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.

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.

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.

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.

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.

1,25-Dihydroxyvitamin D3 exerts opposing effects to IL-4 on MHC class-II antigen expression, accessory activity, and phagocytosis of human monocytes.

To investigate the differentiation and activation of monocytes, the combined effects of 1,25-dihydroxyvitamin D3 (D3) and IL-4 on human blood monocytes were examined with respect to expression of MHC class-II antigens, accessory activity, and phagocytic capacity. IL-4 was reported to upregulate the expression of MHC class-II antigens and accessory activity of monocytes. The experiments described here demonstrate that D3 inhibits the expression of all three subtypes of MHC class-II antigens (HLA-DR, -DP and -DQ) as well as the accessory activity of monocytes, both in a dose- and time-dependent manner. However, D3 enhances the immunoglobulin- and complement-dependent phagocytosis by monocytes in a dose- and time-dependent manner. When monocytes are treated with both IL-4 and D3, the effects of D3 are reverted by IL-4, suggesting that IL-4 induces the development of monocytes into accessory cells, whereas D3 stimulates differentiation of monocytes into classical macrophages. These findings provide further evidence for the contention that, depending on defined stimuli, monocytes may develop either into accessory cells or into classical macrophages.

The adaptive response of the rat small intestine after resection and segmental transplantation during the early postoperative phase.

Organ harvesting from a living donor or spatial constraints in the recipient's abdominal cavity are the main factors to be considered in the segmental transplantation of the small intestine. It was the aim of the following study to gain insight into the functional characteristics of different portions of the small intestine either after partial resection or syngeneic and allogeneic transplantation during the early postoperative period. Nutritional parameters (serum albumin levels, serum triglyceride levels, maltose absorption, excretion of fecal fat) and fat-stimulated neurotensin release were determined in Lewis rats that underwent small bowel resection (n = 21), syngeneic (Lewis-->Lewis, n = 21), or allogeneic transplantation (Brown Norway-->Lewis, n = 24). The length of the remnant, isograft, or allograft was 27 cm (i.e. one third of the rat small intestine) and consisted of the proximal (n = 7), middle (n = 7), or distal (n = 7) portion. Three postoperative deaths were due to ileus or pneumonia. After allotransplantation, cyclosporine (15 mg/kg BW s.c.) was administered for graft acceptance. Controls were unoperated, weight- and age-matched Lewis rats (n = 7). We found that resection of two-thirds of the small intestine led to significantly lower levels of albumin and triglycerides in all the three portions investigated (P < 0.01) but did not affect maltose absorption. Excretion of fecal fat was elevated after distal resection (P < 0.05). When compared to resected animals, syngeneic transplantation did not affect the nutritional parameters, but caused a significantly higher hormone release (P < 0.05) in all three different intestinal grafts. Allogeneic transplantation was successful when the middle or distal portion was grafted. All recipients of proximal allografts showed a severe loss of body weight and died between day 8 and 10 after transplantation. Postmortem examination revealed no signs of acute rejection. When transplantation of short intestinal segments is considered, it is of vital importance to take into account the functional differences and the influence of immunosuppressive drug therapy in the regulatory bowel function.

Fat-stimulated cholecystokinin release following transplantation of the entire small bowel or of different intestinal segments in rats.

This study presents data on the fat-stimulated release of cholecystokinin (CCK) in conscious rats 11 and 84 days after one-stage transplantation of the entire small bowel, or of jejunal, jejunoileal, or ileal segments, under syngeneic and allogenic conditions. After allotransplantation, ciclosporin (CsA) was administered for graft acceptance. The results were compared with those in unoperated controls and in animals that had undergone small-bowel resections leaving jejunal, jejunoileal, or ileal remnants. When the entire small bowel was grafted under syngeneic (92.5 +/- 8.3; 106.6 +/- 7.5) or allogeneic (110.5 +/- 5.5; 101.2 +/- 6.9) conditions, CCK release (pg/ml per 60 min) was similar (P > 0.05) to that of the controls (110.3 +/- 9.0; 94.7 +/- 6.8) at both measurement points. Recipients of jejunal or ileal segmental isografts showed a significantly elevated (P < 0.05) output of CCK (jejunal graft: 176.4 +/- 18.5; 125.5 +/- 10.1 -ileal graft: 55.9 +/- 9.0; 30.1 +/- 5.4) compared with corresponding small-bowel resections (jejunal remnant: 69.0 +/- 7.9; 93.5 +/- 3.9-ileal remnant: 16.7 +/- 3.7; 6.6 +/- 1.3). In contrast, the difference was not significant (P > 0.05) when jejunoileal segments were grafted (jejunoileal graft: 74.4 +/- 19.6; 47.0 +/- 10.4-jejunoileal remnant: 50.7 +/- 11.0; 47.0 +/- 11.9). All recipients of jejunal allografts died between day 8 and day 10 after transplantation, due to functional impairment. Two-stage segmental jejunal allotransplantation, with insertion of the graft into the continuation of the gastrointestinal tract in an accessory, non-functional position after 28 days was successful. Due to this technique, we could gather data on day 84. Recipients of jejunal (118.2 +/- 7.6), jejunoileal (87.1 +/- 19.7; 48.6 +/- 9.3), or ileal (48.1 +/- 6.7; 21.6 +/- 4.6) allografts showed no significant (P < 0.05) differences in CCK output compared with isografts, either on day 11 or on day 84. Our data indicate that transplantation of the entire small bowel affects the fat-stimulated CCK release neither in the early postoperative period nor 3 months after transplantation. In contrast, transplantation of jejunal or ileal segmental isografts caused a significantly elevated output of CCK compared with corresponding resection remnants. Immunosuppression with CsA did not affect CCK release after transplantation, but led to functional impairment with fatal outcome when a short jejunal segment was grafted. This could be prevented by applying the two-stage technique.

