CXCR3阳性自然杀伤细胞在MHV-3诱导暴发性肝炎小鼠组织分布动态变化

目的检测CXC趋化因子受体3(CXCR3)阳性自然杀伤(NK)细胞在3型鼠肝炎病毒诱导的暴发性肝炎小鼠肝脏、脾脏、骨髓及外周血中百分比的动态变化。方法腹腔注射3型鼠肝炎病毒诱导BALB/cJ小鼠暴发性肝炎,利用流式细胞术检测小鼠肝脏、骨髓、外周血及脾脏CXCR3阳性NK细胞百分比的变化。结果肝脏CX-CR3阳性NK细胞百分比在感染后12小时(20.47±1.54%)和24小时(31.53±2.53%)较感染前(14.0±0.25%)均显著升高(P均<0.05),至感染48小时下降至感染前水平;骨髓CXCR3阳性NK细胞百分比随感染时间延长而逐渐减少,由初始水平(25.53±3.50%)的高点至72小时时降至较低水平(1.2±0.17%,P<0.05);脾脏CXCR3阳性NK细胞的百分比在感染后12小时增高(13.63±3.167%),随后下降,至72小时达到较低水平(2.20±0.53%,P<0.05);外周血CXCR3阳性NK细胞的百分比在24小时增高,在72小时下降至感染前水平。结论在MHV-3感染后,CXCR3阳性NK细胞可能逐渐迁移至肝脏,从而在小鼠暴发性肝炎发生发展中发挥重要作用。     

Effectiveness of combined plasma exchange treatment in fulminant viral hepatitis

The plasma exchange is an effective measure in the treatment of acute liver failure. The authors report their own first experiences in this field in the management of fulminant viral hepatitis. Four out of 7 treated patients survived. So, the survival rate was augmented from 25 per cent in a control group of 8 patients without plasma exchange to 57.1 per cent. Other necessary therapeutic measures are high energetic nutrition, branched chained amino acids, stimulation of liver cell regeneration, correction of coagulation alterations, and intensive care. The success depends on coma stage in the beginning of treatment. Patients with signs of praecoma should be admitted to specialized centers. The number of such centers should be limited because of the necessary experiences and the high costs of combined plasma exchange treatment of this rare hepatitis complication.     

Attempted treatment of fulminant viral hepatitis with human fibroblast interferon

Summary -interferon was administered by intravenous infusion to 16 patients affected with fulminant hepatitis B virus infection in third or fourth-grade coma. Ten patients presented a superinfection or a co-infection due to the delta ()-agent. None had detectable interferon (IFN) activity before therapy was begun. Besides fever, no significant side-effects were observed during treatment. Both the IFN-treated group as well as the historical control group, made up of 70 cases of fulminant virus hepatitis, not treated with IFN and observed during a previous ten year-period, received supportive therapy; survival rates were similar in both groups. Furthermore, the presence or absence of the -agent did not appear to affect survival rates significantly.

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Zusammenfassung -Interferon wurde 16 Patienten i.v. infundiert, die an einem Leberkoma dritten oder vierten Grades infolge fulminanter Hepatitis-B-Virus-Infektion litten. Zehn von ihnen wiesen eine durch -Agens bedingte Super- oder Koinfektion auf. Bei keinem Patienten konnte eine meßbare Interferon (IFN)-Aktivität vor Therapie-Beginn nachgewiesen werden. Außer Fieber wurden während der Behandlung keine nennenswerten Nebenwirkungen beobachtet. Die Überlebensraten waren nahezu gleich bei den mit Interferon behandelten Patienten und bei einer aus 70 Fällen von fulminanter Virus-B-Hepatitis gebildeten historischen Kontrollgruppe, welche in den vorherigen zehn Jahren lediglich mit unterstützenden Maßnahmen behandelt worden war. Außerdem konnten keine statistisch signifikanten Unterschiede in der Überlebenstrate -positiver und -negativer Patienten festgestellt werden.

