Hepatocyte apoptotic bodies encasing nonstructural HCV proteins amplify hepatic stellate cell activation: implications for chronic hepatitis C.

Chronic hepatitis C infection leads to increased hepatocyte apoptosis. Because engulfment of apoptotic bodies (ABs) by hepatic stellate cells (HSC) is profibrogenic, we compared the effects of ABs derived from hepatitis C virus (HCV)-negative vs HCV-infected (Con1+) Huh7 hepatoblastoma cells on fibrogenic and activation-related mRNA expression by a human HSC line (LX2). Uptake of Huh7(Con1+) ABs by LX2 cells dose dependently upregulated profibrotic genes (COL1A1, TGFB1; TIMP1; TIMP2). When normalized to the apoptotic cytokeratin-18 M30 neoepitope, HCV(+) ABs exhibited a more pronounced effect than HCV(-) ABs. In contrast, neither noningested ABs nor nucleic acids obtained from Huh7, Huh7(Con1+) or HepG2 cells triggered those AB-dependent effects. Both the engulfment of Huh7(Con1+) ABs and their effects were partially blocked by masking of phosphatidylserine with annexin V and completely inhibited by the class-A scavenger receptor ligand, polyinosinic acid. Our findings demonstrate that AB uptake stimulates HSCs and indicate that HCV infection leads to amplified fibrogenic mRNA expression and enhanced HSC activation.

Acute liver failure in a metropolitan area in Germany: a retrospective study (2002 - 2008).

OBJECTIVES: To determine current etiologies of acute liver failure (ALF) and clinical and laboratory parameters associated with the outcome upon ALF, so as to identify the frequency of present causes of ALF in Germany as well as potential new prognostic parameters. PATIENTS: 134 adult patients (63 % females / 37 % males) aged 41 +/- 16 years (median: 38 years) with established ALF criteria. DESIGN AND SETTING: A retrospective study (1 / 2002 - 4 / 2008) on ALF patients from the Ruhr Area, the largest urban region located in northwestern Germany. Clinical and laboratory data were collected for a period of four weeks after study admission. RESULTS: Etiologies of ALF were identified as drug toxicity (39.6 % of the cases); combined viral hepatitides (23.1 %); or miscellaneous (16.4 %). In 20.9 % of the cases, the etiology remained indeterminate. Overall patient survival at four weeks was 81.3 %. While 89 patients (66.4 %) recovered under best supportive therapy, 26 patients (19.4 %) had to undergo liver transplantation. Increased body mass indices were significantly (p < 0.003) associated with a poor outcome. Intriguingly, high levels of cholestatic enzymes significantly (p < 0.01) correlated with a positive outcome. CONCLUSIONS: In providing first data on current ALF etiologies Germany, this study reveals that drug toxicity - in particular due to acetaminophen - has replaced viral hepatitis as the most single frequent cause of ALF in a densely populated urban area; this correlates with similar findings in the USA, the UK and Scandinavia. Lower body mass indices and elevated cholestatic enzyme levels had statistically significant prognostic power.

Dysregulation of TNF/TNFR superfamily members: a systemic link between intra- and extrathyroidal manifestations in Graves' disease.

Graves' disease (GD) coincides with the occurrence of disease-associated intrathyroidal dendritic cells (DC) and intraorbital inflammatory macrophages (Mphi). Physiologically, tumour necrosis factor-alpha (TNF-alpha) strongly affects the differentiation of DC and Mphi from monocytic precursors; we thus hypothesized that dysregulation of the TNF/TNFR superfamilies may provide a systemic pathogenic link in GD. In patients without eye symptoms, percentages of TNF-alpha-stimulated blood monocytes were highly significantly (P < 0.001) elevated, corresponding to both intrathyroidal DC maturation as well as increases in mature blood DC (MHC-II(hi)/CD40+/RFD1(hi)) and B cells (CD20(hi)/CD40+). GD patients also displaying eye symptoms revealed a striking reduction in blood monocytes, yet significantly (P < 0.05) increased CD40(hi) and TNF-alpha(hi) leucocytes. These findings suggest for GD that excess TNF-alpha induces monocytes to differentiate into hyperactivated thyroidal DC that, once emigrated, initiate systemic humoral autoimmunity associated with CD40/TNF-alpha upregulation. Such overexpression may instigate differentiation of periorbital inflammatory Mphi from CD14(hi)/CD16+ monocytes as a likely precursor subset. These results indicate that dysregulation of TNF/TNFR superfamily members provides a systemic pathogenic link in GD in that hyperactivated circulating monocytic precursors give rise to locally restricted, disease-associated DC and Mphi. Monocytes, therefore, may serve as a suitable target to therapeutically address the common precursor of key promoters of GD.

