N. Muhanna

Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis.

Increased CD8‐T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence‐activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co‐cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha‐smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV‐derived CD8‐subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM‐1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho‐guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV‐derived lymphocytes. Conclusion: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease‐associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis. (HEPATOLOGY 2008.)

Amelioration of hepatic fibrosis by NK cell activation

Background and aims Interactions between hepatic stellate cells (HSCs) and immune cell subsets have emerged as important determinants of liver fibrosis progression and regression. Natural killer (NK) cells have an antifibrotic activity through killing of activated HSCs. In liver injury NK cell expression of activating/inhibitory killer immunoglobulin-related receptors (aKIR/iKIR) and their ratio are significantly increased, while class I major histocompatibilty (MHC) expression by activated HSCs is decreased. The aim of this study was to amplify the antifibrotic activity of NK cells and ameliorate hepatic fibrosis by iKIR silencing. Methods Human lymphocytes from patients with hepatitis C virus (HCV) infection were transfected with specific iKIR small interfering RNAs (siRNAs) or non-silencing control siRNAs, then co-cultured with a human HSC line and assessed for fibrogenic activity. To induce hepatic fibrosis, carbon tetrachloride was administrated to BALBc SCID-Beige male mice (lacking B/T/NK cells) for 4 weeks. Splenocytes from naive SCID donors (lacking B/T cells but with preserved NK cells) were transfected in vitro with either iKIR siRNA or non-silencing control siRNA, and then were transferred to the fibrotic SCID-Beige recipients. Results Transfection with iKIR or positive control siRNAs (mice and human) decreased mRNA expression of iKIR and mitogen-acivated protein kinase 1 (MAPK1). Consequently, total NK cells and NK cell degranulation were increased (p=0.01), consistent with NK cell stimulation. Compared with healthy lymphocytes, when HCV lymphocytes were transfected with non-silencing control siRNA and co-cultured with HSCs there was increased α-smooth muscle actin (αSMA) expression, reflecting HSC activation. Expression of αSMA in co-cultures was attenuated when HCV lymphocytes were transfected with iKIR siRNA. In SCID-Beige recipients, hepatic fibrosis and serum alanine aminotransferase (ALT) levels were significantly attenuated as a result of receiving iKIR siRNA. Conclusions iKIR knockdown stimulates NK cells and promotes their antifibrogenic activity in mice and human co-cultures. These findings have implications for possible immune therapeutic strategies in patients with advanced liver disease.

Lymphocyte–hepatic stellate cell proximity suggests a direct interaction

Recent functional research studies suggest an anti‐fibrotic role for natural killer (NK) cells coupled with a profibrotic role for CD8 cells. However, the morphological cellular interplay between the different cell types is less clear. To investigate lymphocyte/hepatic stellate cell (HSC) interactions, hepatic fibrosis was induced by administering carbon tetrachloride (CCl4) intraperitoneally (i.p.) for 4 weeks in C57Bl/6 mice. Animals were killed at 0, 1, 2, 3 and 4 weeks. Liver sections were stained for Sirius red. Confocal microscopy was used to evaluate alpha smooth‐muscle actin (αSMA) and lymphocyte subsets in liver sections. At weeks 0 and 4, liver protein extracts were assessed for αSMA by Western blotting and isolated liver lymphocytes as well as HSC were analysed by fluorescence activated cell sorter (FACS). Similar to the results obtained from classical Sirius red staining and αSMA blotting, analysis of liver sections by confocal microscopy revealed a marked and continuous accumulation of αSMA staining along sequential experimental check‐points after administering CCl4. Although the number of all liver lymphocyte subsets increased following fibrosis induction, FACS analysis revealed an increase in the distribution of liver CD8 subsets and a decrease of CD4 T cells. Confocal microscopy showed a significant early appearance of CD8 and NK cells, and to a lesser extent CD4 T cells, appearing only from week 2. Lymphocytes were seen in proximity only to HSC, mainly in the periportal area and along fibrotic septa, suggesting a direct interaction. Notably, lymphocyte subsets were undetectable in naive liver sections. Freshly isolated HCS show high expression of major histocompatibility complex (MHC) class II and CD11c. In the animal model of hepatic fibrosis, lymphocytes infiltrate into the liver parenchyma and it is thought that they attach directly to activated HSC. Because HSCs express CD11c/class II molecules, interactions involving them might reflect that HSCs have an antigen‐presenting capacity.

