, or viral RNA). Then, via their adaptor molecules (i.e., MyD88 for TLR2, 3, 4, 5, 7, 8, 9, 11, and toll-interleukin [IL]-1 receptor domain-containing adaptor inducing interferon [IFN]-ß [TRIF] for TLR3 and 4), they trigger a cascade of down-stream molecules leading to NF-κB and AP-1 activation that ultimately induces the transcription of genes promoting the activities of innate immune cells (cytokine production, maturation, differentiation, migration, etc.). The intracellular NOD-like PRRs recognize dangerous compounds (damage-associated molecular patterns [DAMPs]), such as exogenous crystals (e.g., asbestos causing mesothelioma or asbestosis, silica dust causing silicosis, etc.), or endogenous DAMPs including proteins associated with stressed or dying cells (e.g., uric acid, nucleic acids and their degradation products, such as high-mobility group box 1 protein, oligonucleotides and nucloesides) [7], [9]. It is reasonable hence to postulate that DAMPs derived from hepatocyte necrosis may play a pivotal role in the HCV-dependent liver inflammation.
Importantly, these ancestral signals are also involved in alarming all non-lymphoid nucleated cells (including hepatocytes) that express a more limited repertoire of PRRs than immune cells. Indeed, they quickly respond to infections via the IFN-ß production that provides both an antiviral effect to the infected cells themselves and limits infection of neighboring non-infected cells.
Interferences of HCV with endogenous type I IFN by infected cells
HCV is a single-stranded (ss)RNA virus and, therefore induces type I IFN production in infected cells (i.e., hepatocytes) either upon contact with TLR3 in the endosomal compartments, or upon recognition of the polyuridine motif of HCV 3' untranslated region (UTR) by the retinoid acid-inducible gene I (RIG-I) in the cytoplasm (reviewed in [1]). These processes may be affected by HCV. In vitro studies demonstrated that endogenous HCV-NS3/4A protein cleaves both TRIF and IFN-ß promoter stimulator protein 1 (IPS-1) (adaptor molecules of TLR3 and RIG-I, respectively), thus blocking the down-stream pathway leading to IFN-ß production in transfected hepatocyte cultures (reviewed in [1]). HCV-core protein directly inhibits the down-stream IFN regulatory factor 3 molecule, which in concert with NF-κB, activates IFN-ß gene transcription (reviewed in [1]). This data has been emphasized by observations in vivo revealing that liver biopsies from HCV patients express an inactive form of IPS-1, consistent with it being cleaved [10]. However, patients with acute or chronic HCV infection show normal levels of circulating IFN-α/ß, leading the hypothesis that the latter are not produced by HCV-infected hepatocytes, but by non-infected (likely plasmocytoid [p]DCs) cells. Several HCV proteins interfere with the antiviral signals provided by the cell-surface type I IFN receptors upon engagement by circulating IFNs. The overexpression of HCV-core protein in cell culture interferes with Janus kinases (JAK)/Signal Transducers and Activators of Transcription (STAT) pathway, HCV-envelope (E)2 or non-structural (NS)5A with the function of protein kinase R. HCV-NS5A inhibits 2'-5' oligoadenylate synthetase and induces IL-8 which inhibits induction of the IFN-stimulated genes (reviewed in [1]). In synthesis, HCV seems both to affect the capacity to produce type I IFNs by infected cells and to make the latter less sensitive to the antiviral effect of the same cytokines via disturbing the signals provided by type I IFN receptors (Fig. 1). In vivo models of HCV infection are required to ascertain the importance of these selective defects in providing profound impairment of innate responses in infected cells and ultimately in restraining the priming of adequate adaptive immune responses. Should these mechanisms be demonstrated in vivo, they may take part in the establishment of viral persistence. They may also be amplified by the absence of the genetic polymorphism near the IL28B gene encoding IFN-lambda-3, recently related to both the successful treatment of genotype 1 HCV with IFN-α [11], and the spontaneous resolution in the natural course of HCV infection [12].
Fig. 1. Immune activities in resolving HCV infection. Innate immunity can be principally affected by HCV at the level of both type I IFN production by infected hepatocytes and the signals provided by the relative receptors (IFNAR-1/2) once they are engaged by soluble type I IFNs (mainly produced by pDCs). If these defects are combined with low viral load or infection by HCV strains that are highly susceptible to antiviral IFN effects, HCV viral spread would be contained, and the functions by DCs, NK, B, and T cells should not be heavily affected. This possibility might account for the evidence that the HCV-specific CD4 T cells efficiently differentiate into protective TEM (with Th1 phenotype) and TCM cells, despite the fact that HCV-specific CD8 TEM cells that result are dysfunctional. Since DCs are not susceptible to HCV infection, they could activate CD8 T cells through the phenomenon of cross-presentation of apoptotic hepatocyte bodies containing HCV products. This phenomenon might not be enough to induce efficient primary or secondary CD8 T cell responses, in the absence of direct HCV presentation by infected DCs. In addition, HCV-specific PD-1+ CD8 T cells (simultaneously recognizing MHC class I/viral epitope complexes and PD-L1 on infected hepatocytes) should acquire an exhausted/dysfunctional phenotype in the site of infection, more than PD-1+ CD4 cells. CD4 T cells might guarantee resolution, by producing protective cytokines, helping antigen-specific B cells, and finally sparing some virus-specific CD8 cells from becoming dysfunctional. Under these conditions, the negative loop leading to T cell exhaustion by the interaction between PD-1 expressed on activated T cells and PD-L1 expressed on all lymphoid and non-lymphoid cells would have the ability to switch off unwanted responses, once the virus has been cleared.
HCV interference in the functions of innate immune cells
Plasmocytoid and conventional dendritic cells
pDCs deriving from the lymphoid lineage represent the most important source of type I IFNs (reviewed in [3]). They produce IFN-α/ß upon engagement of TLR7 and nine by ssRNA and dsRNA, respectively, making them critical players in fighting viruses, particularly in the early phases of infection. However, how HCV can induce IFN-α/ß production by pDCs is unclear [3]. Indeed, TLR7 and nine harbor the endosomal compartments and pDCs (as well as conventional [c]DCs) do not seem permissive to HCV infection, likely because they express CD81 but not claudin-1 that are simultaneously required to allow HCV entry into hepatocytes [13], [14]. Moreover, DC infection by HCV has not been shown by using highly sensitive infection systems, such as recombinant engineer reporter HCV [3]. Another debated question is if pDCs are functionally competent in HCV infection. Despite the contrasting evidence that has been reported on this topic (reviewed in [3]), recent studies that measured functions per pDC basis and not within total PBMCs, revealed no defect in response to TLR stimulation by circulating pDCs of chronically-infected individuals [15], [16]. The functional defects of circulating pDCs, which have been observed upon contact with different non-infecting HCV products in vitro (reviewed in [3]), are difficult to reconcile with the fact that chronically-infected individuals do not display a generalized immuno-dysfunction (they normally respond to other viruses or recall antigens!) and have high levels of endogenous type I IFNs (reviewed in [3]). Therefore, we favor the hypothesis that HCV does not interfere with the pDC functions, but it makes infected hepatocytes non-susceptible to the high levels of circulating pDC-derived type I IFNs, because of its capacity to affect the signals triggered by their own specific type I IFN receptors (Fig. 1).
The second fundamental DC population in humans is constituted by cDCs deriving from the myeloid lineage [17], [18]. Given the critical role of cDCs in priming T cell responses (see Box 1), they have been extensively studied in HCV infection, with the idea that HCV-mediated inhibition of cDC functions could result in inefficient antiviral T cell responses. Contrasting evidence resulted from these analyses. cDCs from chronically-infected individuals have not been found to be numerically decreased in the peripheral blood or even dysfunctional in vitro in terms of pro-inflammatory or antigen-presentation capacities, in all studies or patients [19], [20]. As well as in the case of pDC studies, the relevance of both the analysis on circulating cDCs from patients, and those showing the capability of some recombinant HCV proteins to affect the functions of normal cDCs in vitro [21], [22], is strongly restrained by the evidence that chronically-infected individuals are not globally immuno-compromised. In vivo models of HCV infection are required to determine the possibility of a selective impairment of DCs or HCV-specific T cells infiltrating HCV-infected livers due to the high concentrations of viral proteins produced in the site of infection. The selective dysfunction of liver-infiltrating DCs or T cells might result relevant by profoundly affecting the adaptive immune responses against HCV at the level of the infection site (Fig. 2).
Fig. 2. Immune dysfunctions related to chronic HCV infection. High viral load and/or infection by HCV strains that are not susceptible to antiviral effects of endogenous type I IFNs, facilitating HCV viral spread, might strongly affect the function of DCs, NK, B, and T cells. The misfunction of CD4 and CD8 T cells will result in inefficient effector and memory responses and will result in the development of a state of viral persistence. This would also be conditioned by the emergence of several, non-mutually exclusive factors, such as viral epitope escape, viral subversion, the host immunological mechanisms (PD-1, Treg cells, etc.) addressed to control immunopathology, at the cost of the acquiring side-effects that limit protection. Under these conditions, a state of chronic low-level inflammation will take place and will be instrumental in limiting liver immunopathology, viral spread, and finally to ensure long-lasting survival of the host.
Natural killer (NK) cells
Genetic studies demonstrated the association of some HLA and NK cell inhibitory receptor (KIR) genes with resolution of HCV infection [23]. Functional and molecular studies on HLA-KIR interactions are required to determine if these genetic associations result in blocking particular KIRs expressed by NK cells and hence in NK cell-mediated protection in animal models of HCV infection. New studies have also shown an increased proportion of NK cells expressing activating receptors, enhanced cytotoxic function, and defective cytokine production in chronic HCV infection [24]. Additional investigations should be brought forth to verify if they participate in the establishment of chronic inflammation, on the one hand, and viral persistence, on the other hand [24]. In regards to the reports showing induction of NK cell defects upon exposure to some HCV proteins in vitro [25], [26], in vivo models are needed to ascertain if they effectively play a major role in chronic HCV disease development.
Adaptive immunity and HCV infection
As described above, prompt and efficient innate immune responses are mandatory to prime naïve T or B lymphocytes that will then fight, eliminate, and permanently remember the pathogens encountered, via the specific recognition of microbial epitopes. Successful effector responses and memory establishment by CD4 Th cells are dependent on the presence during priming of a wide array of stimulatory signals: those provided by professional APCs (e.g., DCs) in primis, duration of antigenic stimulus, the cytokine milieu, etc. Priming of protective (cytotoxic) CD8 T cell responses requires the same conditions, but the long-lasting CD8 T cell memory seems to be conditioned by the constant presence of memory CD4 T cells [27]. These mechanisms guarantee the prompt emergence of high frequencies of competent effector T cells that are essential for recovery. Upon infection resolution, effector cells disappear, whereas memory cells remain numerically constant because of the expression of receptors specific for the homeostatic (IL-7 and IL-15) cytokines [28]. The homeostatic proliferation of memory cells in the absence of antigen, is critical for prompt differentiation into effector cells, should they re-encounter the original infecting pathogen.
The immunologic scenario promoting infection resolution is only partially respected in acute HCV infection. However, the common conviction that HCV induces chronic infection in the majority of infected individuals has been challenged by the observation that T cells against multiple HCV epitopes persist in a considerable proportion of healthy (non-infected) individuals accidentally exposed to HCV [29], [30]. This data strongly suggests that recovery from asymptomatic form of HCV infection, and that the generation of efficient virus-specific T cell responses clearing HCV are far more frequent than commonly believed [31].