The relationship between apoptosis and non-alcoholic fatty liver disease: an evolutionary cornerstone turned pathogenic.

The data currently available favor a model for the pathogenesis of non-alcoholic fatty liver disease that is based on an apparent sequential relationship of intrahepatic apoptosis, inflammation and fibrogenesis. Based on both hepatic and peripheral insulin resistance, the hepatocellular accumulation of triglycerides, termed steatosis, initially leads to an altered metabolism of glucose and free fatty acids in the liver. In response, increased expression of death receptors in simple steatosis enhances the hepatocytes' susceptibility for pro-apoptotic stimuli, thus eliciting excessive hepatocyte apoptosis and inflammation. Evidence indicates that these processes, if prolonged, activate both hepatic stellate and Kupffer cells, thus leading to a vicious circle in which apoptosis, inflammation, cellular activation, and collagen deposition are upregulated even further.

Thyroid associated ophthalmopathy: evidence for CD4(+) gammadelta T cells; de novo differentiation of RFD7(+) macrophages, but not of RFD1(+) dendritic cells; and loss of gammadelta and alphabeta T cell receptor expression.

AIM: To characterise periorbital immune cells (stages, kinetics) in active and inactive thyroid associated ophthalmopathy (A-TAO; I-TAO). METHODS: In orbital tissue cryosections of patients with A-TAO (n = 15), I-TAO (n = 11), and healthy controls (n = 14), adipose and fibrovascular areas were evaluated for MHC II(+) cells, CD45(+) total leukocytes, myeloid cells (CD33(+) monocytes; CD14(+) macrophages; mature RFD7(+) macrophages; RFD1(+) dendritic cells (DCs)), and lymphoid cells (CD4(+) T cells; alphabeta and gammadelta T cells; CD20(+) B cells). Results are expressed as medians and 5% confidence intervals. RESULTS: In fibrovascular septae, a surge of CD33(+) immigrants clearly correlating with disease activity generated significantly increased (p<0.05) percentages of CD14(+) and RFD7(+) macrophages. Intriguingly, CD4(+) cells were mostly gammadelta T cells, while alphabeta T helper cells were much less frequent. Successful treatment rendering TAO inactive apparently downregulates monocyte influx, macrophage differentiation, and T cell receptor expression. Similar trends were recorded for adipose tissue. Interestingly, RFD1(+) DCs were completely absent from all conditions examined. CONCLUSION: A-TAO coincides with periorbital monocyte infiltration and de novo differentiation of macrophages, but not DCs. The authors discuss a novel potential role for inflammatory CD4(+) gammadelta T cells in TAO. Successful treatment apparently downregulates orbital monocyte recruitment and effects functional T cell knockout.

DC-SIGN-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for HIV disease.

Myeloid dendritic cells (MyDCs), prime stimulators of antigen-specific immunity, can serve as one of the major reservoirs for human immunodeficiency virus type-1 (HIV-1). Utilizing mature monocyte-derived MyDCs generated with granulocyte/macrophage colony-stimulating factor, interleukin-4, and tumour necrosis factor-alpha as an in vitro model, we here present the first proof of concept for liposomal compound delivery to these cells by specifically addressing CD209, i.e. DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a MyDC-associated C-type lectin implicated in the transmission of HIV-1 to T helper cells. By employing a liposomally entrapped tracer, calcein, we demonstrate by flow cytometry and mathematics a superior targeting efficacy for DC-SIGN, as compared with select other MyDC markers (CD1a, CD4, CD45R0, and CD83). Fluorescence microscopy reveals time-dependent surface binding and intracellular uptake of DC-SIGN-specific liposomes by both immature and mature MyDCs. This pilot study implies that liposomal targeting to CD209 and related C-type lectins may afford therapeutic intracellular drug delivery to MyDCs and other reservoir and nonreservoir cells susceptible to infection with HIV-1.