Immunomodulatory effects of plasminogen activators on hepatic fibrogenesis

Tissue‐type plasminogen activators (tPA) and urokinase‐type plasminogen activators (uPA) are involved in liver repair. We examined the potential immunomodulatory actions of uPA, tPA and uPA‐receptor (uPAR) in carbon‐tetrachloride‐induced hepatic fibrosis in wild‐type (WT), tPA−/−, uPA−/− and uPAR−/− mice. Carbon‐tetrachloride treatment increased fibrosis in four groups but significantly less in three knock‐out models. Serum cytokines and intrahepatic T cells elevated significantly following fibrosis process in WT animals but not in the knock‐out groups. In culture, uPA increased lymphocyte proliferation significantly in WT and uPA−/− but not uPAR−/− animals. Following uPA exposure in vivo, there was CD8 predominance. To isolate uPAs effect on lymphocytes, WT mice were irradiated sublethally and then reconstituted with WT or uPA−/− lymphocytes. In these animals fibrosis was decreased and T cells were reduced in the uPA−/− recipients. Based on these data we postulate that plasminogen activators affect fibrosis in part by liver‐specific activation of CD8 subsets that govern the fibrogenic activity of hepatic stellate cells.

Amelioration of hepatic fibrosis via Padma Hepaten is associated with altered natural killer T lymphocytes

Hepatic fibrosis is the end‐stage consequence of chronic liver disease, affecting many people worldwide. Unlike the anti‐fibrotic effect of natural killer (NK) cells, CD8 and NK T subsets are considered as profibrogenic subsets. Padma Hepaten is a multi‐compound herbal preparation derived from Tibetan medicine and has proven efficacy in some clinical trials and tests at the cellular level. In this study, we evaluate the immune efficacy of Padma Hepaten administered intraperitoneally (i.p.) and/or orally in a mice model of hepatic fibrosis. Hepatic fibrosis was induced by 6 weeks of biweekly i.p. carbon tetrachloride (CCl4) injections in male C57Bl6 mice. There were four groups, including naive mice, non‐treated fibrotic mice and fibrotic mice treated by Padma Hepaten at weeks 5–6 of fibrosis induction either orally or by i.p. injections. Padma Hepaten was prepared at 10 mg/ml in saline and 250 µl (2·5 mg) were administered four times per week. After week 6, animals were killed. To isolate a Padma Hepaten‐associated effect on lymphocytes, splenocytes were harvested from either naive or Padma Hepaten‐treated non‐fibrotic donors. Isolated splenocytes were therefore reconstituted into two groups of irradiated recipients. Recipients were then administered the same CCl4 regimen. Hepatic fibrosis was determined by sirius red staining of liver sections and by assessment of alpha smooth muscle actin expression compared with β‐actin (both by mRNA as well as the protein liver extract western blotting). Hepatic fibrosis and alanine aminotransferase serum levels were decreased significantly in both Padma Hepaten‐treated groups compared with the non‐treated fibrotic group. Padma Hepaten treatment was associated with attenuation of lymphocyte subsets in both treated groups. Using a chemiluminescence technique to assess total anti‐oxidant capacities (TAC), it was found that both the plasmas and livers of mice treated by CCl4 had significantly higher TAC compared with controls. However, the levels of TAC in animals treated either by CCl4 alone or CCl4 with Padma Hepaten were similar. Adoptive transfer of Padma Hepaten‐treated lymphocytes was associated with fibrosis amelioration compared with recipients with naive lymphocytes. CCl4 generates higher levels of anti‐oxidant capacities, probably as a response to oxidative stress. Padma Hepaten administration attenuated hepatic fibrogenesis significantly, accompanied by attenuation of lymphocyte but not anti‐oxidant capacities.