HCV-specific B and T cell responses
The finding that agammaglobulinemic patients can resolve acute HCV infection upon IFN-α treatment, leads to the hypothesis that HCV-specific T cells may compensate for the lack of neutralizing antibodies to obtain HCV clearance [32]. However, recent data suggested that the prompt emergence of neutralizing antibodies in the early phases of infection could play a major role in clearing HCV in immunocompetent patients. Indeed, they have been detected at high levels both during the early phase of infection in association with spontaneous resolution of HCV [32], and once chronic HCV infection is established (reviewed in [1]). Therefore, the availability of neutralizing antibodies or of appropriate vaccines eliciting them may have a central role in the prophylaxis of HCV infection. It is possible that the role of antibodies may have been underestimated in the past due to methodical difficulties in neutralization assays before the HCVpp system was developed.
The emergence of HCV-specific T cells can be detectable in the peripheral blood or in the liver compartment several weeks after infection in humans or experimental chimpanzee models (reviewed in [1], [3]), corresponding with the initial peak of transaminases and irrespective of clinical outcome (resolution vs. chronicity). Despite the delayed appearance of antigen-specific responses, the latter are essential for the HCV control (reviewed in [1]). The majority of studies have been addressed to analyze CD8 T cells in HCV infection, because of their pivotal role in clearing intracellular pathogens. These studies revealed that the magnitude of CD8 T cell responses does not correlate with the clinical or viral outcome in acute HCV infection [34], [35], [36] (Fig. 1, Fig. 2). HCV-specific CD8 T cells are at a relatively high frequency, but express a dysfunctional phenotype (weak proliferation, IFN-γ production, and cytotoxicity) and increased levels of programmed death-1 receptor (PD-1), known to be associated with the exhausted phenotype, irrespective of infection progression [37], [38], [39], [40], [41], [42]. In contrast to HCV-specific CD8, vigorous responses of HCV-specific CD4 T cells producing IFN-γ and IL-2 (Th1 cell profile) are detectable in the peripheral blood at the time of peak of ALT levels, in patients with acute HCV infection undergoing resolution [35], [36], [43], [44] (Fig. 1). The protective effects of CD4 T cells seem to be due, not only to the antiviral cytokines produced, but also to their capacity to help antiviral B cells and to maintain CD8 T cell memory. Indeed, work in experimental animal models support the idea that the CD4-dependent memory HCV-specific CD8 T cells are indispensable both for HCV control and for providing long-term protection [45], [46]. On the contrary, weak, absent, or transient CD4 responses are correlated with chronic infection progression [35], [36], [43], [44] (Fig. 2), suggesting hence that the simultaneous dysfunctions of both CD8 and CD4 cells are associated with disease progression in the majority of infected individuals. Thus, the combination of functional HCV-specific CD4 and CD8 T cells obviously should be the right recipe for recovery, as it is in resolving flu, CMV, or EBV infections. This scenario may contribute to HCV clearance in a considerable proportion of asymptomatic infected individuals, which have been exposed to a different source of HCV infection [29], [30], [31]. Another aspect to consider is the possibility that other T cell subsets or functions may intervene in dictating the fate of HCV infection. In this context, the role of HCV-specific T (CD4 and/or CD8) cells with a Th17 profile in HCV protection, chronic evolution, or pathogenesis is an important topic requiring more in-depth investigations [47]. Indeed, it has been recently reported that Th17 cells play a key role in establishing chronic viral infections [48], [49]. In the following section, the possible mechanisms that may affect the HCV-specific adaptive immune response will be analyzed.
Mechanisms affecting adaptive immune cells in acute HCV infection
The mechanism of cross-presentation
It would be relevant to determine if the selective impairment of HCV-specific CD8 T cell responses may be related to the observation that DCs are not susceptible to HCV infection, and thus in principle, they cannot process endogenous HCV antigens and directly present the resulting epitopes on class I molecules [3], [13], [14]. As a result, HCV-specific CD8 T cells might be primed only via the mechanism of cross-presentation (see Box 1). In cross-presentation, non-infected DCs capture exogenous HCV antigens or apoptotic liver cells carrying HCV and then cross-present the related HCV epitopes on class I molecules [50], [51], [52], [53] (Fig. 1). This mechanism might not be enough to prime efficient CD8 T cell responses (See Box 2).
PD-1/PD-L1 interaction
A special emphasis has been recently placed on PD-1, a death receptor expressed by T or B cells in the late phases of activation [37], [38]. PD-1 induces peripheral T or B cell tolerance or turns off unwanted immune responses, upon the simultaneous interaction of the T cell receptor (TCR) or BCR with antigens and of PD-1 with its own ligands (L): PD-L1, which is virtually expressed on all somatic cells (particularly from inflamed tissues) and PD-L2, and is mainly expressed by DCs [37], [54]. Inflamed/infected hepatocytes up-regulate expression of both PD-L1 [55] and class I molecules bearing viral epitopes (whereas the class II are undetectable or only barely expressed) [56]. Consequently, HCV-specific effector PD-1+ CD8 (recognizing class I/epitope complexes on infected hepatocytes) more than PD-1+ CD4 cells should acquire an exhausted/dysfunctional phenotype in the site of infection. This may account for the conserved functional capacities of HCV-specific CD4 T cells shown in patients undergoing infection resolution, despite dysfunctional HCV-specific CD8 T cells (Fig. 1). Otherwise, the lack of functionally competent HCV-specific CD4, associated with exhausted CD8 T cells, would unavoidably lead towards chronic infection in the majority of patients (Fig. 2). The yet unresolved question is what makes the total HCV-specific adaptive (both CD4 and CD8) T cell responses "not-functionally-competent". This is likely conditioned by the emergence of several, non-mutually exclusive factors, such as high viral load, viral epitope escape, viral subversion, and host immunological mechanisms (PD-1, T regulatory [Treg] cells, etc.) addressed to control immunopathology, at the cost of the side effect of limiting protection. All these factors will induce the generation of non-protective virus-specific CD4 and CD8 T cells, which might even become harmful (Fig. 2).
Viral mutations
HCV's strong capability to mutate B or T cell epitopes and possibly to escape related responses at several levels (antigen processing, MHC binding, TCR or BCR recognition, etc.) is due to its high replication rate and the lack of proofreading capacity of its polymerase (reviewed in [1]). The evidence that about 50% of the CD8 epitopes continue to escape [57], [58] leaving another 50% that do not mutate, renders the role of mutational escape in HCV persistence unclear. Despite both the huge T or B cell repertoire and the fact that several viral epitopes do not mutate due to fitness constraints (reviewed in [1]), there are some CD8 escape mutations associated with fitness costs [59], [60], [61]. These, in synergy with additional mechanisms (high viral load, viral subversion, host immune-suppressive mechanisms, etc.), may participate in the establishment of viral persistence, particularly during the course of the acute phase of infection when the highest level of selective pressure occurs (reviewed in [1]).
Virus-induced immune-subversion
As mentioned above, the studies revealing a general subversion of both T cell and DC functions, upon exposure to some HCV proteins in vitro [21], [22], [62], [63], are difficult to reconcile with the fact that chronically-infected individuals are not globally immuno-compromised. However, if in vivo models of HCV infection demonstrate a selective impairment of T cells infiltrating HCV-infected livers due to the high concentrations of viral proteins produced at the site of infection, this may participate in establishing HCV persistence by affecting the local adaptive immune responses.
Regulatory cytokines
A recent report demonstrated that peripheral HCV-specific CD4 and CD8 T cells producing IL-10 are detectable in the early phases of acute HCV infection [64]. These cells seem to suppress antiviral effector responses, promoting chronic evolution of infection, while limiting progressive liver damage [64]. These suggestions are reminiscent of our previous studies showing that IL-10 producing HCV-specific CD8 T cells infiltrate the liver of chronically-infected individuals [65]. They inversely correlated with both the frequency of HCV-specific T cells producing IFN-γ and the inflammatory staging at the level of liver biopsies, suggesting that they modulate excessive liver immunopathology [65]. Accordingly, it has been reported that intrahepatic HCV-specific IL-10 producing CD8 T cells prevent liver damage during chronic infection [66]. TGF-ß-producing virus-specific CD4 and CD8 T cells have been related to antiviral immune suppression and chronic HCV infection evolution [67]. Taken together, these data suggest that regulatory cytokines such as IL-10 or TGF-ß minimize liver inflammation, at the cost of the protective immune responses clearing the virus (Fig. 2).
CD25+Foxp3+ Treg cells
Treg cells expressing the transcription factor forkhead box P (Foxp)3 develop either in the thymus (natural) or in the periphery from conventional CD4+ T cells (induced) [68], [69], [70], [71]. A lack of Foxp3 expression results in the complete absence of Treg cells, which leads to the development of severe autoimmunity, as observed in immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome [68], [69], [70], [71]. The main physiological functions of Treg cells are as follows: (a) to participate in the establishment of peripheral tolerance by inhibiting autoreactive T or B lymphocytes that escaped either thymus or bone marrow checkpoints, respectively (central tolerance), (b) to suppress ongoing protective immune responses once they are no longer necessary or become harmful after the elimination of the pathogen and (c) to limit excessive immunopathology during chronic inflammatory diseases. As a result of the expression of the Il-2 gene-inhibitory Foxp3 transcription factor, Treg cells do not produce IL-2 and are unable to respond to antigens (anergy) [72], [73]. However, Foxp3 activity maintains high levels of IL-2 receptors (CD25hi) on Treg cells, hence compensating for the incapacity of producing IL-2. Indisputably, CD25hi Treg cells promptly proliferate both in vitro and in vivo in response to relevant antigens in the presence of paracrine IL-2, which is mainly produced by responder (effector) T-lymphocytes, but it is dominantly absorbed by Treg cells expressing higher CD25 levels than responder T cells [74], [75], [76]. This appears to represent a key suppression mechanism, because CD25hi Treg cells steal the majority of IL-2 produced by responder T cells that in turn will be deprived of their most important growth factor. Furthermore, Treg cells suppress via different, likely non-mutually exclusive mechanisms, involving membrane molecules (such as cytotoxic T-lymphocyte antigen-4 or adenosine receptors) and suppressive cytokine production (such as TGF-ß or IL-10) [68], [69], [70], [71]. Treg cells are induced and proliferate in response to HCV and seem to modulate liver inflammation in the course of chronic infection [77], [78]. Therefore, the model of HCV infection supports the idea that Treg cells participate in the establishment of a fine equilibrium between immunopathology and immune protection, ultimately resulting in the long-lasting survival of the host during chronic infections [69], [70], [79], [80], [81], [82], [83] (Fig. 2). This would be dependent on a compromise between a status of chronic low-level hepatic inflammation and the generation of antiviral responses that, although unable to clear HCV, are enough to limit excessive viral spread. It is unclear how Treg cells control unwarranted inflammation without completely suppressing the protective immune responses. High CD25 expression by Treg cells drives a positive feedback loop, as the dominant IL-2 capture increases STAT-5 phosphorylation (pSTAT-5) that in turn drives Treg cell proliferation and function. We recently showed that PD-1 is over-expressed on Foxp3+ Treg cells and limits Treg cell proliferation and function during chronic HCV infection. The expression of PD-1, upon contact with its own ligands, inhibits pSTAT-5 via the activation of Src homology 2-containing tyrosine phosphatases (SHPs) [78] (Fig. 3). As a consequence, responder T cells can escape from excessive expansion of Treg cells and render them available for responding to possible novel waves of infection. This negative feedback loop assumes a different significance during chronic infections, such as HCV. The incapacity to clear HCV by the immune system (due to the various mechanisms emphasized above) perpetuates a vicious spiral, whereby responder T cells are chronically stimulated to produce IL-2 that will be dominantly adsorbed by CD25hi Treg cells that in turn will continuously suppress the effector responses. PD-1 up-regulation limits the excessive expansion of Treg cells by controlling pSTAT-5 and fine-tunes Treg function in order to minimize the immunopathology without completely switching off those intended to limit excessive viral spread (Fig. 3). This may represent a critical contra-suppression mechanism that has evolved to assure that Treg cells have limited suppression. Homeostatic balance participates in establishing a status of chronic low-level liver inflammation that is in turn instrumental to ensure long-lasting survival of the host.
