Kurt Grüngreiff

Evaluation of liver disease progression in the German hepatitis C virus (1b)‐contaminated anti‐D cohort at 35 years after infection

The natural course of HCV infection remains controversial. The German HCV (1b)‐contaminated anti‐D cohort provides an ideal population to investigate the natural course of HCV infection in a large, homogenous cohort of young women from the date of HCV inoculation. Our previous follow‐up studies at 20 and 25 years after infection suggested slow fibrosis progression rates in this unique cohort. The aim of our prospective, community‐based, multicenter study was to reevaluate the liver disease progression in 718 patients of the original anti‐D cohort at 35 years after infection. Patients with self‐limited HCV infection (n = 189) were compared to those who failed to eliminate the virus spontaneously (n = 529), comprising patients who were treatment naïve (n = 197) or achieved a sustained virological response (SVR; n = 149), respectively, failed to clear the virus (non‐SVR; n = 183) after antiviral therapy. In the overall cohort, 9.3% of patients showed clinical signs of liver cirrhosis at 35 years after infection. Liver disease progression largely depended on HCV infection status. The highest proportion of patients with clinical signs of end‐stage liver disease was observed in the non‐SVR group (15.3%), whereas decreased cirrhosis rates were detected in the SVR group (6%) and in patients with self‐limited HCV infection (1.1%; P = 6.2 × 10−6). Overall survival was significantly enhanced after SVR, compared to treatment‐naïve patients or non‐SVR (P = 0.027). Conclusion: The present study provides further evidence for a mild, but significant, disease progression at 35 years after infection in the German HCV (1b)‐contaminated anti‐D cohort. Patients with self‐limited HCV infection or SVR after antiviral treatment were protected from progressive liver disease and showed the best clinical long‐term outcome. (Hepatology 2014;58:49–57)

The predictive value of IL28B gene polymorphism for spontaneous clearance in a single source outbreak cohort is limited in patients carrying the CCR5Δ32 mutation

BACKGROUND & AIMS The CCR5Δ32 mutation has been suspected to adversely affect outcomes of HCV infection, although reports have remained controversial. Here, we investigated the relative genetic contributions of the CCR5Δ32 deletion and the IL28B rs12979860 polymorphisms to spontaneous clearance of hepatitis C in a single-source outbreak. METHODS We retrieved 396 Caucasian women (119 women with spontaneous HCV clearance) who had been infected with HCV genotype 1-contaminated anti-D immunoglobulin in 1978, and determined their IL28B and CCR5 alleles. RESULTS IL28B CC, CT, and TT genotypes were found in 35.4%, 50%, and 14.6% of patients and corresponded to spontaneous clearance rates of 50%, 21.2%, and 12.1% (Chi(2)=38.7, p=5.0×10(-10)), respectively. CCR5 WT/WT, WT/Δ32, and Δ32/Δ32 genotypes were observed in 76%, 22.7%, and 1.3% of patients and corresponded to clearance rates of 33.2%, 21.2%, and 0% (Chi(2)=6.9, p=0.009), respectively. In a stepwise forward-conditional multivariate regression model both CCR5 (OR 2.1, p=0.01 for WT/WT) and IL28B genetic variants (OR 4.3, p=4.6×10(-10) for the C/C genotype) were identified as independent predictors of spontaneous HCV clearance. Importantly, favorable response rates were associated with the IL28B CC genotype only in CCR5 wild-type homozygous women, while HCV clearance in CCR5Δ32 carriers remained poor even in patients with the rs12979860 CC genotype. CONCLUSIONS Both IL28B rs1297860 and CCR5Δ32 allelic variants are independent genetic determinants of spontaneous HCV clearance. The variable relative distribution between IL28B rs1297860 and CCR5Δ32 allelic variants in different populations may have masked the role of the CCR5Δ32 mutation in some studies.