Persistently altered visceral perception after resection of an esophageal granular cell myoblastoma: a therapeutic concept revisited.

Granular cell tumors (GCTs) are rare and usually benign gastrointestinal tumors. Their most frequent symptoms are dysphagia and epigastric or retrosternal discomfort. We here report a case of esophageal GCT with continued symptoms of retrosternal discomfort, postprandial feeling of fullness, and early satiety despite complete thoracoscopic resection of the tumor. In contrast, all functional tests were in the normal range. We thus suggest that, due to their neuroectodermal origin, GCTs may affect neuronal alterations leading to a persistently disturbed visceral mechanosensory perception. Consequently, this case also cautions the therapeutic concept to solely relieve GCT symptoms by resection if the tumor is less than 20 mm in diameter.

Maturation of thyroidal dendritic cells in Graves' disease.

Because thyroidal dendritic cells (t-DC) may be implicated in the pathogenesis of Graves' disease (GD), we compared t-DC in thyroid sections of patients with GD (n = 15) and control patients with toxic (TG; n = 12) or non-toxic goitre (NG; n = 12). Goitres in GD, but not TG or NG, were populated with three discernible t-DC phenotypes. (i) Immature t-DC (major histocompatibility complex (MHC) II+/CD40-/CD80-) were located perifollicularly (95% of the patients with GD, but only 55% of TG and 51% of NG patients); numbers of such t-DC were significantly elevated in GD (P < 0.001). (ii) Partially matured CD80+ t-DC were present in connective tissue (73% of the patients) and focal interstitial clusters (40% of the patients). In 53% of the patients with GD, single as well as clustered interstitial t-DC expressed CD40. (iii) However, phenotypically mature t-DC (MHC II+/CD40+/CD80+/RFD1+) were only present in clusters and colocalized with activated CD4+/MHC class II+ T-helper (Th) cells. Expression of CD54 and CD83 did not significantly differ among the groups. The phenotype of intrathyroidal DC in GD thus supports their role as potential (co)stimulators of thyroid autoimmunity.

[Generalized pustulous psoriasis: A novel extraintestinal manifestation of Crohn's disease?]. / Psoriasis pustulosa generalisata: neue extraintestinale Manifestation des Morbus Crohn?

A 66-year-old female patient suffering for 10 years from Crohn's disease firstly presented with a parallel outbreak of generalized pustulous psoriasis and Crohn's disease. A second synchronous exacerbation of both disorders occurred after discontinuation of treatment with prednisolone, methotrexate, and mesalazine. As to their pathogenetic concepts, both disease entities reveal similar immunologic alterations, i. e. comparable patterns of cytokines, chemokines, and inflammatory cells (T cells and neutrophils). Generalized pustulous psoriasis, therefore, might develop as hitherto undescribed, more rare extraintestinal manifestation of Crohn's disease.

[Etiology and pathophysiology of Graves' disease]. / Atiopathogenese des Morbus Basedow.

Infectious agents are considered as etiologic factors for the onset of Graves' disease, while psychosocial stress and smoking may amplify the pathogenic cascade. Furthermore, smoking increases the risk of developing Graves' ophthalmopathy. Occasional evidence indicates that the autoimmune process may primarily be induced by foreign antigens, leading to the synthesis of antibodies which, in part, cross-react with thyrocytic TSH receptors. Once initiated, autoimmunity appears sustained within the thyroid by the cooperation of immunomodulated thyrocytes and antigen-presenting dendritic cells, which may, moreover, cause humoral responses against further thyrocytic antigens. Owing to a local increase in cytokine and integrin expression, lymphocytes and monocytes are attracted to the organ. The synthesis of autoantibodies is mainly propagated by T-helper-2 cells, while, at the same time, T-suppressor cells appear to be functionally deficient. Proof for this hypothetical scenario may allow for novel immunotherapeutic onsets in the near future.