Fig. 3. PD-1 controls Treg cells in HCV infection. (A) Responder (CD25lowFoxp3-) TEM cells proliferate in response to HCV antigens, and produce IL-2, which through IL-2R (CD25) signaling, induces pSTAT-5. This leads to the development of the genetic program dictating their effector phenotype. In parallel, the same phenomena occurs for the (CD25hiFoxp3+) Treg cells that do not proliferate to viral antigens alone because of the expression of the Il-2 inhibitory gene Foxp3. Their proliferation is dependent on the dominant capture of paracrine IL-2 that is initially produced by responder T cells. CD25hiFoxp3+ Treg cells can proliferate by the engagement of the Jak3/STAT-5 pathway, and extrinsically down-regulate the TEM cell responses. (B) In the late phases of T cell activation, the death receptors intrinsically deliver negative signals to activated T cells (including Fas, CTLA-4, and PD-1) in order to terminate the T cell responses. PD-1 is up-regulated on both responder and Treg cells and upon contact with PD-L1/2 inhibits pSTAT-5 possibly via SHP2. This mechanism results in limiting both TEM cell responses and excessive Treg cell function. Under conditions resulting in HCV resolution, this loop is self-limited because of the disappearance of the viral antigenic stimuli. During a chronic HCV infection, in which responder T cells have been unable to clear HCV, the negative loop is maintained by the persisting HCV antigens that chronically stimulate IL-2 producing responder T cells. Chronic PD-1 expression on both TEM and Treg cells modulate the potential excessive pSTAT-5-dependent cell proliferation. The resulting contra-regulation of Treg cells will have an important role in limiting excessive suppression of immune responses, controlling the spreading virus at the cost inability to maintain chronic low-level liver immunopathology. This mechanism establishes long-lasting survival of the host.
Conclusions
Through the different (non-mutually exclusive) mechanisms illustrated above, the host survives for a long time in parallel with both the persistent HCV infection and a low-grade liver inflammation that can degenerate into liver failure after several decades. The ambiguous co-existence of virus and inflammation results in an advantage for the evolutionary process and hence for human species survival. If immune responses were invariantly strong and aggressive during a persistent infection such as HCV, they would be unable to eliminate that infection, because of its acquired capacity to escape or to subvert them. In such a situation, exuberant (but non-protective) responses would produce prompt irreversible tissue (hepatic failure) damage, leading to catastrophic epidemic infections. Considering this point of view, chronic (low-level) inflammatory diseases seem to represent a sort of safeguard for the human survival. We can assume that chronic inflammation may be defined as the "Yin and Yang" of the immune system. On the one hand, it guarantees the long-term survival of human hosts despite pathogen persistence. On the other hand, the imbalance of the homeostatic mechanisms maintaining chronic inflammation may degenerate into severe "side-effects" (i.e., the development of either autoimmune diseases or tumours) in a minority of infected individuals. From an evolutionary point of view, the onset of autoimmune diseases or the development of some tumours might be the price to pay following the establishment of chronic inflammation. Indeed, a status of pre-existing chronic inflammation can contribute to the development of cancer, by the production of growth and angiogenic factors eventually promoting cancer-cell survival, implantation, and growth. In addition, chronic inflammation can affect the immune-surveillance directly via its own intrinsic mechanisms (i.e., expansion of Treg cells, T cell exhaustion, etc.), and indirectly by the incapacity to limit the immunosuppressive effects of tumours. The production of soluble factors (i.e., pro-inflammatory or cell growth cytokines) that favor cell proliferation, generally needed for the immune system to defend the host efficaciously, can also facilitate the mitotic cycle of non-lymphoid cells. In the long run, this prolonged stimulation can induce, as in the case of liver cirrhosis by both HBV and HCV, necrosis, cell renewal, and even neoplastic transformation [84]. A further example in HCV infection, is the chronic stimulation of B lymphocytes that can induce the monoclonal expansion of anti-IgG antibodies, which are responsible for the formation of cryoglobulins, autoantibodies, or even the establishment of follicular B cell lymphomas [85].
The immune system simultaneously expresses different strategies that are seemingly opposite but eventually result in an evolutionary advantage. On the one hand, the immune response contributes to species survival; on the other hand it can lead to the sacrifice of single individuals. During the evolutionary process, selective pressure has led to the generation of multiple ambiguous mechanisms to help counteract aggressive infectious agents. Although this is obtained at the cost of severe side-effects (tumour development, autoimmune diseases) in some individuals, these side-effects are considered irrelevant in terms of the survival of the species.
The challenge for scientists is to eliminate the side-effects that emerge in the chronic HCV-host relationship (i.e., cirrhosis, liver failure, HCC, autoimmunity, etc.), possibly via ad hoc modeling and production of new antiviral drugs, immuno-modulatory molecules, therapeutic antiviral antibodies, antiviral small interference (si)RNAs, systems restoring T cell exhaustion (by inhibiting PD-1, Treg cell function, IL-10, or TGF-ß, etc.), and new vaccination strategies.
Source
Articles in Press
Jnl of Hepatology
Vincenzo Barnaba
Departimento of Medicina Interna, Sapienza Universita di Roma, Fondazione Andrea Cesalpino, Fondazione Cenci Bolognetti, Italy
Received 28 February 2010; received in revised form 20 May 2010; accepted 9 June 2010. published online 02 July 2010.
Uncorrected Proof
ABSTRACT: Herein, various ambiguous aspects of the immune system that render this complex biological network so highly flexible and able to defend the host from persisting infections such as that induced by the hepatitis C virus (HCV) are reviewed. This ambiguity stems mainly from the property of the immune system to be both protective and harmful. Immunity cannot be fully protective without producing a certain degree of damage (acute hepatitis resulting in resolving HCV infection). In addition, the balance between protection and tissue damage is critical for the development of chronic HCV infection. The establishment of a state of chronic low-level inflammation is instrumental to limit liver immunopathology, to limit viral spread, and ultimately to ensure a long-lasting survival of the host. It is dictated by a fine equilibrium maintained by multiple immunologic mechanisms, including: sensory perception of innate immunity, virus-specific T and B cell functions, control of immune responses, and finally the balance between immunity and immunopathology that has principally evolved to favor the species survival.
Abbreviations: HCV, hepatitis C virus, pDCs, plasmocytoid dendritic cells, cDCs, conventional dendritic cells, PRRs, pattern-recognition receptors, TLRs, toll-like receptors, NOD, intracellular nuclear oligomerisation domain, PAMPs, pathogen-associated molecular patterns, LPS, lipopolysaccharide, IL, interleukin, TRIF, toll-IL-1 receptor domain-containing adaptor inducing IFN-ß, IFN, interferon, DAMPs, damage-associated molecular patterns, UTR, untraslated region, RIG-I, retinoid acid-inducible gene I, IPS-1, adapter molecule IFN-ß promoter stimulator protein 1, p, plasmocytoid, JAK, Janus kinases, E, envelope, NS, non-structural, c, conventional, CCR7, Cys-Cys chemokine receptor 7, TGF, transforming growth factor, Th, T helper, NK, natural killer, KIR, NK cell inhibitory receptor, PD-1, programmed death-1 receptor, TCR, T cell receptor, L, ligand, Treg, T regulatory, Foxp, forkhead box P, IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked, SHPs, Src homology 2-containing tyrosine phosphatases, pSTAT-5, STAT-5 phosphorylation
Conclusions
Through the different (non-mutually exclusive) mechanisms illustrated above, the host survives for a long time in parallel with both the persistent HCV infection and a low-grade liver inflammation that can degenerate into liver failure after several decades. The ambiguous co-existence of virus and inflammation results in an advantage for the evolutionary process and hence for human species survival. If immune responses were invariantly strong and aggressive during a persistent infection such as HCV, they would be unable to eliminate that infection, because of its acquired capacity to escape or to subvert them. In such a situation, exuberant (but non-protective) responses would produce prompt irreversible tissue (hepatic failure) damage, leading to catastrophic epidemic infections. Considering this point of view, chronic (low-level) inflammatory diseases seem to represent a sort of safeguard for the human survival. We can assume that chronic inflammation may be defined as the "Yin and Yang" of the immune system. On the one hand, it guarantees the long-term survival of human hosts despite pathogen persistence. On the other hand, the imbalance of the homeostatic mechanisms maintaining chronic inflammation may degenerate into severe "side-effects" (i.e., the development of either autoimmune diseases or tumours) in a minority of infected individuals. From an evolutionary point of view, the onset of autoimmune diseases or the development of some tumours might be the price to pay following the establishment of chronic inflammation. Indeed, a status of pre-existing chronic inflammation can contribute to the development of cancer, by the production of growth and angiogenic factors eventually promoting cancer-cell survival, implantation, and growth. In addition, chronic inflammation can affect the immune-surveillance directly via its own intrinsic mechanisms (i.e., expansion of Treg cells, T cell exhaustion, etc.), and indirectly by the incapacity to limit the immunosuppressive effects of tumours. The production of soluble factors (i.e., pro-inflammatory or cell growth cytokines) that favor cell proliferation, generally needed for the immune system to defend the host efficaciously, can also facilitate the mitotic cycle of non-lymphoid cells. In the long run, this prolonged stimulation can induce, as in the case of liver cirrhosis by both HBV and HCV, necrosis, cell renewal, and even neoplastic transformation [84]. A further example in HCV infection, is the chronic stimulation of B lymphocytes that can induce the monoclonal expansion of anti-IgG antibodies, which are responsible for the formation of cryoglobulins, autoantibodies, or even the establishment of follicular B cell lymphomas [85].
The immune system simultaneously expresses different strategies that are seemingly opposite but eventually result in an evolutionary advantage. On the one hand, the immune response contributes to species survival; on the other hand it can lead to the sacrifice of single individuals. During the evolutionary process, selective pressure has led to the generation of multiple ambiguous mechanisms to help counteract aggressive infectious agents. Although this is obtained at the cost of severe side-effects (tumour development, autoimmune diseases) in some individuals, these side-effects are considered irrelevant in terms of the survival of the species.
The challenge for scientists is to eliminate the side-effects that emerge in the chronic HCV-host relationship (i.e., cirrhosis, liver failure, HCC, autoimmunity, etc.), possibly via ad hoc modeling and production of new antiviral drugs, immuno-modulatory molecules, therapeutic antiviral antibodies, antiviral small interference (si)RNAs, systems restoring T cell exhaustion (by inhibiting PD-1, Treg cell function, IL-10, or TGF-ß, etc.), and new vaccination strategies.
Article Outline
Introduction
Hepatitis C virus (HCV) is a positive-stranded RNA virus belonging to the Flaviviridae family (reviewed in [1]). HCV eludes host defenses in a considerable portion of infected individuals, developing a status of viral persistence, representing the major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (reviewed in [1], [2], [3]). This review considers the various mechanisms of HCV persistence, and mainly concentrates on those by which T cell responses have been evolved to favor long-term host survival, in spite of chronic HCV-dependent liver disease.