Plasma concentrations of zinc, copper, interleukin-6 and interferon-γ, and plasma dipeptidyl peptidase IV activity in chronic hepatitis C

Copper and zinc are essential trace elements which play an important role in various biological processes. Along with various cytokines, glucocorticoids, glucagon and insulin, the acute-phase protein metallothionein is involved in the regulation of immune cell functions. Metallothionein is the central protein regulating zinc concentration. Zinc deficiency is often found in patients with chronic liver disease, chronic kidney disease and diabetes mellitus, and in those with acute infectious diseases. In contrast, copper deficiency is rarely reported. In order to determine whether there is a correlation between zinc and/or copper and selected immunological parameters in patients with chronic liver disease, we investigated plasma levels of zinc and copper, concentrations of interleukin-6 (IL-6) and interferon-γ (IFN-γ), and the enzymatic activity of dipeptidyl peptidase IV (DP IV, CD26) in patients with chronic hepatitis C and in healthy control subjects. Whereas zinc plasma levels did not differ between patients and control subjects, copper concentrations revealed gender-specific differences. The mean copper concentration was higher in female patients with chronic hepatitis C and in female controls compared with the respective male groups. The immunological parameters of IFN-γ concentration and DP IV activity showed similar levels in the patient and control groups. Of note, the mean IL-6 plasma concentrations were significantly elevated in patients with chronic hepatitis C compared with healthy control subjects. In summary, there was no correlation between either zinc or copper concentrations and the immunological parameters measured (IL-6 and IFN-γ levels and DP IV activity) in patients with chronic hepatitis C and in healthy control subjects.

The role of zinc in liver cirrhosis.

Zinc is an essential trace element playing fundamental roles in cellular metabolism. It acts mostly by binding a wide range of proteins, thus affecting a broad spectrum of biological processes, which include cell division, growth and differentiation. Zinc is critical to a large number of structural proteins, enzymatic processes, and transcription factors. Zinc deficiency can result in a spectrum of clinical manifestations, such as poor of appetite, loss of body hair, altered taste and smell, testicular atrophy, cerebral and immune dysfunction, and diminished drug elimination capacity. These are common symptoms in patients with chronic liver diseases, especially liver cirrhosis. The liver is the main organ responsible for the zinc metabolism which can be affected by liver diseases. On the other hand, zinc deficiency may alter hepatocyte functions and also immune responses in inflammatory liver diseases. Liver cirrhosis represents the most advanced stage of chronic liver diseases and is the common outcome of chronic liver injury. It is associated with energy malnutrition, with numerous metabolic disorders, such as hypoalbuminemia, with imbalance between branched-chain amino acids and aromatic amino acids, and with reduced zinc serum concentrations. All these processes can influence the clinical outcome of patients, such ascites, hepatic encephalopathy and hepatocellular carcinoma. In the present review, we summarize the emerging evidence on the pitoval role of zinc in the pathogenesis of liver cirrhosis.Zinc is an essential trace element playing fundamental roles in cellular metabolism. It acts mostly by binding a wide range of proteins, thus affecting a broad spectrum of biological processes, which include cell division, growth and differentiation. Zinc is critical to a large number of structural proteins, enzymatic processes, and transcription factors. Zinc deficiency can result in a spectrum of clinical manifestations, such as poor of appetite, loss of body hair, altered taste and smell, testicular atrophy, cerebral and immune dysfunction, and diminished drug elimination capacity. These are common symptoms in patients with chronic liver diseases, especially liver cirrhosis. The liver is the main organ responsible for the zinc metabolism which can be affected by liver diseases. On the other hand, zinc deficiency may alter hepatocyte functions and also immune responses in inflammatory liver diseases. Liver cirrhosis represents the most advanced stage of chronic liver diseases and is the common outcome of chronic liver injury. It is associated with energy malnutrition, with numerous metabolic disorders, such as hypoalbuminemia, with imbalance between branched-chain amino acids and aromatic amino acids, and with reduced zinc serum concentrations. All these processes can influence the clinical outcome of patients, such ascites, hepatic encephalopathy and hepatocellular carcinoma. In the present review, we summarize the emerging evidence on the pitoval role of zinc in the pathogenesis of liver cirrhosis.