In-vitro differentiation of mature dendritic cells from human blood monocytes.

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-gamma to differentiate monocyte-derived DC in vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class I++/ , HLA-DR++/ , HLA-DP+, and HLA-DQ+. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD11c+ CD45R0+ blood DC subset identified earlier, their differentiation in the presence of the Th1 and Th2 cytokines IFN-gamma and IL-4 indicates that these DC may conform to mature mucosal DC.

Specific autologous anti-melanoma T cell response in vitro using monocyte-derived dendritic cells.

Dendritic cells (DC) are antigen-presenting cells initiating primary and secondary immune responses. Since malignant tumors are able to escape immunologic control, DC might be prime candidates to activate the immune system against tumor cells. In an autologous system, a dynamic interaction among monocyte-derived DC (MoDC), T lymphocytes, and tumor cells obtained from melanoma patients could be noted. MoDC were generated from blood monocytes in the presence of GM-CSF, IL-4, and IFN-gamma. T cells were isolated either from peripheral blood or from lymph nodes. Melanoma cells were harvested from surgically removed tumor metastases. They were then gamma-irradiated and co-cultured with autologous MoDC and T lymphocytes. After 5 days, the lymphocytes showed a high proliferative activity and the majority of them were CD8-positive. In five cases tested, they revealed a high cytotoxic activity resulting in apoptosis of tumor cells. These findings suggest that MoDC are capable of initiating an effective specific anti-tumor response in a strictly autologous mixed lymphocyte tumor culture (MLTC), even though tumor-specific antigens had not been individually defined. Therefore (I) whole melanoma cells can serve as a source of antigen, (II) monocyte-derived dendritic cells may process and present melanoma-specific antigens resulting in a strong lymphocyte proliferation, (III) the majority of responding T lymphocytes are CD8-positive, and (IV) an acquired cytotoxic response eventually leads to apoptosis of the melanoma cells. The reaction demonstrated here permits to in vitro and quantitatively monitoring the effect of T cell directed immunotherapies such as the adoptive immunotherapy of tumors.

[Allogenic small intestine transplantation in the rat model with initial CsA therapy: first detection of Th2-induced immune tolerance]. / Allogene Dünndarmtransplantation im Rattenmodell unter initialer CsA-Therapie: Erste Nachweise für Th2-vermittelte Immuntoleranz.

It is not clear how initial therapy with CsA effects indefinite allograft acceptance after small bowel transplantation in the rat. Our study of post-transplant alterations of immune parameters within donor-type mesenteric lymph nodes suggests CsA-dependent, Th2-mediated induction of graft-specific T-cell tolerance.

Apoptosis in the small intestinal allograft of the rat.

BACKGROUND: Necrosis and apoptosis are different cell death mechanisms. Necrosis is a pathological process that occurs after destruction of the cell membrane. Apoptosis is a DNA-dependent cell death mechanism, which occurs under physiological and pathological conditions. Although necrosis is a well-defined phenomenon in an acute graft rejection, the occurrence and relevance of apoptosis during this process is largely unknown. METHODS: The enterocyte apoptosis rates in allografted (n=24) and isografted (n=24) small intestines of the rat were compared using the in situ end-labeling technique. RESULTS: In situ end-labeling showed a dramatically increased number of apoptotic enterocytes in allografted small intestines, whereas increased labeling could not be observed in isogeneic small intestinal grafts. CONCLUSIONS: We suggest that graft rejection-associated apoptosis, in addition to necrosis, plays an important role in the course of organ failure, and that the degree of apoptosis represents another reliable indicator for the diagnosis and prognosis of transplant rejection.

Enrichment and characterization of dendritic cells from rat renal mesangium.