Innate immunity and HCV infection
Resolution of acute infections is dependent on a complex interplay between innate and adaptive immunity. Innate immune cells and molecules play a central role in promptly controlling infections in the early phases and providing the environment required for priming efficient adaptive immune responses. Innate immune cells (mainly monocytes, neutrophiles, dendritic cells [DCs]) are promptly activated upon the recognition of infecting agents by a wide array of pattern-recognition receptors (PRRs), such as the toll-like receptors (TLRs), or the intracellular nuclear oligomerisation domain (NOD)-like receptors [4], [5], [6], [7], [8]. TLRs identify infectious signals derived by molecular patterns common to different pathogens (pathogen-associated molecular patterns [PAMPs], such as lipopolysaccharide [LPS], bacterial DNA, or viral RNA). Then, via their adaptor molecules (i.e., MyD88 for TLR2, 3, 4, 5, 7, 8, 9, 11, and toll-interleukin [IL]-1 receptor domain-containing adaptor inducing interferon [IFN]-ß [TRIF] for TLR3 and 4), they trigger a cascade of down-stream molecules leading to NF-κB and AP-1 activation that ultimately induces the transcription of genes promoting the activities of innate immune cells (cytokine production, maturation, differentiation, migration, etc.). The intracellular NOD-like PRRs recognize dangerous compounds (damage-associated molecular patterns [DAMPs]), such as exogenous crystals (e.g., asbestos causing mesothelioma or asbestosis, silica dust causing silicosis, etc.), or endogenous DAMPs including proteins associated with stressed or dying cells (e.g., uric acid, nucleic acids and their degradation products, such as high-mobility group box 1 protein, oligonucleotides and nucloesides) [7], [9]. It is reasonable hence to postulate that DAMPs derived from hepatocyte necrosis may play a pivotal role in the HCV-dependent liver inflammation.
Importantly, these ancestral signals are also involved in alarming all non-lymphoid nucleated cells (including hepatocytes) that express a more limited repertoire of PRRs than immune cells. Indeed, they quickly respond to infections via the IFN-ß production that provides both an antiviral effect to the infected cells themselves and limits infection of neighboring non-infected cells.
Interferences of HCV with endogenous type I IFN by infected cells
HCV is a single-stranded (ss)RNA virus and, therefore induces type I IFN production in infected cells (i.e., hepatocytes) either upon contact with TLR3 in the endosomal compartments, or upon recognition of the polyuridine motif of HCV 3' untranslated region (UTR) by the retinoid acid-inducible gene I (RIG-I) in the cytoplasm (reviewed in [1]). These processes may be affected by HCV. In vitro studies demonstrated that endogenous HCV-NS3/4A protein cleaves both TRIF and IFN-ß promoter stimulator protein 1 (IPS-1) (adaptor molecules of TLR3 and RIG-I, respectively), thus blocking the down-stream pathway leading to IFN-ß production in transfected hepatocyte cultures (reviewed in [1]). HCV-core protein directly inhibits the down-stream IFN regulatory factor 3 molecule, which in concert with NF-κB, activates IFN-ß gene transcription (reviewed in [1]). This data has been emphasized by observations in vivo revealing that liver biopsies from HCV patients express an inactive form of IPS-1, consistent with it being cleaved [10]. However, patients with acute or chronic HCV infection show normal levels of circulating IFN-α/ß, leading the hypothesis that the latter are not produced by HCV-infected hepatocytes, but by non-infected (likely plasmocytoid [p]DCs) cells. Several HCV proteins interfere with the antiviral signals provided by the cell-surface type I IFN receptors upon engagement by circulating IFNs. The overexpression of HCV-core protein in cell culture interferes with Janus kinases (JAK)/Signal Transducers and Activators of Transcription (STAT) pathway, HCV-envelope (E)2 or non-structural (NS)5A with the function of protein kinase R. HCV-NS5A inhibits 2'-5' oligoadenylate synthetase and induces IL-8 which inhibits induction of the IFN-stimulated genes (reviewed in [1]). In synthesis, HCV seems both to affect the capacity to produce type I IFNs by infected cells and to make the latter less sensitive to the antiviral effect of the same cytokines via disturbing the signals provided by type I IFN receptors (Fig. 1). In vivo models of HCV infection are required to ascertain the importance of these selective defects in providing profound impairment of innate responses in infected cells and ultimately in restraining the priming of adequate adaptive immune responses. Should these mechanisms be demonstrated in vivo, they may take part in the establishment of viral persistence. They may also be amplified by the absence of the genetic polymorphism near the IL28B gene encoding IFN-lambda-3, recently related to both the successful treatment of genotype 1 HCV with IFN-α [11], and the spontaneous resolution in the natural course of HCV infection [12].
Fig. 1. Immune activities in resolving HCV infection. Innate immunity can be principally affected by HCV at the level of both type I IFN production by infected hepatocytes and the signals provided by the relative receptors (IFNAR-1/2) once they are engaged by soluble type I IFNs (mainly produced by pDCs). If these defects are combined with low viral load or infection by HCV strains that are highly susceptible to antiviral IFN effects, HCV viral spread would be contained, and the functions by DCs, NK, B, and T cells should not be heavily affected. This possibility might account for the evidence that the HCV-specific CD4 T cells efficiently differentiate into protective TEM (with Th1 phenotype) and TCM cells, despite the fact that HCV-specific CD8 TEM cells that result are dysfunctional. Since DCs are not susceptible to HCV infection, they could activate CD8 T cells through the phenomenon of cross-presentation of apoptotic hepatocyte bodies containing HCV products. This phenomenon might not be enough to induce efficient primary or secondary CD8 T cell responses, in the absence of direct HCV presentation by infected DCs. In addition, HCV-specific PD-1+ CD8 T cells (simultaneously recognizing MHC class I/viral epitope complexes and PD-L1 on infected hepatocytes) should acquire an exhausted/dysfunctional phenotype in the site of infection, more than PD-1+ CD4 cells. CD4 T cells might guarantee resolution, by producing protective cytokines, helping antigen-specific B cells, and finally sparing some virus-specific CD8 cells from becoming dysfunctional. Under these conditions, the negative loop leading to T cell exhaustion by the interaction between PD-1 expressed on activated T cells and PD-L1 expressed on all lymphoid and non-lymphoid cells would have the ability to switch off unwanted responses, once the virus has been cleared.
HCV interference in the functions of innate immune cells
Plasmocytoid and conventional dendritic cells
pDCs deriving from the lymphoid lineage represent the most important source of type I IFNs (reviewed in [3]). They produce IFN-α/ß upon engagement of TLR7 and nine by ssRNA and dsRNA, respectively, making them critical players in fighting viruses, particularly in the early phases of infection. However, how HCV can induce IFN-α/ß production by pDCs is unclear [3]. Indeed, TLR7 and nine harbor the endosomal compartments and pDCs (as well as conventional [c]DCs) do not seem permissive to HCV infection, likely because they express CD81 but not claudin-1 that are simultaneously required to allow HCV entry into hepatocytes [13], [14]. Moreover, DC infection by HCV has not been shown by using highly sensitive infection systems, such as recombinant engineer reporter HCV [3]. Another debated question is if pDCs are functionally competent in HCV infection. Despite the contrasting evidence that has been reported on this topic (reviewed in [3]), recent studies that measured functions per pDC basis and not within total PBMCs, revealed no defect in response to TLR stimulation by circulating pDCs of chronically-infected individuals [15], [16]. The functional defects of circulating pDCs, which have been observed upon contact with different non-infecting HCV products in vitro (reviewed in [3]), are difficult to reconcile with the fact that chronically-infected individuals do not display a generalized immuno-dysfunction (they normally respond to other viruses or recall antigens!) and have high levels of endogenous type I IFNs (reviewed in [3]). Therefore, we favor the hypothesis that HCV does not interfere with the pDC functions, but it makes infected hepatocytes non-susceptible to the high levels of circulating pDC-derived type I IFNs, because of its capacity to affect the signals triggered by their own specific type I IFN receptors (Fig. 1).
The second fundamental DC population in humans is constituted by cDCs deriving from the myeloid lineage [17], [18]. Given the critical role of cDCs in priming T cell responses (see Box 1), they have been extensively studied in HCV infection, with the idea that HCV-mediated inhibition of cDC functions could result in inefficient antiviral T cell responses. Contrasting evidence resulted from these analyses. cDCs from chronically-infected individuals have not been found to be numerically decreased in the peripheral blood or even dysfunctional in vitro in terms of pro-inflammatory or antigen-presentation capacities, in all studies or patients [19], [20]. As well as in the case of pDC studies, the relevance of both the analysis on circulating cDCs from patients, and those showing the capability of some recombinant HCV proteins to affect the functions of normal cDCs in vitro [21], [22], is strongly restrained by the evidence that chronically-infected individuals are not globally immuno-compromised. In vivo models of HCV infection are required to determine the possibility of a selective impairment of DCs or HCV-specific T cells infiltrating HCV-infected livers due to the high concentrations of viral proteins produced in the site of infection. The selective dysfunction of liver-infiltrating DCs or T cells might result relevant by profoundly affecting the adaptive immune responses against HCV at the level of the infection site (Fig. 2).
Fig. 2. Immune dysfunctions related to chronic HCV infection. High viral load and/or infection by HCV strains that are not susceptible to antiviral effects of endogenous type I IFNs, facilitating HCV viral spread, might strongly affect the function of DCs, NK, B, and T cells. The misfunction of CD4 and CD8 T cells will result in inefficient effector and memory responses and will result in the development of a state of viral persistence. This would also be conditioned by the emergence of several, non-mutually exclusive factors, such as viral epitope escape, viral subversion, the host immunological mechanisms (PD-1, Treg cells, etc.) addressed to control immunopathology, at the cost of the acquiring side-effects that limit protection. Under these conditions, a state of chronic low-level inflammation will take place and will be instrumental in limiting liver immunopathology, viral spread, and finally to ensure long-lasting survival of the host.
Natural killer (NK) cells
Genetic studies demonstrated the association of some HLA and NK cell inhibitory receptor (KIR) genes with resolution of HCV infection [23]. Functional and molecular studies on HLA-KIR interactions are required to determine if these genetic associations result in blocking particular KIRs expressed by NK cells and hence in NK cell-mediated protection in animal models of HCV infection. New studies have also shown an increased proportion of NK cells expressing activating receptors, enhanced cytotoxic function, and defective cytokine production in chronic HCV infection [24]. Additional investigations should be brought forth to verify if they participate in the establishment of chronic inflammation, on the one hand, and viral persistence, on the other hand [24]. In regards to the reports showing induction of NK cell defects upon exposure to some HCV proteins in vitro [25], [26], in vivo models are needed to ascertain if they effectively play a major role in chronic HCV disease development.
Adaptive immunity and HCV infection
As described above, prompt and efficient innate immune responses are mandatory to prime naïve T or B lymphocytes that will then fight, eliminate, and permanently remember the pathogens encountered, via the specific recognition of microbial epitopes. Successful effector responses and memory establishment by CD4 Th cells are dependent on the presence during priming of a wide array of stimulatory signals: those provided by professional APCs (e.g., DCs) in primis, duration of antigenic stimulus, the cytokine milieu, etc. Priming of protective (cytotoxic) CD8 T cell responses requires the same conditions, but the long-lasting CD8 T cell memory seems to be conditioned by the constant presence of memory CD4 T cells [27]. These mechanisms guarantee the prompt emergence of high frequencies of competent effector T cells that are essential for recovery. Upon infection resolution, effector cells disappear, whereas memory cells remain numerically constant because of the expression of receptors specific for the homeostatic (IL-7 and IL-15) cytokines [28]. The homeostatic proliferation of memory cells in the absence of antigen, is critical for prompt differentiation into effector cells, should they re-encounter the original infecting pathogen.
The immunologic scenario promoting infection resolution is only partially respected in acute HCV infection. However, the common conviction that HCV induces chronic infection in the majority of infected individuals has been challenged by the observation that T cells against multiple HCV epitopes persist in a considerable proportion of healthy (non-infected) individuals accidentally exposed to HCV [29], [30]. This data strongly suggests that recovery from asymptomatic form of HCV infection, and that the generation of efficient virus-specific T cell responses clearing HCV are far more frequent than commonly believed [31].