Immunobiology of zinc and zinc therapy

In a recent article in Immunology Today, Wellinghausen et al.1xWellinghausen, N., Kirchner, H., and Rink, L. Immunol. Today. 1997; 18: 519–521Abstract | Full Text PDF | PubMed | Scopus (164)See all References1 reviewed the various interactions between zinc and the immune system. The authors clearly pointed out that zinc (zinc ions and/or chelated zinc) plays an important role in the maintenance of immune function. Zinc deficiency results in hypoplasia of the immune system, impaired immune response, poor wound healing, diminished T-cell dependent reactions, and attenuated chemotaxis by neutrophils and monocytes. It should be underlined, however, that zinc therapy has to be discussed very carefully and a number of important factors have to be addressed.Recent observations indicate that zinc doses as low as 50 mg/day over a prolonged period in healthy individuals can induce subtle impairment of immunologic responses2xChandra, R.K. and McBean, L.D. Nutrition. 1994; 10: 79–80PubMedSee all References2. The pharmacological interaction of zinc with the immune system has implications for the long-term administration of zinc supplements. In a study of 11 healthy young men, 150 mg zinc given twice daily decreased the lymphocyte proliferation response to phytohemagglutinin and reduced the chemotaxis and phagocytosis of neutrophils3xChandra, R.K. J. Am. Med. Assoc. 1984; 252: 1443–1447Crossref | Scopus (322)See all References3. In a clinical trial in which elderly subjects were given 100 mg zinc per day, the delayed-hypersensitivity response was reduced4xBogden, J.D., Olesky, J.M., and Lavenhaar, M.A. J. Am. Coll. Nutr. 1990; 9: 214–219Crossref | PubMed | Scopus (99)See all References4.Our studies show that zinc affects DNA synthesis and cytokine production [interleukin 2 (IL-2), IL-6, IL-10] by pokeweed mitogen-stimulated peripheral blood mononuclear cells in a concentration-dependent manner5xReinhold, D., Ansorge, S., and Grungreiff, K. J. Trace Elem. Exp. Med. 1997; 10: 19–27Crossref | Scopus (12)See all References5. Both functions of immune cells were strongly suppressed by zinc at a concentration of 0.2 mm. These findings are in accordance with the observations of Wellinghausen et al. (toxic dose for T cells: 96–128 μm; for monocytes: 38–512 μm)1xWellinghausen, N., Kirchner, H., and Rink, L. Immunol. Today. 1997; 18: 519–521Abstract | Full Text PDF | PubMed | Scopus (164)See all References1.Furthermore, we found that zinc levels higher than 0.5 mm – equivalent to a daily dose of ∼45 mg zinc salt – had a toxic effect on immune cells5xReinhold, D., Ansorge, S., and Grungreiff, K. J. Trace Elem. Exp. Med. 1997; 10: 19–27Crossref | Scopus (12)See all References5. Prasad6xPrasad, A.S. Scand. J. Work Environ. Health. 1993; 19: 134–136PubMedSee all References6 suggests that orally ingested zinc at doses up to 45 mg/day (elemental zinc) is virtually nontoxic in adults.We have also examined the effect of long-term, low-dose zinc supplementation [zinc-hydrogenaspartate; UNIZINK 50 three times daily (29.76 mg/day)] on serum levels of IL-6 and IL-10 in 16 patients with chronic liver disease: all had reduced serum zinc levels, in most due to liver cirrhosis5xReinhold, D., Ansorge, S., and Grungreiff, K. J. Trace Elem. Exp. Med. 1997; 10: 19–27Crossref | Scopus (12)See all References5. The liver, which plays an important role in zinc metabolism, contains two pools of exchangeable zinc in the cells, one rapidly exchangeable, the other slowly exchangeable. A reduction in liver zinc content can hinder both regeneration and recovery of liver cells. Patients with chronic liver disease, particularly liver cirrhosis, frequently have endotoxemia, increased serum concentrations of cytokines, notably IL-6, and reduced serum zinc levels. In liver disease, the decline in serum zinc levels is due to diminished hepatic extraction, portosystemic shunts, alcohol-induced disturbed absorption and is possibly influenced by cytokines, such as IL-6 (Ref. 5xReinhold, D., Ansorge, S., and Grungreiff, K. J. Trace Elem. Exp. Med. 1997; 10: 19–27Crossref | Scopus (12)See all ReferencesRef. 5). In our study, we showed that in most patients zinc supplementation decreases serum levels of certain cytokines, especially IL-6, but also IL-10, and leads to a normalization of serum zinc concentrations.In conclusion, our results show that zinc affects a functional activation or inhibition of isolated immune cells in a concentration-dependent manner. The critical concentration is 0.5 mm, equivalent to a daily dose of ∼45 mg zinc salt. In patients with diminished serum zinc levels due to chronic liver disease, zinc supplementation also appears to influence cell growth and cytokine production. These findings suggest new uses for zinc supplementation, but they also reveal the potential risks of zinc therapy.