Signals required for differentiating dendritic cells from human monocytes in vitro.

Human peripheral blood monocytes (Mo) can quantitatively be differentiated into potent accessory cells which exhibit dendritic cell (DC) function and phenotype. This alternative differentiation of Mo into DC rather than into macrophages (M phi) will be triggered when signals leading to M phi differentiation are omitted from the culture. Serum contains such stimulatory signals and was therefore omitted from the cultures. The cells were cultured on solid agarose surfaces. This newly developed technique allows for the attachment-free differentiation of DC. In the absence of signals, Mo do not survive in culture. IL-1 and IL-6 are endogenously produced by Mo and create an autokrine stimulatory milieu which increases the accessory function. However, also mature Mph will respond by an increased accessory activity upon stimulation by these cytokines. Cyclic AMP is the most likely second messenger to trigger an increase in accessory activity. IL-4 plus GM-CSF further act to upregulate dendritic cell properties and function. By action of these mediators, virtually all markers and functions of Mo/M phi are lost, and the cells convert to the phenotype and function of dendritic cells.

Human blood dendritic cells exhibit a distinct T-cell-stimulating mechanism and differentiation pattern.

In this study, the mechanisms underlying stimulation of T-cell proliferation by human blood dendritic cells (BDC) and their differentiation have been defined with a panel of monoclonal antibodies (MoAbs). It was found that the MoAbs against LFA-1 (CD11a), CD11c, LFA-3 (CD58), ICAM-1 (CD54) or HLA-DR could significantly suppress T-cell proliferation in an allogeneic mixed lymphocyte reaction (P < 0.05), while being unable to inhibit clustering of BDC with T cells. Addition of anti-CD18 or CD45 MoAbs increased the size of clusters after 18 h of culture, but had no effect on the proliferation of T cells (P < 0.05). The suppressive effect of the MoAbs may be viewed not as an inhibition of contact between BDC and T cells, but rather as a blocking of co-stimulatory signals for T-cell activation, which are mediated by interaction of the adhesion molecules. After depleting the BDC preparations of monocytes, we used a double staining in FACS analysis to demonstrate that BDC do not express specific T (CD3), B (CD20 and CD21) and myeloid cell markers (CD11b, CD13 and CD14), but abundant class II antigens. This pattern remained unaltered after 8 days of culture in the presence of 100 U/ml GM-CSF, although a threefold increase of HLA-DQ and ICAM-1 molecules on the cultured cells was observed.

Inhibition of human immunodeficiency virus-1 proliferation by liposome-encapsulated sense DNA to the 5' tat splice acceptor site.

A liposome formulation containing a distearoylphosphatidylethanolamine analog was developed that was endocytosed by both lymphocytes and monocytes. This formulation was used to encapsulate sense and antisense 20-mer oligodeoxynucleotides to the 5' tat splice acceptor site of human immunodeficiency virus type 1. At a DNA concentration of 140 nM, the liposome-encapsulated sense DNA inhibited p24 production by as much as 84% in human peripheral blood leukocytes infected with "wild-type" virus. This treatment also reduced the number of peripheral blood leukocytes producing intracellular viral antigen by 71%. Of interest, no reduction in either parameter was observed for the antisense-containing liposomes. The results demonstrate the promise of a new liposomal delivery vehicle to inhibit human immunodeficiency virus replication by an entrapped oligodeoxynucleotide.