HCV-specific B and T cell responses
The finding that agammaglobulinemic patients can resolve acute HCV infection upon IFN-α treatment, leads to the hypothesis that HCV-specific T cells may compensate for the lack of neutralizing antibodies to obtain HCV clearance [32]. However, recent data suggested that the prompt emergence of neutralizing antibodies in the early phases of infection could play a major role in clearing HCV in immunocompetent patients. Indeed, they have been detected at high levels both during the early phase of infection in association with spontaneous resolution of HCV [32], and once chronic HCV infection is established (reviewed in [1]). Therefore, the availability of neutralizing antibodies or of appropriate vaccines eliciting them may have a central role in the prophylaxis of HCV infection. It is possible that the role of antibodies may have been underestimated in the past due to methodical difficulties in neutralization assays before the HCVpp system was developed.
The emergence of HCV-specific T cells can be detectable in the peripheral blood or in the liver compartment several weeks after infection in humans or experimental chimpanzee models (reviewed in [1], [3]), corresponding with the initial peak of transaminases and irrespective of clinical outcome (resolution vs. chronicity). Despite the delayed appearance of antigen-specific responses, the latter are essential for the HCV control (reviewed in [1]). The majority of studies have been addressed to analyze CD8 T cells in HCV infection, because of their pivotal role in clearing intracellular pathogens. These studies revealed that the magnitude of CD8 T cell responses does not correlate with the clinical or viral outcome in acute HCV infection [34], [35], [36] (Fig. 1, Fig. 2). HCV-specific CD8 T cells are at a relatively high frequency, but express a dysfunctional phenotype (weak proliferation, IFN-γ production, and cytotoxicity) and increased levels of programmed death-1 receptor (PD-1), known to be associated with the exhausted phenotype, irrespective of infection progression [37], [38], [39], [40], [41], [42]. In contrast to HCV-specific CD8, vigorous responses of HCV-specific CD4 T cells producing IFN-γ and IL-2 (Th1 cell profile) are detectable in the peripheral blood at the time of peak of ALT levels, in patients with acute HCV infection undergoing resolution [35], [36], [43], [44] (Fig. 1). The protective effects of CD4 T cells seem to be due, not only to the antiviral cytokines produced, but also to their capacity to help antiviral B cells and to maintain CD8 T cell memory. Indeed, work in experimental animal models support the idea that the CD4-dependent memory HCV-specific CD8 T cells are indispensable both for HCV control and for providing long-term protection [45], [46]. On the contrary, weak, absent, or transient CD4 responses are correlated with chronic infection progression [35], [36], [43], [44] (Fig. 2), suggesting hence that the simultaneous dysfunctions of both CD8 and CD4 cells are associated with disease progression in the majority of infected individuals. Thus, the combination of functional HCV-specific CD4 and CD8 T cells obviously should be the right recipe for recovery, as it is in resolving flu, CMV, or EBV infections. This scenario may contribute to HCV clearance in a considerable proportion of asymptomatic infected individuals, which have been exposed to a different source of HCV infection [29], [30], [31]. Another aspect to consider is the possibility that other T cell subsets or functions may intervene in dictating the fate of HCV infection. In this context, the role of HCV-specific T (CD4 and/or CD8) cells with a Th17 profile in HCV protection, chronic evolution, or pathogenesis is an important topic requiring more in-depth investigations [47]. Indeed, it has been recently reported that Th17 cells play a key role in establishing chronic viral infections [48], [49]. In the following section, the possible mechanisms that may affect the HCV-specific adaptive immune response will be analyzed.
Mechanisms affecting adaptive immune cells in acute HCV infection
The mechanism of cross-presentation
It would be relevant to determine if the selective impairment of HCV-specific CD8 T cell responses may be related to the observation that DCs are not susceptible to HCV infection, and thus in principle, they cannot process endogenous HCV antigens and directly present the resulting epitopes on class I molecules [3], [13], [14]. As a result, HCV-specific CD8 T cells might be primed only via the mechanism of cross-presentation (see Box 1). In cross-presentation, non-infected DCs capture exogenous HCV antigens or apoptotic liver cells carrying HCV and then cross-present the related HCV epitopes on class I molecules [50], [51], [52], [53] (Fig. 1). This mechanism might not be enough to prime efficient CD8 T cell responses (See Box 2).
PD-1/PD-L1 interaction
A special emphasis has been recently placed on PD-1, a death receptor expressed by T or B cells in the late phases of activation [37], [38]. PD-1 induces peripheral T or B cell tolerance or turns off unwanted immune responses, upon the simultaneous interaction of the T cell receptor (TCR) or BCR with antigens and of PD-1 with its own ligands (L): PD-L1, which is virtually expressed on all somatic cells (particularly from inflamed tissues) and PD-L2, and is mainly expressed by DCs [37], [54]. Inflamed/infected hepatocytes up-regulate expression of both PD-L1 [55] and class I molecules bearing viral epitopes (whereas the class II are undetectable or only barely expressed) [56]. Consequently, HCV-specific effector PD-1+ CD8 (recognizing class I/epitope complexes on infected hepatocytes) more than PD-1+ CD4 cells should acquire an exhausted/dysfunctional phenotype in the site of infection. This may account for the conserved functional capacities of HCV-specific CD4 T cells shown in patients undergoing infection resolution, despite dysfunctional HCV-specific CD8 T cells (Fig. 1). Otherwise, the lack of functionally competent HCV-specific CD4, associated with exhausted CD8 T cells, would unavoidably lead towards chronic infection in the majority of patients (Fig. 2). The yet unresolved question is what makes the total HCV-specific adaptive (both CD4 and CD8) T cell responses "not-functionally-competent". This is likely conditioned by the emergence of several, non-mutually exclusive factors, such as high viral load, viral epitope escape, viral subversion, and host immunological mechanisms (PD-1, T regulatory [Treg] cells, etc.) addressed to control immunopathology, at the cost of the side effect of limiting protection. All these factors will induce the generation of non-protective virus-specific CD4 and CD8 T cells, which might even become harmful (Fig. 2).
Viral mutations
HCV's strong capability to mutate B or T cell epitopes and possibly to escape related responses at several levels (antigen processing, MHC binding, TCR or BCR recognition, etc.) is due to its high replication rate and the lack of proofreading capacity of its polymerase (reviewed in [1]). The evidence that about 50% of the CD8 epitopes continue to escape [57], [58] leaving another 50% that do not mutate, renders the role of mutational escape in HCV persistence unclear. Despite both the huge T or B cell repertoire and the fact that several viral epitopes do not mutate due to fitness constraints (reviewed in [1]), there are some CD8 escape mutations associated with fitness costs [59], [60], [61]. These, in synergy with additional mechanisms (high viral load, viral subversion, host immune-suppressive mechanisms, etc.), may participate in the establishment of viral persistence, particularly during the course of the acute phase of infection when the highest level of selective pressure occurs (reviewed in [1]).
Virus-induced immune-subversion
As mentioned above, the studies revealing a general subversion of both T cell and DC functions, upon exposure to some HCV proteins in vitro [21], [22], [62], [63], are difficult to reconcile with the fact that chronically-infected individuals are not globally immuno-compromised. However, if in vivo models of HCV infection demonstrate a selective impairment of T cells infiltrating HCV-infected livers due to the high concentrations of viral proteins produced at the site of infection, this may participate in establishing HCV persistence by affecting the local adaptive immune responses.
Regulatory cytokines
A recent report demonstrated that peripheral HCV-specific CD4 and CD8 T cells producing IL-10 are detectable in the early phases of acute HCV infection [64]. These cells seem to suppress antiviral effector responses, promoting chronic evolution of infection, while limiting progressive liver damage [64]. These suggestions are reminiscent of our previous studies showing that IL-10 producing HCV-specific CD8 T cells infiltrate the liver of chronically-infected individuals [65]. They inversely correlated with both the frequency of HCV-specific T cells producing IFN-γ and the inflammatory staging at the level of liver biopsies, suggesting that they modulate excessive liver immunopathology [65]. Accordingly, it has been reported that intrahepatic HCV-specific IL-10 producing CD8 T cells prevent liver damage during chronic infection [66]. TGF-ß-producing virus-specific CD4 and CD8 T cells have been related to antiviral immune suppression and chronic HCV infection evolution [67]. Taken together, these data suggest that regulatory cytokines such as IL-10 or TGF-ß minimize liver inflammation, at the cost of the protective immune responses clearing the virus (Fig. 2).
CD25+Foxp3+ Treg cells
Treg cells expressing the transcription factor forkhead box P (Foxp)3 develop either in the thymus (natural) or in the periphery from conventional CD4+ T cells (induced) [68], [69], [70], [71]. A lack of Foxp3 expression results in the complete absence of Treg cells, which leads to the development of severe autoimmunity, as observed in immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome [68], [69], [70], [71]. The main physiological functions of Treg cells are as follows: (a) to participate in the establishment of peripheral tolerance by inhibiting autoreactive T or B lymphocytes that escaped either thymus or bone marrow checkpoints, respectively (central tolerance), (b) to suppress ongoing protective immune responses once they are no longer necessary or become harmful after the elimination of the pathogen and (c) to limit excessive immunopathology during chronic inflammatory diseases. As a result of the expression of the Il-2 gene-inhibitory Foxp3 transcription factor, Treg cells do not produce IL-2 and are unable to respond to antigens (anergy) [72], [73]. However, Foxp3 activity maintains high levels of IL-2 receptors (CD25hi) on Treg cells, hence compensating for the incapacity of producing IL-2. Indisputably, CD25hi Treg cells promptly proliferate both in vitro and in vivo in response to relevant antigens in the presence of paracrine IL-2, which is mainly produced by responder (effector) T-lymphocytes, but it is dominantly absorbed by Treg cells expressing higher CD25 levels than responder T cells [74], [75], [76]. This appears to represent a key suppression mechanism, because CD25hi Treg cells steal the majority of IL-2 produced by responder T cells that in turn will be deprived of their most important growth factor. Furthermore, Treg cells suppress via different, likely non-mutually exclusive mechanisms, involving membrane molecules (such as cytotoxic T-lymphocyte antigen-4 or adenosine receptors) and suppressive cytokine production (such as TGF-ß or IL-10) [68], [69], [70], [71]. Treg cells are induced and proliferate in response to HCV and seem to modulate liver inflammation in the course of chronic infection [77], [78]. Therefore, the model of HCV infection supports the idea that Treg cells participate in the establishment of a fine equilibrium between immunopathology and immune protection, ultimately resulting in the long-lasting survival of the host during chronic infections [69], [70], [79], [80], [81], [82], [83] (Fig. 2). This would be dependent on a compromise between a status of chronic low-level hepatic inflammation and the generation of antiviral responses that, although unable to clear HCV, are enough to limit excessive viral spread. It is unclear how Treg cells control unwarranted inflammation without completely suppressing the protective immune responses. High CD25 expression by Treg cells drives a positive feedback loop, as the dominant IL-2 capture increases STAT-5 phosphorylation (pSTAT-5) that in turn drives Treg cell proliferation and function. We recently showed that PD-1 is over-expressed on Foxp3+ Treg cells and limits Treg cell proliferation and function during chronic HCV infection. The expression of PD-1, upon contact with its own ligands, inhibits pSTAT-5 via the activation of Src homology 2-containing tyrosine phosphatases (SHPs) [78] (Fig. 3). As a consequence, responder T cells can escape from excessive expansion of Treg cells and render them available for responding to possible novel waves of infection. This negative feedback loop assumes a different significance during chronic infections, such as HCV. The incapacity to clear HCV by the immune system (due to the various mechanisms emphasized above) perpetuates a vicious spiral, whereby responder T cells are chronically stimulated to produce IL-2 that will be dominantly adsorbed by CD25hi Treg cells that in turn will continuously suppress the effector responses. PD-1 up-regulation limits the excessive expansion of Treg cells by controlling pSTAT-5 and fine-tunes Treg function in order to minimize the immunopathology without completely switching off those intended to limit excessive viral spread (Fig. 3). This may represent a critical contra-suppression mechanism that has evolved to assure that Treg cells have limited suppression. Homeostatic balance participates in establishing a status of chronic low-level liver inflammation that is in turn instrumental to ensure long-lasting survival of the host.