     

Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival

Most current cancer therapies focus on killing malignant cells, but these cells are often genetically unstable and can become resistant to chemotherapy. Tumor-associated macrophages (TAMs) facilitate disease progression by promoting angiogenesis and tumor cell growth, as well as by suppressing the adaptive immune response. TAMs are therefore potential targets for adjuvant anticancer therapies. However, resident macrophages are critical to host defense, and preferential ablation of TAMs remains challenging. Macrophage activation is broadly categorized as classically activated, or M1, and alternatively activated, or M2, and TAMs in the tumor microenvironment have been shown to adopt the anti-inflammatory, M2-like phenotype. To date, there are no methods for specific molecular targeting of TAMs. In this work, we report the discovery of a unique peptide sequence, M2pep, identified using a subtractive phage biopanning strategy against whole cells. The peptide preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes. Confocal imaging demonstrates the accumulation of M2pep in TAMs in vivo after tail vein injection. Finally, tail vein injection of an M2pep fusion peptide with a proapoptotic peptide delays mortality and selectively reduces the M2-like TAM population. This work therefore describes a molecularly targeted construct for murine TAMs and provides proof of concept of this approach as an anticancer treatment. In addition, M2pep is a useful tool for murine M2 macrophage identification and for modulating M2 macrophages in other murine models of disease involving M2 cells.Macrophages are phagocytic cells of our immune system that are found in almost all tissues. Originating from progenitor cells in bone marrow, circulating blood monocytes extravasate into tissues, where they differentiate into macrophages and can subsequently be shifted to diverse functional phenotypes by local environmental cues (1). These polarization states are broadly categorized as classically activated M1 macrophages or alternatively activated M2 macrophages. The M1 macrophage phenotype is induced by mediators, such as IFN-γ or LPS, resulting in a proinflammatory and microbicidal functional phenotype (2). In contrast, M2 macrophages are activated by IL-4 and IL-13, and they possess functional roles in resolution of inflammation and tissue remodeling (3).There are several chronic pathological findings associated with increased tissue levels of M2 macrophages, including cancer, late-stage atherosclerosis, fibrosis, and asthma (4). In cancer, populations of tumor-associated macrophages (TAMs) mediate immunosuppression and possess M2-like qualities, such as poor antigen presentation, promotion of angiogenesis, and tissue remodeling and repair, although there is heterogeneity in pathways and phenotypes in different tumors (5). TAMs also secrete the factors milk fat globule-EGF factor 8 and IL-6 that lead to tumorigenicity and drug resistance of cancer stem/initiating cells (6). The extent of TAM accumulation within tumors generally correlates with poor disease prognosis (7, 8). Indeed, the role of M2 macrophages in chronic inflammation and disease is increasingly appreciated, and the ability to modulate specific subsets of macrophage populations is a major focus of macrophage-targeted therapy (4). However, there is a dearth of available targeting entities that are specific for M2 macrophages.Several ligands have been used to target macrophage populations. The small molecules mannose and folate, which are ligands for the mannose receptor and folate receptor β, respectively, have been conjugated to drugs or drug carriers for macrophage delivery (9–11). However, receptors for both molecules are highly expressed in other cell types. Mannose receptor is a pathogen recognition receptor that is also used by dendritic cells (12). In addition to activated macrophages, folate binds to receptors on normal epithelial cells and tumor cells (13). Segers et al. (14) reported a peptide that binds the scavenger receptor-A on macrophages found within atherosclerotic plaques, but scavenger receptor-A is also expressed on all dendritic cells.Our goal is to develop an M2-targeted construct that can be used to ablate TAMs selectively but not other leukocytes. To achieve this goal, an M2-selective ligand was first identified by a library selection approach. Peptide phage library screening is a common method of identifying novel targeting ligands, and it has been used to select peptides for targeting tumor vasculature (15) and colon cancer (16) among several others (17). We therefore designed a subtractive phage panning strategy that uses bone marrow-derived, in vitro-activated macrophage subpopulations to increase binding specificity of a selected phage to the M2 macrophage subpopulation. We report here the discovery and evaluation of a peptide, called M2pep, which shows selective binding and internalization in M2 macrophages but with minimal M1 macrophage interaction. We demonstrate this peptide’s ability to identify murine M2-like TAMs within mixed cell populations isolated from mouse colon carcinoma tumors and to accumulate in TAMs after i.v. injection into tumor-bearing mice. We show that i.v. administration of M2pep fusion peptides with KLAKLAKKLAKLAK (KLA), a proapoptotic peptide, to tumor-bearing mice selectively reduces TAM populations and prolongs survival.     