Zinc: A complementary factor in the treatment of chronic hepatitis C? (Review).

Chronic hepatitis C (HCV) infection persists in more than 170 million people worldwide and is one of the major causes of hepatic failure and liver transplantation. Current treatment of chronic HCV, consisting of pegylated interferon and ribavirin, is associated with a wide range of side effects, contraindications and costs, and leads to viral clearance in only 50-55% (genotype 1) to 80% (genotype 3) of cases. Thus, the development of more efficient treatment regimes with fewer side effects and costs is of high priority. It is generally accepted that the cellular immune response plays the most important role in determining the outcome of HCV infection. Moreover, oxidative stress is considered to be an important pathogenic factor. Zinc is an essential nutrient for a broad range of biological activities. It is necessary for normal liver function, and vice versa the liver plays a central role in zinc homeostasis. Zinc ions are crucial for multiple aspects of the immune system, including the normal development, differentiation and function of cells belonging to both innate and acquired immunity. Among the immune cells that are affected by zinc deficiency, T lymphocytes are noted to have the highest susceptibility. Zinc deficiency causes substantial impairment of cellular immunity, oxidation and damage to DNA. Several studies have investigated the effect of zinc supplementation in chronic HCV patients. Following zinc supplementation, decreases in the incidence of gastrointestinal disturbances, body weight loss and hair loss were found in patients with chronic HCV, along with improved fingernail health. In addition, zinc administered in combination with IFN-α was more effective against chronic HCV than treatment with IFN-α alone. Finally, in addition to the effects of zinc on immune functions and viral defence, its role as an antioxidant may be important in HCV. To conclude, the controlled application of zinc, particularly in a deficient state, is recommended as a complementary therapy for chronic hepatitis C.

Zinc aspartate suppresses proliferation and Th1/Th2/Th17 cytokine production of pre-activated human T cells in vitro

The essential trace element zinc, necessary for many biological processes of living organisms, plays a regulatory role in the maintenance of immune functions. Zinc deficiency affects components of both the innate and the adaptive immune system. On the other side, zinc is capable of suppressing activation and proliferation of human T cells. In the present study, we investigated the effect of zinc aspartate (Unizink®), an approved drug to treat zinc deficiency, on pre-activated human T cells (T cell blasts) in vitro. T cells of healthy donors were stimulated for 48 h with anti-CD3/CD28 antibodies. After this time period, zinc aspartate or the immunosuppressive drugs cyclosporin A, dexamethasone, and rapamycin were added for additional 24 h to these cell cultures. Subsequently, T cell proliferation and cytokine production was measured. In contrast to cyclosporine A and dexamethasone, only zinc aspartate and rapamycin were capable of suppressing the proliferation and Th1 (IFN-γ), Th2 (IL-5), and Th17 (IL-17) cytokine production of pre-activated T cells. This data suggest that zinc aspartate has the capacity to suppress proliferation and cytokine production of pre-activated human T cells in vitro. Thus, administration of zinc aspartate may have beneficial effects on T cell-mediated autoimmune diseases.