Fig. 3. PD-1 controls Treg cells in HCV infection. (A) Responder (CD25lowFoxp3-) TEM cells proliferate in response to HCV antigens, and produce IL-2, which through IL-2R (CD25) signaling, induces pSTAT-5. This leads to the development of the genetic program dictating their effector phenotype. In parallel, the same phenomena occurs for the (CD25hiFoxp3+) Treg cells that do not proliferate to viral antigens alone because of the expression of the Il-2 inhibitory gene Foxp3. Their proliferation is dependent on the dominant capture of paracrine IL-2 that is initially produced by responder T cells. CD25hiFoxp3+ Treg cells can proliferate by the engagement of the Jak3/STAT-5 pathway, and extrinsically down-regulate the TEM cell responses. (B) In the late phases of T cell activation, the death receptors intrinsically deliver negative signals to activated T cells (including Fas, CTLA-4, and PD-1) in order to terminate the T cell responses. PD-1 is up-regulated on both responder and Treg cells and upon contact with PD-L1/2 inhibits pSTAT-5 possibly via SHP2. This mechanism results in limiting both TEM cell responses and excessive Treg cell function. Under conditions resulting in HCV resolution, this loop is self-limited because of the disappearance of the viral antigenic stimuli. During a chronic HCV infection, in which responder T cells have been unable to clear HCV, the negative loop is maintained by the persisting HCV antigens that chronically stimulate IL-2 producing responder T cells. Chronic PD-1 expression on both TEM and Treg cells modulate the potential excessive pSTAT-5-dependent cell proliferation. The resulting contra-regulation of Treg cells will have an important role in limiting excessive suppression of immune responses, controlling the spreading virus at the cost inability to maintain chronic low-level liver immunopathology. This mechanism establishes long-lasting survival of the host.
Conclusions
Through the different (non-mutually exclusive) mechanisms illustrated above, the host survives for a long time in parallel with both the persistent HCV infection and a low-grade liver inflammation that can degenerate into liver failure after several decades. The ambiguous co-existence of virus and inflammation results in an advantage for the evolutionary process and hence for human species survival. If immune responses were invariantly strong and aggressive during a persistent infection such as HCV, they would be unable to eliminate that infection, because of its acquired capacity to escape or to subvert them. In such a situation, exuberant (but non-protective) responses would produce prompt irreversible tissue (hepatic failure) damage, leading to catastrophic epidemic infections. Considering this point of view, chronic (low-level) inflammatory diseases seem to represent a sort of safeguard for the human survival. We can assume that chronic inflammation may be defined as the "Yin and Yang" of the immune system. On the one hand, it guarantees the long-term survival of human hosts despite pathogen persistence. On the other hand, the imbalance of the homeostatic mechanisms maintaining chronic inflammation may degenerate into severe "side-effects" (i.e., the development of either autoimmune diseases or tumours) in a minority of infected individuals. From an evolutionary point of view, the onset of autoimmune diseases or the development of some tumours might be the price to pay following the establishment of chronic inflammation. Indeed, a status of pre-existing chronic inflammation can contribute to the development of cancer, by the production of growth and angiogenic factors eventually promoting cancer-cell survival, implantation, and growth. In addition, chronic inflammation can affect the immune-surveillance directly via its own intrinsic mechanisms (i.e., expansion of Treg cells, T cell exhaustion, etc.), and indirectly by the incapacity to limit the immunosuppressive effects of tumours. The production of soluble factors (i.e., pro-inflammatory or cell growth cytokines) that favor cell proliferation, generally needed for the immune system to defend the host efficaciously, can also facilitate the mitotic cycle of non-lymphoid cells. In the long run, this prolonged stimulation can induce, as in the case of liver cirrhosis by both HBV and HCV, necrosis, cell renewal, and even neoplastic transformation [84]. A further example in HCV infection, is the chronic stimulation of B lymphocytes that can induce the monoclonal expansion of anti-IgG antibodies, which are responsible for the formation of cryoglobulins, autoantibodies, or even the establishment of follicular B cell lymphomas [85].
The immune system simultaneously expresses different strategies that are seemingly opposite but eventually result in an evolutionary advantage. On the one hand, the immune response contributes to species survival; on the other hand it can lead to the sacrifice of single individuals. During the evolutionary process, selective pressure has led to the generation of multiple ambiguous mechanisms to help counteract aggressive infectious agents. Although this is obtained at the cost of severe side-effects (tumour development, autoimmune diseases) in some individuals, these side-effects are considered irrelevant in terms of the survival of the species.
The challenge for scientists is to eliminate the side-effects that emerge in the chronic HCV-host relationship (i.e., cirrhosis, liver failure, HCC, autoimmunity, etc.), possibly via ad hoc modeling and production of new antiviral drugs, immuno-modulatory molecules, therapeutic antiviral antibodies, antiviral small interference (si)RNAs, systems restoring T cell exhaustion (by inhibiting PD-1, Treg cell function, IL-10, or TGF-ß, etc.), and new vaccination strategies.
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Ten Liver-Friendly Tips for Repelling Mosquitoes
by Nicole Cutler, L.Ac.
The arrival of summer puts more people outside to enjoy the warm weather than any other time of year. Although most of us consider summer’s picnics, hikes, swimming and other outdoor- activities to be fun and carefree, this season also has a dark side. Especially for those who tend to be targets for buzzing, biting nuisances, the itchy welts left by mosquitoes can put a damper on any summer celebration. To prevent being bitten by hordes of mosquitoes, many people rely on commercially available insect repellents. Unfortunately, most of these products are too toxic to be safely used by those with a compromised liver.
Any kind of chemical that must be removed from the body’s blood supply puts an added burden on the liver. Those with chronic liver disease are either at risk of or already have a liver with limited capacity to function. Thus, introducing unnecessary chemicals to the liver’s daily workload could put too much stress on the hepatic system and result in subsequent liver cell damage.
DEET
To keep mosquito annoyances at bay, the most popular solution is the topical application of DEET-containing mosquito repellents. While DEET (diethyl-meta-toluamide) is fairly effective at repelling mosquitoes, its toxicity is a major concern for those who have chronic liver disease.
Preventing insect bites from mosquitoes, biting flies, fleas and other small, flying insects, DEET was developed by the U.S. Army in 1946 for protecting soldiers in insect-infested areas. Despite reports confirming DEET’s toxicity, this chemical has been used by the general public in the United States since 1957.
Metabolized by the liver, DEET can find its way into the bloodstream in several ways:
· Skin Absorption – DEET-containing products are usually applied topically – directly on the skin. After being applied, DEET is found in the blood for up to 12 hours. Greater quantities of this chemical are absorbed when it is in a product containing alcohol or when it is combined with a sunscreen.
· Inhalation – DEET can be unintentionally inhaled when insect-repelling sprays are used, especially when applied in indoor spaces where the vapors can linger.
· Ingestion – Although no one purposefully ingests mosquito repellent, accidental ingestion occurs easily when hands are not washed thoroughly after using DEET on the skin.
Regardless of its ability to gain entry into the bloodstream, DEET-based insect repellents represent big business. However, even the Environmental Protection Agency (EPA) hints at DEET’s toxicity. According to the EPA, DEET should not be used frequently. Confirming the need for caution, The American Academy of Pediatrics recommends no more than one application of DEET per day for children. These positions assume that a person’s liver is functioning at 100 percent. Because of the variety of ways DEET can gain access to the bloodstream, those with chronic liver disease are urged to find alternative ways to repel mosquitoes.
Alternative Repellents
Listed below are ten, liver-friendly suggestions for reducing the risk of being a mosquito’s buffet:
1. Wind Power – Because mosquitoes are known, weak fliers, they don’t have the strength to battle wind. Thus, many people are placing powerful fans in outdoor areas, not just to keep cool, but also to blow away biting insects.
2. No Standing Water – Mosquitoes are attracted to and breed in standing water. Therefore, make sure you are not encouraging their presence. Replace all standing water regularly as found in birdbaths, ponds and unfiltered pools and make sure receptacles that could catch water have drainage.
3. Time it Right – Avoid going outdoors during dusk and dawn when mosquitoes are most active.
4. Skip Bananas – When heading outside, choose foods other than bananas and other high-potassium foods. Experts believe eating such foods attracts mosquitoes, because they are attracted to the lactic acid given off after consuming potassium-rich foods.
5. Go for Garlic – Mosquitoes appear to be naturally repelled by its smell, so include fresh garlic in your summer menu planning. Others insist that pinching a garlic clove and rubbing its juice over exposed skin is the best natural mosquito repellent.
6. Screen Repair – Check and repair all screens (doors and windows) for holes or tears that mosquitoes can use to enter a home. Experts suggest putting mesh screening or hardware cloth over bathroom and other vent outlets on the roof to keep insects out.
7. Befriend Bats – One small brown bat can catch about 600 mosquitoes per hour. Many people benefit from installing a bat house on their property to attract these useful insect eaters.
8. Lighten Up – When dressing for the outdoors, opt for white or light colored clothing, because mosquitoes are attracted to dark colors.
9. Try Essential Oils – Sold individually or in combinations as natural insect repellents, there are several essential oils that deter flying pests, including citronella, eucalyptus, geranium, catnip, lavender and clove. Essential oils can be rubbed or sprayed on the skin or clothing, burned in a coil or incense, or infused into a special bracelet.
10. Weed Whack – Remove overgrown and unneeded vegetation from your outdoor space. This eliminates having a desirable place for mosquitoes to rest.
Being outdoors during warm weather involves taking a mosquito gamble – there is no guarantee you will escape their bite. This even applies to those applying DEET-based repellents on a regular basis. However, many people successfully escape mosquito bites with the insect repelling suggestions above without putting their liver health at risk.
References:
http://npic.orst.edu/factsheets/DEETgen.pdf, DEET: General Fact Sheet, Retrieved July 1, 2010, National Pesticide Information Center, 2010.
http://www.care2.com/greenliving/mosquito-free-naturally.html, Mosquito-Free Naturally, Michelle Schroffo Cook, Retrieved July 1, 2010, care2.com, 2010.
http://www.hepatitis-central.com/mt/archives/2009/08/mosquito_repell.html, Mosquito Repellant Warning for Hepatitis C, Nicole Cutler, L.Ac., Retrieved June 30, 2010, Natural Wellness, 2010.
http://www.mosquito.org/resources/summer-safety.aspx, Summer Mosquito Safety, Retrieved July 1, 2010, American Mosquito Control Association, 2010.
http://www.naturalnews.com/001586.html, Chemical Mosquito Repellant DEET Causes Neurological Damage, Gets Absorbed Through The Skin, Mike Adams, Retrieved July 1, 2010, Natural News Network, 2010.