肝脏自然杀伤细胞在小鼠急性肝衰竭中的作用

目的 探讨肝脏自然杀伤(NK)细胞在3型鼠肝炎病毒(MHV-3)诱导的小鼠急性肝衰竭中的作用.方法 取6～8周龄雌性Balb/cJ小鼠,腹腔注射100 pfu MHV-3,采用流式细胞术检测感染MHV-3 0、24、48、70 h后的Balb/cJ小鼠肝脏、脾脏、外周血和骨髓中NK细胞的百分率及肝脏NK细胞表面活化分子CD69表达的百分率.细胞内细胞因子染色法检测肝脏NK细胞分泌干扰素γ的水平.非放射性细胞毒试验检测肝脏NK细胞的杀伤活性. 结果Balb/cJ小鼠感染MHV-3后,肝脏NK细胞的比例显著升高,在感染48 h后达到峰值(43.9%±2.3%),约为感染前的4倍,随后仍维持在较高水平至小鼠死亡;外周血NK细胞比例同样明显升高,在感染48 h后达到峰值(18.0%±5.4%),但随后显著同落,至70 h仅为1.3%±0.6%,脾脏和骨髓NK细胞比例均先明显减少后又有所上升.肝脏NK细胞在MHV-3感染48 h后其表面活化分子CD69表达明显上调,杀伤活性显著增强,同时分泌干扰素Y的水平也显著增加. 结论 Balb/cJ小鼠感染MHV-3后,来自骨髓和脾脏的NK细胞在肝脏迅速大量募集和活化,且杀伤活性显著增强,分泌干扰素Y水平也显著增加,表明肝脏NK细胞在MHV-3导致的急性肝衰竭中可能发挥着关键作用.     

Interferon and cyclosporin A in the treatment of fulminant viral hepatitis

The prognosis of fulminant hepatitis due to non-A, non-B virus infection and acute reactivation of hepatitis B virus in HB carriers is generally poor, and the treatment of choice in Western countries is recognized as liver transplantation. In countries such as Japan where liver transplantation is not readily available, however, these intractable types of fulminant hepatitis have to be treated medically. Based on the assumption that persistent replication of causal viruses and enhanced host immune responses, especially cellular immunity, to eradicate the viruses are the key mechanism in progressive liver cell destruction and the poor prognosis, we attempted a combination treatment with interferon and cyclosporin A for these types of fulminant viral hepatitis. Subjects in the present study consisted of 1 patient with acute severe hepatitis without coma and 13 patients with coma (13 with fulminant hepatic failure) due to non-A, non-B virus and acute reactivation of hepatitis B virus. The patients were given interferon-beta, 300 × 10⁴U daily, and cyclosporin A, at an initial dose of 3 mg/kg, with tapering. Fourteen patients with coma received artificial liver support that we devised. The patient with acute severe hepatitis survived, showing histologically remarkable liver regeneration. Eight of the 14 patients with hepatic coma, all of whom were indications for liver transplantation according to the criteria of the King's College group, survived. Decreased transaminase level, increased liver volume, and histological liver regeneration were observed in all the survivors. The combination of interferon and cyclosporin A is worth attempting in fulminant hepatitis caused by non-A, non-B virus and acute reactivation of hepatitis B virus in HB carriers.     