Expression of zinc transporters ZIP4, ZIP14 and ZnT9 in hepatic carcinogenesis—An immunohistochemical study

INTRODUCTION Dysregulation of both, systemic zinc levels and tissue-specific zinc transporters, is reported in chronic inflammatory and malignant liver disease (hepatocellular carcinoma, HCC). Aim of this study is to assess the expression level of three zinc transporters in liver tissue and HCC: ZIP4, ZIP14 and ZnT9. METHODS The study is based on tissue samples obtained from 138 patients with histologically proven HCC. Tissue specimens from tumor (n = 138) and extra-lesional specimens (n = 72) were assessed immunohistochemically for the expression of the three zinc transporters. Expression levels were semi-quantitatively scored and statistically analyzed with respect to the etiology of HCC (alcohol, AFLD; non-alcoholic fatty liver disease, NAFLD; virus-hepatitis, VH) and survival. RESULTS Overall, expression levels of ZIP4, ZIP14 and ZnT9 were significantly higher in HCC tissue than in adjacent extra-lesional liver tissue. Expression levels in tumor tissue and survival time revealed a negative correlation for ZIP4 and ZIP14, and in part for ZnT9 (nuclear staining) (p < 0.05), whereas cytoplasmic staining of ZnT9 did not correlate with survival. Furthermore, the expression level of ZIP4 in extra-lesional tissue showed inverse correlation with survival time. CONCLUSION The upregulation of zinc transporters in hepatic carcinogenesis and its negative correlation with survival time implies a regulatory/functional link between zinc-homeostasis and development/progression of HCC that deserves to be further explored.

Combined Treatment with Zinc Aspartate and Intravenous Immunoglobulins (IVIGs) Ameliorates Experimental Autoimmune Encephalomyelitis (EAE)

Intravenous immunoglobulins (IVIGs) are widely used in replacement therapy of primary and secondary immunodeficiency disorders and in approved autoimmune indications. In addition, IVIG application is used off-label for treatment of other autoimmune diseases, e.g., multiple sclerosis (MS), an inflammatory autoimmune disorder with a clear T cell-mediated immune pathogenesis. The trace element zinc is shown to play a regulatory role in the maintenance of immune functions. Changes of zinc homeostasis affect both the innate and the adaptive immune system. On one hand, therapeutic zinc supplementation can normalize impaired immune functions due to zinc deficiency. On the other hand, therapeutic zinc supplementation is under consideration as a possible option to treat T cell-mediated autoimmune diseases. The aim of the present study was to investigate the influence of IVIG (Octagam®), zinc aspartate (Unizink®), and the combined application of both preparations in the experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Therapeutic intraperitoneal application of zinc aspartate significantly diminished clinical signs during the relapsing-remitting phase of EAE in SJL/J mice. In contrast, IVIG given in a therapeutic manner did not influence the course of EAE. Interestingly, the combined application of both, IVIG and zinc aspartate, significantly reduced the severity of the disease during the acute and the relapsing-remitting phase of the EAE. Our data suggest that the combination of IVIG and zinc aspartate may have beneficial effects in autoimmune diseases, like MS. Further studies should verify the benefit of a controlled immunosuppressive therapy with IVIG and zinc for such diseases.

Der Einfluss des Spurenelements Zink auf das Immunsystem

Zusammenfassung Klinische und experimentelle Untersuchungen haben gezeigt, dass enge Wechselwirkungen zwischen dem essentiellen Spurenelement Zink und dem Immunsystem existieren. Zink beeinflusst die zellulären und humoralen Komponenten sowohl des angeborenen als auch des erworbenen Immunsystems. Zinkmangelzustände beim Menschen sind häufig mit einer gestörten Immunfunktion, also einem sekundären Immundefekt vergesellschaftet. Je nach zugrundeliegender Ursache des Zinkmangels führt eine kontrollierte Zinksubstitution zu einer Normalisierung des Serumzinks, der Zinkhomöostase des Körpers und zu einer Verbesserung der Immunfunktion. In vitro wirken geringe Zinkkonzentrationen stimulierend auf funktionelle Parameter von Immunzellen, höhere Zinkkonzentrationen sind supprimierend oder zytotoxisch für diese Zellen. Arbeiten der letzten Jahre belegen die immunsuppressive Wirkung von Zink in T-Zell-vermittelten autoimmunen Tiermodellen, wie der Experimentellen Autoimmunen Enzephalomyelitis (EAE). Zudem wurde bei einer Reihe von Autoimmunerkrankungen eine Abnahme der Serum-/Plasma-Zinkkonzentrationen nachgewiesen. In zukünftigen klinischen Studien sollte bei diesen Erkrankungen der mögliche Einsatz einer kontrollierten immunsuppressiven Zinktherapie geprüft werden.