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Small Molecule Modifiers of MicroRNA miR-122 Function for the Treatment of Hepatitis C Virus Infection and Hepatocellular Carcinoma
Douglas D. Young‡, Colleen M. Connelly‡, Christoph Grohmann and Alexander Deiters*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695
J. Am. Chem. Soc., 2010, 132 (23), pp 7976–7981
DOI: 10.1021/ja910275u
Publication Date (Web): May 19, 2010
Copyright © 2010 American Chemical Society
alex_deiters@ncsu.edu, ‡ These authors contributed equally to this work
Abstract
MicroRNAs are a recently discovered new class of important endogenous regulators of gene function. Aberrant regulation of microRNAs has been linked to various human diseases, most importantly cancer. Small molecule intervention of microRNA misregulation has the potential to provide new therapeutic approaches to such diseases. Here, we report the first small molecule inhibitors and activators of the liver-specific microRNA miR-122. This microRNA is the most abundant microRNA in the liver and is involved in hepatocellular carcinoma development and hepatitis C virus (HCV) infection. Our small molecule inhibitors reduce viral replication in liver cells and represent a new approach to the treatment of HCV infections. Moreover, small molecule activation of miR-122 in liver cancer cells selectively induced apoptosis through caspase activation, thus having implications in cancer chemotherapy. In addition to providing a new approach for the development of therapeutics, small molecule modifiers of miR-122 function are unique tools for exploring miR-122 biogenesis.
Source
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695
J. Am. Chem. Soc., 2010, 132 (23), pp 7976–7981
DOI: 10.1021/ja910275u
Publication Date (Web): May 19, 2010
Copyright © 2010 American Chemical Society
alex_deiters@ncsu.edu, ‡ These authors contributed equally to this work
Abstract
MicroRNAs are a recently discovered new class of important endogenous regulators of gene function. Aberrant regulation of microRNAs has been linked to various human diseases, most importantly cancer. Small molecule intervention of microRNA misregulation has the potential to provide new therapeutic approaches to such diseases. Here, we report the first small molecule inhibitors and activators of the liver-specific microRNA miR-122. This microRNA is the most abundant microRNA in the liver and is involved in hepatocellular carcinoma development and hepatitis C virus (HCV) infection. Our small molecule inhibitors reduce viral replication in liver cells and represent a new approach to the treatment of HCV infections. Moreover, small molecule activation of miR-122 in liver cancer cells selectively induced apoptosis through caspase activation, thus having implications in cancer chemotherapy. In addition to providing a new approach for the development of therapeutics, small molecule modifiers of miR-122 function are unique tools for exploring miR-122 biogenesis.
Source
A new opportunity for hepatitis C research
This image shows infected human cells with miceCD81.
Public release date: 5-Jul-2010
Contact: Dr. Thomas Pietschmann
thomas.pietschmann@twincore.de
49-511-220-027-130
Helmholtz Association of German Research Centres
Scientists at TWINCORE develop new model approaches for HCV research
The hepatitis C virus is highly specialised. We humans are its natural hosts. The only other living organisms that could be infected with the hepatitis C virus in the lab are chimpanzees. Nevertheless it is – from the viewpoint of the virus – highly successful: around 170 million people are chronically infected with the virus. And with the chronic infection the risk of developing liver cancer also increases.
Researchers worldwide are working to develop vaccines and medication to combat the virus. The problem is that although they are able to research in liver cell cultures, when they want to find out how the immune system controls an infection or whether possible vaccines are effective research comes up against a brick wall: tests at such an early stage are unthinkable for humans or chimpanzees.
At TWINCORE researchers are now adapting the HCV to mice, thus enabling immunologists and vaccine researchers to take the next steps against this illness in the future. Because the immune system of mice is very similar to that of humans and it is only when vaccines are successful and safe in animal experiments that researchers can take the risk of transferring them to humans.
The fact that HCV can only infect humans and chimpanzees is partly down to the highly complicated mechanism with which it accesses the cell. The virus has to first bind four different molecules on the surface of our liver cells. This triggers a mechanism in our cells that transports the virus into the liver cells. "Mice also have these receptors on their liver cells in principle," says scientist Julia Bitzegeio of the Department of Experimental Virology at TWINCORE, "however, they do not fit those on the surface of the virus."
The two molecules that cause particular difficulty are called CD81 and occludin – these need to be human, otherwise the virus has no chance of infecting the cell. To make the HCV "mouse-capable" the researchers resorted to a trick: they have removed the CD81 receptor from human liver cells and replaced it with mouse CD81. In an electrical field they then tore tiny holes in the cell membrane before inserting the HC virus artificially through these holes. "The virus reproduced inside the cells and we repeatedly inserted the virus into the altered liver cells," explains Julia Bitzegeio. This led to the highly transformable virus gradually changing until it was able to penetrate the cells with mouse CD81 receptor even without assistance.
"In this selection process the surface of the virus altered so much that it continued to infect human cells very quickly, but also simple mouse cells containing the four mouse variants of the HCV receptors," says Research Group Leader Professor Thomas Pietschmann. The mouse-adapted virus is able to penetrate the mouse cells; however, the human specialisation of the HC virus is so high that it is unable to reproduce in the cells. "Successful infiltration is the first step towards a new small animal model, one that is urgently required for immunological investigations and the development of vaccines against HCV."
TWINCORE is an joint venture between Helmholtz-Center for Infection Research at Braunschweig an the Hannover Medical School.
Literature: Bitzegeio J, Bankwitz D, Hueging K, Haid S, Brohm C, et al. (2010) Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors. PLoS Pathog 6(7): e1000978. doi:10.1371/journal.ppat.1000978
Further information: http://www.twincore.de/
Pictures: presse@twincore.de
Legend: Infected human cells with miceCD81(Copyright: TWINCORE)
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Three Rivers Pharmaceuticals Announces FDA Approval Of Expanded Labeling of INFERGEN®
July 06, 2010 08:00 AM Eastern Daylight Time.
New Labeling Provides Alternative for Hepatitis C Patients Who Need Retreatment
WARRENDALE, Pa.--(BUSINESS WIRE)--Three Rivers Pharmaceuticals, LLC, today received expanded labeling from the U.S. Food and Drug Administration (FDA) to include daily use of INFERGEN (Consensus Interferon) in combination with ribavirin (RBV) for retreatment of chronic hepatitis C patients. The expanded labeling targets hepatitis C patients who need retreatment. In the clinical trial leading to the expanded labeling, the primary endpoint of increased sustained virological response (SVR) was achieved demonstrating that INFERGEN provides a second chance for patients to clear their hepatitis C virus.
“The FDA’s recognition of this expanded label allows patients failing therapy a safe and efficacious retreatment strategy. The results from this study legitimize how we properly treat these patients helping them to achieve SVR.”
“Approximately 50 percent of patients with chronic hepatitis C do not respond to their initial course of therapy,” stated Dr. Bruce Bacon, the lead investigator for the registration trial. “The FDA’s recognition of this expanded label allows patients failing therapy a safe and efficacious retreatment strategy. The results from this study legitimize how we properly treat these patients helping them to achieve SVR.”
The data reported and published in Hepatology (2009) from the U.S.-based, randomized, DIRECT clinical trial (Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy) led to the approval of the expanded label. Results from the DIRECT trial had shown that the use of INFERGEN and RBV is a safe and effective retreatment strategy for patients failing initial therapy with PEG-IFN/RBV. This was especially apparent with interferon-sensitive patients with lower baseline fibrosis scores. In fact, up to 38 percent of non-cirrhotic patients (in the 15mcg arm) who were sensitive to Peg-IFN/RBV and who did not modify their INFERGEN and RBV dosages achieved an SVR. Additionally, patients with cirrhosis were less likely to benefit from retreatment with INFERGEN and RBV unless they displayed previous interferon sensitivity or at least 1-log10 drop in viral levels on prior therapy.
“The expanded labeling for INFERGEN is a significant step forward for retreatment of hepatitis C patients who deserve a second chance to overcome their HCV,” stated Patrick Kerrish, R.Ph, M.B.A., President of Three Rivers Pharmaceuticals, LLC. “Our hope is that INFERGEN will become the standard of care for the retreatment of chronic hepatitis C patients.”
Three Rivers Pharmaceuticals, LLC is expanding its INFERGEN and hepatitis C education efforts via physician outreach and online patient support programs. An instructional video and guide about proper at-home injections of INFERGEN, tips for patient compliance, access to reimbursement specialists and a hotline staffed by nurse counselors are available at http://www.infergen.com/.
About Consensus Interferon (INFERGEN®)
Consensus Interferon or INFERGEN is a unique, bio-optimized, selective and highly potent type 1 interferon alpha. Because it is a bioengineered interferon, it is also different from other interferons currently used to treat chronic hepatitis C virus. INFERGEN is indicated for the treatment of chronic hepatitis C infection in patients 18 years of age or older with compensated liver disease. Additionally, INFERGEN is now FDA approved for daily use in combination with ribavirin in HCV patients who need retreatment. More prescribing information regarding INFERGEN, including the product’s safety profile and the box warning for all interferon alphas regarding neuropsychiatric, autoimmune, ischemic and infectious disorders, are available by visiting the product website at www.infergen.com.
About Hepatitis C
Hepatitis means inflammation of the liver. Hepatitis C is a liver disease that results from infection with the hepatitis C virus. It can range in severity from a mild illness lasting a few weeks to a serious, lifelong illness. Hepatitis C virus can be either "acute" or "chronic." Acute hepatitis C virus infection is a short-term illness that occurs within the first 6 months after someone is exposed to the hepatitis C virus. Seventy five-85% of acute HCV infections become chronic HCV infections. Chronic hepatitis C virus is a serious disease than can result in long-term health problems, or even death. There is currently no vaccine available for prevention of hepatitis C virus.
About Three Rivers Pharmaceuticals
Three Rivers Pharmaceuticals is a privately held company headquartered in Warrendale, Pennsylvania and focuses on specialized therapies including hepatitis C therapies. With its unique experience and understanding of the complex challenges of treating chronic, difficult diseases, Three Rivers is a valuable partner in the healthcare community. The company's mission is to develop, manufacture, and market the highest quality branded and generic drug products for patients with serious diseases. More information on the company is available at http://www.3riverspharma.com/.
Contacts
Pascale Communications, LLC
Audra B. Friis
631-462-1726 (office)
917-519-9577 (mobile)
audra@pascalecommunications.com
New Labeling Provides Alternative for Hepatitis C Patients Who Need Retreatment
WARRENDALE, Pa.--(BUSINESS WIRE)--Three Rivers Pharmaceuticals, LLC, today received expanded labeling from the U.S. Food and Drug Administration (FDA) to include daily use of INFERGEN (Consensus Interferon) in combination with ribavirin (RBV) for retreatment of chronic hepatitis C patients. The expanded labeling targets hepatitis C patients who need retreatment. In the clinical trial leading to the expanded labeling, the primary endpoint of increased sustained virological response (SVR) was achieved demonstrating that INFERGEN provides a second chance for patients to clear their hepatitis C virus.
“The FDA’s recognition of this expanded label allows patients failing therapy a safe and efficacious retreatment strategy. The results from this study legitimize how we properly treat these patients helping them to achieve SVR.”
“Approximately 50 percent of patients with chronic hepatitis C do not respond to their initial course of therapy,” stated Dr. Bruce Bacon, the lead investigator for the registration trial. “The FDA’s recognition of this expanded label allows patients failing therapy a safe and efficacious retreatment strategy. The results from this study legitimize how we properly treat these patients helping them to achieve SVR.”
The data reported and published in Hepatology (2009) from the U.S.-based, randomized, DIRECT clinical trial (Daily-Dose Consensus Interferon and Ribavirin: Efficacy of Combined Therapy) led to the approval of the expanded label. Results from the DIRECT trial had shown that the use of INFERGEN and RBV is a safe and effective retreatment strategy for patients failing initial therapy with PEG-IFN/RBV. This was especially apparent with interferon-sensitive patients with lower baseline fibrosis scores. In fact, up to 38 percent of non-cirrhotic patients (in the 15mcg arm) who were sensitive to Peg-IFN/RBV and who did not modify their INFERGEN and RBV dosages achieved an SVR. Additionally, patients with cirrhosis were less likely to benefit from retreatment with INFERGEN and RBV unless they displayed previous interferon sensitivity or at least 1-log10 drop in viral levels on prior therapy.