Targeted delivery of antisense DNA in woodchuck hepatitis virus-infected woodchucks

Summary. An asialoglycoprotein-based DNA delivery system containing an antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of woodchuck hepatitis virus (WHV) was prepared. Experimental woodchucks were inoculated neonatally with the woodchuck virus 23 weeks before initiating the study, and all animals subsequently developed hepatitis as evidenced by the presence of measurable levels of circulating viral DNA. Animals were injected intravenously (i.v.) with asialoorosomucoid (AsOR)-poly- l -lysine complexes containing 0.1 mg kg^-1 antisense DNA for five consecutive days. Levels of surface antigen did not differ substantially between treated and control animals. However, intravenous administration of complexed antisense DNA significantly decreased viraemia, as shown by a five-to 10-fold decrease in circulating viral DNA 25 days post treatment. The decline lasted for at least 2 weeks, after which there was a gradual increase in DNA levels. Antisense DNA alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels. We conclude that antisense oligo DNA can be targeted to the liver in vivo, resulting in a substantial and prolonged decrease in viral DNA levels in WHV-infected woodchucks.     

Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B

AIM: To evaluate the expression of fibrinogen-like protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury. RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2) was detected in 21 of 23 patients (91.30%) with severe AOC hepatitis B, while only 1 of 13 patients (7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.     

Effect of exogenous mouse interferon on murine fulminant hepatitis induced by mouse hepatitis virus type 2

We investigated the effect of exogenous mouse alpha- + beta-interferon produced by mouse L cells on the growth of mouse hepatitis virus type 2 (MHV-2) in the liver, the development of liver cell necrosis, and survival in murine fulminant hepatitis induced by MHV-2. Murine fulminant hepatitis was induced in 4-week-old male ICR mice by intraperitoneal inoculation of MHV-2. Mouse interferon (10(3) IU/mouse/day) was intraperitoneally injected every day. Exogenous mouse interferon suppressed both the growth of MHV-2 in the liver tissue and development of liver cell necrosis, and prolonged the survival. It was also found that the earlier mouse interferon was administered, the greater was the prolongation of survival.     

Aetiology and prognosis of fulminant viral hepatitis in Japan: A multicentre study

In 236 patients with fulminant viral hepatitis (FVH), type B (FBH) was most common (47.5%), followed by non-A non-B hepatitis (FNANB, 44.9%) and hepatitis type A (FAH, 7.6%). The survival rate was significantly higher in the FAH group than in the FBH and FNANB groups (61.1, 36.6 and 18.9% respectively), and was significantly higher in the FBH group than in the FNANB group. In spite of screening for hepatitis B virus (HBV), FBH was prevalent (27 of 41) in post-transfusion cases; this phenomenon is discussed in relation to a recently revealed mutation of HBV. Within a month after the onset of hepatitis symptoms all cases in the FAH, 93% in the FBH and 79% in the FNANB group, developed encephalopathy. When the duration of illness before the onset of encephalopathy was more than 10 days (a subacute form), the survival rate was significantly lower than when encephalopathy developed in less than 11 days (an acute form). This difference could be accounted for by the difference in the relative frequency of aetiological viruses in the two forms and the higher survival rate in the acute, than the subacute, form in the FNANB group.     

Aetiology and outcome of acute viral hepatitis in pregnancy

The aetiologic types of sporadic acute viral hepatitis in 169 pregnant women were compared with those of 70 non-pregnant women and 287 adult men. The majority of pregnant women (87.6%) came with acute hepatitis in the last trimester of pregnancy. Non-A, non-B (NANB) hepatitis accounted for 81.6% of hepatitis during pregnancy in comparison with 48.6% in non-pregnant women and 57.1% in adult men. Hepatitis A was extremely uncommon during pregnancy. Hepatitis B infection accounted for 17% of all cases in pregnant women compared with 45% in controls. Acute viral hepatitis in pregnancy had a poor outcome as assessed by maternal and/or fetal mortality (28.5%). The outcome was equally bad in hepatitis NANB and hepatitis B. Pregnant women generally had significantly lower immunoglobulin levels in comparison with non-pregnant women. In acute NANB hepatitis during pregnancy, serum IgG and IgM levels were lower and higher, respectively, compared with those in non-pregnant women and pregnant women with acute hepatitis B. It is suggested that an immune suppression during pregnancy might be responsible for increased susceptibility to acute NANB viral hepatitis, which, by itself, seems to induce only a transient acute phase IgM response.     