Harmonic Scalpel Assisted Superficial Parotidectomy

Objective: The Harmonic Scalpel (HS) has been recently widely used to perform a variety of surgical procedures. We reviewed our experience with the use of HS in superficial parotidectomy to determine the safety and efficacy of this procedure, with regard to operative time, postoperative facial nerve function, and drainage output. Study Design: Nonrandomized retrospective review. Materials and Methods: The medical records of all patients who underwent superficial parotidectomy for benign pathology at Shaare Zedek Medical Center from January 2006 to July 2009 were retrospectively reviewed. Patients with prior facial nerve weakness or prior parotid surgery or who had undergone concurrent neck dissection or total parotidectomy were excluded. Results: Fifty-eight patients were reviewed; 26 patients underwent HS parotidectomy and 32 patients underwent conventional (cold knife) parotidectomy (control group). Harmonic Scalpel assisted parotidectomy was associated with significantly decreased length of surgery from 163.12 ± 21.8 minutes for controls to 137.3 ± 18.6 minutes in the HS assisted group (P < .05). The incidence of temporary postoperative facial nerve paresis was significantly reduced from 43% in the controls to 23% in the HS group (P < .05). No permanent facial nerve paralysis was reported. There were differences in the overall postoperative drain output between the HS and control groups, 68 ± 22.3 mL and 73.5 ± 38.2 mL, respectively, but these differences did not achieve significance. Conclusion: This study shows that HS assisted superficial parotidectomy for benign pathology is a safe technique and associated with reduced surgical time and incidence of temporary postoperative facial nerve paresis compared with conventional techniques.

Starplasty tracheostomy: case series and literature review

ObjectivesThe starplasty tracheostomy (SPT) technique has been suggested to reduce the short-term complications of tracheostomy, including accidental decannulation and pneumothorax. The aim of the present study was to conduct a review of key parameters prior to and following treatment of neonates and children with the SPT technique, including indications, complications, perioperative department stay, and overall length of stay in one University-Affiliated Medical Center.MethodsA retrospective chart review of all children under the age of 18 underwent SPT in a single center between February 2006 and January 2012.ResultsAmong the 39 patients reviewed, the median age at the time of surgery was 14.5 months, ranging from 3 days to 8.8 years. The most common indication for SPT was respiratory insufficiency resulting from central nervous system disorders (15, 38.4%) followed by neuromuscular disorders (14, 35.9%). Ten (25.6%) operations were performed on neonatal intensive care unit (NICU) patients and 29 (74.4%) on pediatric intensive care unit (PICU) patients. The median postoperative hospital stay was 19.5 days (range of 3–207 days); however, the median postoperative stay in the PICU was 13.5 days. There were no decannulations or any other short-term complications after SPT, and no SPT-related deaths occurred.ConclusionsIn our series, pediatric SPT was not associated with any major complications. Therefore, we conclude that SPT should be considered as a safe and advantageous alternative for traditional tracheotomy, especially in patients with low probability of future decannulation, and, therefore, at low risk of a persistent tracheocutaneous fistula.