“The expanded labeling for INFERGEN is a significant step forward for retreatment of hepatitis C patients who deserve a second chance to overcome their HCV,” stated Patrick Kerrish, R.Ph, M.B.A., President of Three Rivers Pharmaceuticals, LLC. “Our hope is that INFERGEN will become the standard of care for the retreatment of chronic hepatitis C patients.”
Three Rivers Pharmaceuticals, LLC is expanding its INFERGEN and hepatitis C education efforts via physician outreach and online patient support programs. An instructional video and guide about proper at-home injections of INFERGEN, tips for patient compliance, access to reimbursement specialists and a hotline staffed by nurse counselors are available at http://www.infergen.com/.
About Consensus Interferon (INFERGEN®)
Consensus Interferon or INFERGEN is a unique, bio-optimized, selective and highly potent type 1 interferon alpha. Because it is a bioengineered interferon, it is also different from other interferons currently used to treat chronic hepatitis C virus. INFERGEN is indicated for the treatment of chronic hepatitis C infection in patients 18 years of age or older with compensated liver disease. Additionally, INFERGEN is now FDA approved for daily use in combination with ribavirin in HCV patients who need retreatment. More prescribing information regarding INFERGEN, including the product’s safety profile and the box warning for all interferon alphas regarding neuropsychiatric, autoimmune, ischemic and infectious disorders, are available by visiting the product website at www.infergen.com.
About Hepatitis C
Hepatitis means inflammation of the liver. Hepatitis C is a liver disease that results from infection with the hepatitis C virus. It can range in severity from a mild illness lasting a few weeks to a serious, lifelong illness. Hepatitis C virus can be either "acute" or "chronic." Acute hepatitis C virus infection is a short-term illness that occurs within the first 6 months after someone is exposed to the hepatitis C virus. Seventy five-85% of acute HCV infections become chronic HCV infections. Chronic hepatitis C virus is a serious disease than can result in long-term health problems, or even death. There is currently no vaccine available for prevention of hepatitis C virus.
About Three Rivers Pharmaceuticals
Three Rivers Pharmaceuticals is a privately held company headquartered in Warrendale, Pennsylvania and focuses on specialized therapies including hepatitis C therapies. With its unique experience and understanding of the complex challenges of treating chronic, difficult diseases, Three Rivers is a valuable partner in the healthcare community. The company's mission is to develop, manufacture, and market the highest quality branded and generic drug products for patients with serious diseases. More information on the company is available at http://www.3riverspharma.com/.
Contacts
Pascale Communications, LLC
Audra B. Friis
631-462-1726 (office)
917-519-9577 (mobile)
audra@pascalecommunications.com
Clinical Trial of GeoVax’s AIDS Vaccine Moves Forward
5 July 2010
A year ago, we reported on the push to discover an effective AIDS vaccine. As President Obama prepares to release his U.S. AIDS strategy, at least one pharmaceutical company is readying a crucial clinical trial of its vaccine against HIV/AIDS.
The AIDS Research Consortium of Atlanta recently put out a call for volunteers to test a DNA-based AIDS vaccine developed by GeoVax. Participants must have had a negative HIV test followed by a positive test six months later, and must have started drugs to fight the virus within the past six months. A total of 10 to 12 patients will be enrolled in the Phase I study. Participants will be monitored for up to 77 weeks. Although GeoVax is already testing the vaccine for virus prevention, this will be the first study to test the vaccine in individuals who already have the virus.
Other updates: A lab devoted to developing an HIV/AIDS vaccine has opened in Brooklyn, New York. Researchers at SUNY Downstate’s Incubator will work with the International AIDS Vaccine Initiative, which has discovered two antibodies that are effective against multiple strains of HIV. The facility also has labs devoted to finding a cure for hepatitis C, developing a home blood test for cancer, and more.
Mymetics Corporation is currently testing the first human volunteers in a clinical trial of its preventive AIDS vaccine. The vaccine was well-tolerated by participants. The new study follows an animal trial in which the vaccine demonstrated the ability to prevent infection with the HIV virus. Participants in the human study will be followed for 12 months. The CDC estimates that 1.2 million people in the U.S. have HIV, the virus that causes AIDS.
Do you think the U.S. pharmaceutical industry has made adequate progress over the past year in the search for an HIV/AIDS vaccine? What needs to happen next? Feel free to leave your feedback in comments.
Source
A year ago, we reported on the push to discover an effective AIDS vaccine. As President Obama prepares to release his U.S. AIDS strategy, at least one pharmaceutical company is readying a crucial clinical trial of its vaccine against HIV/AIDS.
The AIDS Research Consortium of Atlanta recently put out a call for volunteers to test a DNA-based AIDS vaccine developed by GeoVax. Participants must have had a negative HIV test followed by a positive test six months later, and must have started drugs to fight the virus within the past six months. A total of 10 to 12 patients will be enrolled in the Phase I study. Participants will be monitored for up to 77 weeks. Although GeoVax is already testing the vaccine for virus prevention, this will be the first study to test the vaccine in individuals who already have the virus.
Other updates: A lab devoted to developing an HIV/AIDS vaccine has opened in Brooklyn, New York. Researchers at SUNY Downstate’s Incubator will work with the International AIDS Vaccine Initiative, which has discovered two antibodies that are effective against multiple strains of HIV. The facility also has labs devoted to finding a cure for hepatitis C, developing a home blood test for cancer, and more.
Mymetics Corporation is currently testing the first human volunteers in a clinical trial of its preventive AIDS vaccine. The vaccine was well-tolerated by participants. The new study follows an animal trial in which the vaccine demonstrated the ability to prevent infection with the HIV virus. Participants in the human study will be followed for 12 months. The CDC estimates that 1.2 million people in the U.S. have HIV, the virus that causes AIDS.
Do you think the U.S. pharmaceutical industry has made adequate progress over the past year in the search for an HIV/AIDS vaccine? What needs to happen next? Feel free to leave your feedback in comments.
Source
Targeting a Master Regulator of Disease
Drugmakers place big bets on the emerging science of microRNA.
By Arlene Weintraub
Tuesday, July 06, 2010
San Diego startup Regulus, founded in 2007, has quietly been working on a new way to target RNA for drug development. The company has been studying a subset of RNA molecules called microRNAs, or miRNAs. First discovered in the 1990s, misbehaving miRNAs have been linked to several diseases, including cancer and heart failure. Drug developers hope these molecules will prove to be particularly effective drug targets because manipulating just one seems to suppress several disease-linked proteins--whereas most biotech drugs only target individual proteins.
Regulus is co-owned by Alnylam and Isis, leaders in RNA-based drug development. While it is just one of a handful of startups developing miRNA therapeutics, it has attracted significant attention from big pharmaceutical companies. Last month, French pharmaceutical giant Sanofi-Aventis announced a research alliance with the company. Sanofi has pledged up to $750 million in payments, including $35 million up front to Regulus--an unusually large investment in such early-stage science. Sanofi and Regulus will work together to target fibrosis, an excessive buildup of hard collagen that can wreak havoc on the heart, kidneys, and other organs. Regulus already has a multimillion-dollar alliance with GlaxoSmithKline to codevelop drugs to treat immune diseases and a hepatitis C treatment.
While many RNAs encode proteins, miRNAs instead regulate the expression of multiple genes by preventing protein-coding RNAs from fulfilling their function. That, in turn, controls everything from metabolism to immune response to muscle development. About 700 miRNAs have been identified so far.
"The analogy we like to use is that miRNA is the maestro conducting the orchestra--the entire biological network," says Zak Zimmerman, Regulus's director of business development. "If something goes wrong with the maestro, the orchestra starts to play off-key."
Regulus has synthesized several compounds that block or modify "oligonucleotides"--the chains of nucleotides that comprise miRNAs. In 2008, the company demonstrated that it could inhibit a particular miRNA in mouse hearts, reversing a fibrotic condition that causes heart failure. Regulus is also researching potential remedies for renal fibrosis, a major cause of kidney failure and common complication among transplant patients. Sanofi had a team of scientists researching miRNA for quite some time, but they lacked the chemistry know-how to transform their discoveries into drugs.
Despite their promise, translating miRNA targeting compounds into safe drugs is likely to prove challenging. MiRNAs control many bodily processes, so altering them can cause unwanted side effects. "They're promiscuous--they affect multiple downstream components," says Sumit Chanda, associate professor at the Sandford Burnham Medical Research Institute in La Jolla, CA. "The more targets you take out, the more toxicities there can be."
Regulus's scientists acknowledge that they'll have to perform extensive toxicity testing before they identify molecules that are safe for testing in people. Funding from Sanofi and GSK will help move that testing forward. The company expects to choose its first drug candidate for clinical trials by the end of this year.
Also See:
Micromanaging RNA Researchers target a tiny strand of RNA to try to treat hepatitis C.
Hepatitis C Drug Targets RNA A new drug suppresses the virus in chimps without generating resistance.
Source
By Arlene Weintraub
Tuesday, July 06, 2010
San Diego startup Regulus, founded in 2007, has quietly been working on a new way to target RNA for drug development. The company has been studying a subset of RNA molecules called microRNAs, or miRNAs. First discovered in the 1990s, misbehaving miRNAs have been linked to several diseases, including cancer and heart failure. Drug developers hope these molecules will prove to be particularly effective drug targets because manipulating just one seems to suppress several disease-linked proteins--whereas most biotech drugs only target individual proteins.
Regulus is co-owned by Alnylam and Isis, leaders in RNA-based drug development. While it is just one of a handful of startups developing miRNA therapeutics, it has attracted significant attention from big pharmaceutical companies. Last month, French pharmaceutical giant Sanofi-Aventis announced a research alliance with the company. Sanofi has pledged up to $750 million in payments, including $35 million up front to Regulus--an unusually large investment in such early-stage science. Sanofi and Regulus will work together to target fibrosis, an excessive buildup of hard collagen that can wreak havoc on the heart, kidneys, and other organs. Regulus already has a multimillion-dollar alliance with GlaxoSmithKline to codevelop drugs to treat immune diseases and a hepatitis C treatment.
While many RNAs encode proteins, miRNAs instead regulate the expression of multiple genes by preventing protein-coding RNAs from fulfilling their function. That, in turn, controls everything from metabolism to immune response to muscle development. About 700 miRNAs have been identified so far.
"The analogy we like to use is that miRNA is the maestro conducting the orchestra--the entire biological network," says Zak Zimmerman, Regulus's director of business development. "If something goes wrong with the maestro, the orchestra starts to play off-key."
Regulus has synthesized several compounds that block or modify "oligonucleotides"--the chains of nucleotides that comprise miRNAs. In 2008, the company demonstrated that it could inhibit a particular miRNA in mouse hearts, reversing a fibrotic condition that causes heart failure. Regulus is also researching potential remedies for renal fibrosis, a major cause of kidney failure and common complication among transplant patients. Sanofi had a team of scientists researching miRNA for quite some time, but they lacked the chemistry know-how to transform their discoveries into drugs.
Despite their promise, translating miRNA targeting compounds into safe drugs is likely to prove challenging. MiRNAs control many bodily processes, so altering them can cause unwanted side effects. "They're promiscuous--they affect multiple downstream components," says Sumit Chanda, associate professor at the Sandford Burnham Medical Research Institute in La Jolla, CA. "The more targets you take out, the more toxicities there can be."
Regulus's scientists acknowledge that they'll have to perform extensive toxicity testing before they identify molecules that are safe for testing in people. Funding from Sanofi and GSK will help move that testing forward. The company expects to choose its first drug candidate for clinical trials by the end of this year.
Also See:
Micromanaging RNA Researchers target a tiny strand of RNA to try to treat hepatitis C.
Hepatitis C Drug Targets RNA A new drug suppresses the virus in chimps without generating resistance.
Source
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