Accurate prediction of fulminant hepatic failure in severe acute viral hepatitis: multicenter study

Background: We have attempted to predict the development of fulminant hepatic failure at the stage of severe acute hepatitis before the onset of coma. This prediction is valuable because it may be used to block the development of fulminant hepatic failure with appropriate medical treatment. Methods: To establish a discrimination formula, we retrospectively compared 13 clinical and laboratory variables in 36 patients with acute viral hepatitis and prothrombin levels of 40% or less of the control value who later developed fulminant hepatic failure with these variables in 12 patients who recovered spontaneously. A prospective study of 58 patients who developed fulminant hepatic failure and 18 who spontaneously recovered confirmed the validity of this formula. Results: In the retrospective study, we established the following discrimination equation: Z = −0.89 + 1.74 × (causal viruses, 1 point for type A or type B in acute hepatitis B virus [HBV] infection, 2 points for others) + 0.056 × (total bilirubin, mg/dl) −0.014 × (cholinesterase, U/ml). A positive Z value indicates that fulminant hepatic failure will develop. In the prospective study, the specificity, sensitivity, predictive accuracy, and positive and negative predictive values were 0.833, 0.983, 0.947, 0.950, and 0.938, respectively. Conclusions: The present study indicated that fulminant hepatic failure can be predicted, by a simple discrimination equation, at the stage of severe acute hepatitis. Received: September 4, 2001 / Accepted: May 31, 2002 Acknowledgments. Supported by a grant from the Ministry of Health and Welfare of Japan. Reprint requests to: M. Yoshiba     

Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon

A dynamic equilibrium between viral production and clearance characterizes untreated chronic hepatitis C viral infection. After initiating antiviral treatment, a typical multiphasic decay of viremia can be observed and analyzed using mathematical models. To elucidate the antiviral mechanism of ribavirin when used in combination with (pegylated) interferon alfa, we investigated kinetic parameters in patients with chronic hepatitis C treated with either peginterferon alpha-2a with or without ribavirin and standard interferon alpha-2b plus ribavirin for 48 weeks. Serum HCV RNA was measured frequently before, during, and at the end-of-treatment and the follow-up period. By using an appropriate model for viral dynamics, kinetic parameters were derived from nonlinear, least square fitting of serum HCV RNA quantifications. The first phase of viral decay (day 1) and the second phase of viral decay (days 2 to 21) were similar for all treatment groups. After about 7 to 28 days, a third phase of viral decay was seen in several patients, and this phase of decay was significantly faster in patients treated with peginterferon alpha-2a plus ribavirin compared with those treated with peginterferon alpha-2a alone. The decay of this third phase was associated with the virologic end-of-treatment response and sustained virologic response. In conclusion, the third-phase decay of initial viral kinetics, which may represent a treatment-enhanced degradation of infected cells, was more pronounced in patients treated with peginterferon alpha-2a plus ribavirin. This finding suggests that combination treatment leads to a better restoration of the patient's immune response.     

Early hepatitis C viral kinetics correlate with long-term outcome in patients receiving high dose induction followed by combination interferon and ribavirin therapy

BACKGROUND/AIMS: The majority of patients with genotype 1 do not respond to interferon (IFN) plus ribavirin. Limited data exist on the use of induction followed by combination therapy. METHODS: In this prospective study of 28 patients infected with genotype 1, randomization involved either daily or twice daily high dose IFN for 6 weeks, followed by standard therapy of 3 million units three times a week in combination with ribavirin for an additional 42 weeks. Hepatitis C virus (HCV) RNA was quantitated before and frequently during treatment. RESULTS: The best correlate of response was delta (the infected cell loss rate). Sixteen patients continued on the study because they had at least a 2 log drop in their HCV RNA levels by week 12; all but one were PCR negative for HCV RNA at 48 weeks, and 14 of these 16 patients continued to be PCR negative at 72 weeks. Both African-Americans in our trial failed to respond to therapy, and differences were evident during the induction phase. CONCLUSIONS: This randomized study of induction IFN therapy followed by combination IFN plus ribavirin yielded the highest rate of sustained response (50%) reported to date in chronically HCV-infected patients with genotype 1. The predictive value of the infected cell loss rate needs to be evaluated prospectively in larger studies, particularly in patients receiving pegylated IFN.