The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis

Background A high prevalence of thyroid papillary cancer was reported in hepatitis-C-virus (HCV) positive patients. However, the mechanistic role of hepatic-fibrosis in thyroid malignancy progressions is still unclear. Aim We aimed to study the immune-modulatory interactions between thyroid papillary carcinoma and hepatic-fibrosis. Methods Hepatic-fibrosis was induced in nude-nu-male mice by intra-peritoneal administration of carbon-tetrachloride. To induce thyroid-tumor, a thyroid papillary carcinoma cell line (NPA) was injected subcutaneously in the backs. Fibrotic profile was estimated by α-smooth-muscle-actin (αSMA) expression in liver tissue extracts using western-blots and RT-PCR. Intra-hepatic NK cells were isolated and stained for NK activity (CD107a) by flow cytometry. Liver histopathology (H&E staining), thyroid tumor mass and serum alanine aminotransferase (ALT), serum vascular endothelial growth factor (VEGF) and free-T4 levels were also assessed. Results Ex-vivo: NPA cells were co-cultured with intra-hepatic NK cells isolated from fibrotic mice with/without the tumor were analyzed for CFSE-proliferations. Both tumor groups (with/without hepatic-fibrosis) excreted higher serum free T4 levels. Hepatic-fibrosis increased tumor weight and size and serum free-T4 levels. In addition, tumor induction increased liver injury (both hepatic-fibrosis, necro-inflammation and serum ALT levels). In addition, tumor-bearing animals with hepatic-fibrosis had increased NK activity. NPA tumor-bearing animals increased fibrosis in spite of increased NK activity; probably due to a direct effect through increased serum free-T4 excretions. Serum VEGF levels were significantly increased in the fibrotic- bearing tumor groups compared to the non-fibrotic groups. In-vitro, NK cells from fibrotic tumor-bearing animals reduced proliferation of NPA cells. This decrease is attributed to increase NK cells activity in the fibrotic animals with the NPA tumors. Conclusions Our results propose that NK cells although were stimulated in advanced fibrosis with tumor, they lost their anti-tumor and anti-fibrotic activity probably due to secretions of T4 and VEFG and may explain increased risk of thyroid tumors in chronic HCV patients.

341 THE HEMOSTASIS EFFECTS OF HEPATIC STELLATE CELLS IN HEPATOCELLULAR CARCINOMA

Background: Chronic liver inflammation is a critical component of hepatocarcinogenesis. Indeed, inflammatory mediators are believed to promote liver cancer by upholding compensatory proliferation of hepatocytes in response to tissue damage. However, we previously showed that mice, which overexpress interferon (IFN)-gamma under the control of a hepatocyte specific promoter, are protected from chemical hepatocarcinogenesis, although they manifest severe liver inflammation, injury and compensatory regeneration. Methods: To explore the molecular mechanism responsible for the observed IFN-gamma-dependent protection from chemical hepatocarcinogenesis despite chronic inflammatory stress, we treated IFNgamma transgenic or non-transgenic mice with chemical carcinogen diethylnitrosamine (DEN). After 6 days, we examined cellular stress response pathways (STAT, MAPK, NF-kappaB, p53) by western blot analysis and hepatic apoptosis by TUNEL assays. Results: We found that DEN-treated IFN-gamma transgenic livers showed increased STAT1 activation, and increased STAT1-induced p53 accumulation. Moreover, IFN-gamma transgenic livers exhibited increased p53 activation in response to genotoxic stress, as indicated by elevated p21 and cleavage of caspase-3. The IFN/STAT1-induced p53 activation was associated with a significant increase in hepatocellular apoptosis, compared to non-transgenic littermates (53 vs. 4 apoptotic cells/mm2; p< 0.0001), as assessed by TUNEL assay. DEN-treated IFN-gamma transgenic livers did not display differences in the levels of JNK or IKKb phosphorylation. These in vivo findings were confirmed in vitro by stimulation of primary hepatocytes with INFgamma. Here too, we found an increase of STAT1 activation, p53 accumulation and p53 activation in response to DEN, as well as increased p53-dependent DEN-induced apoptosis in an MTT assay (34% vs. 83% survival, compared to DEN-treated hepatocytes without IFN-gamma stimmulation). IFN-induced apoptosis was indeed p53-dependent, since it could be blocked by the p53 inhibitor pifithrin-a. Conclusions: Our findings indicate that the inflammatory IFN/STAT1 pathway suppresses liver cancer by activating the tumor-suppressive p53 pathway. These findings challenge the view that chronic inflammation per se promotes carcinogenesis; indeed, the carcinogenic potential of inflammation may be determined by type and composition of its mediators, rather than duration. Thus, manipulation of the type of chronic inflammation may serve the prevention of cancer inflammation may serve the prevention of cancer.