Dendritic cells (DC) initiate primary immune reactions and are distributed throughout most tissues. The most potent DC population of the kidney has long been suggested to reside within the glomerular mesangium. Using LEW.1A rats, we enriched and characterized such low-density cells. Mesangial DC generally exhibited round to oval cell bodies and cytoplasmic veils. Phenotypically, these cells were 100% OX-6++, 45% OX-42++, 35% ED1low, 10% OX-62low, and negative for ED2 and alpha-naphtylbutyrate esterase. Introducing a new monoclonal antibody, R3, which stains a subset of splenic DC, we showed strong antigen expression on 60% of mesangial DC. Correlating cell populations were detected immunohistochemically. Functionally, mesangial DC potently stimulated allogeneic mixed leucocyte reactions, but did not phagocytose opsonized Escherichia coli. In addition to their striking phenotypic similarity with autologous splenic DC, mesangial DC exhibited 88% of the allostimulatory activity of splenic DC. Calculation indicated approximately two mesangial DC per glomerulum. We suggest that these cells comprise different maturation-dependent subsets. The OX-62 integrin especially appears to be expressed only on mature mesangial DC, which may correlate to lymphoid veiled cells or interdigitating DC. An employment of mesangial DC in experimental models of acute allograft rejection or glomerulonephritis is discussed.

Enhanced renal allograft rejection by inhibitors of nitric oxide synthase: a nonimmunologic influence on alloreactivity.

Clinical and experimental studies indicate that nonimmunologic factors may modulate the alloreactivity of a renal transplant. Nitric oxide (NO) is an essential modulator of endothelial function. It was postulated that, in renal allografts, inhibition of constitutive NO synthase may lead to an aggravation of immunologic damage to endothelia and therefore may enhance dysfunction of the graft. Male Lewis (RT1l) rats received syngeneic or allogeneic Brown Norway (RT1n) renal grafts and were treated with cyclosporin A (CyA) or with CyA and an NO synthase blocker (NOS-B): N omega-nitro-L-arginine (L-NNA) or NG-monomethyl-L-arginine (L-NMMA). CyA was given at a dose of 3.5 mg/kg body weight for 14 days and the NOS-B at a dose of 66 mg/L drinking water for up to 28 days postoperatively. Animals (N = 6/group) were studied at 4 to 7, 14, and 28 days posttransplantation. Four to 5 days posttransplantation, renal blood flow and glomerular filtration rate of allogeneic grafts did not differ between animals treated only with CyA and those treated with CyA and NOS-B. Mean arterial pressure was significantly elevated by NOS-B (CyA+L-NNA: 115 +/- 13 versus CyA: 78 +/- 16 mm Hg). Combined NOS-B and CyA administration led to a pronounced increase in vascular and tubulointerstitial damage. The number of mononuclear cells in vessels, glomeruli, and tubulointerstitium increased significantly in allografts upon treatment with NOS-B. During NOS-B administration, adhesion molecules (intracellular adhesion molecule-1; leukocyte-function-associated molecules-1 alpha and-beta) were strongly expressed in endothelial and leukocytic cells of the allograft. A pronounced positivity for mRNA and protein of cytokines tumor necrosis factor-alpha and transforming growth factor-beta could be demonstrated in the inflammatory infiltrate. With L-NNA treatment, the total vascular injury index was 10-fold higher (14 days posttransplantation, CyA+L-NNA: 59.8 +/- 11.7 versus CyA: 6.0 +/- 1.8; p < 0.05). The tubulointerstitial damage score rose more than 2.5-fold after CyA and L-NNA therapy (28 days posttransplantation: CyA+L-NNA: 83 +/- 1 versus CyA:29 +/- 1). L-NNA was more potent than L-NMMA at the dosages used. Thus, pronounced vascular leukostasis, vasculitis, and T-cell and monocyte infiltration of the tubulointerstitium led to a severe damage of the allograft under therapy with CyA and NOS-B. Inhibition of NO synthesis may aggravate alloreactive immunemediated injury in kidney transplants acting primarily by a disturbance of endothelial function.