Human T cell responses to recombinant mite antigens of Dermatophagoides farinae

We studied T cell responses to four glutathione S transferase (GST)-fused mite antigens prepared in our laboratory using peripheral blood lymphocytes from mite-sensitive patients with bronchial asthma. Of the four recombinant antigens, purified GST-Mag3 had the strongest ability to cause patients' lymphocytes to proliferate, and its potency was almost comparable to that of crude mite bodies (Dfb) and faeces (Dff) antigens and a purified major antigen, Der f 2. The responder lymphocytes were mainly T cells, because the proliferative response was depleted by the treatment of lymphocytes with anti-CD3 antibody and complement, but not with anti-CD20 antibody and complement. The responsiveness of lymphocytes to GST-Mag3 correlated with that to Der f 2, but GST-Mag3 displayed slightly higher activity to stimulate lymphocytes than Der f 2. Simultaneously, the levels of Dff- and GST-Mag3-specific IgE antibodies correlated with the responsiveness of lymphocytes to GST-Mag3. These results suggest that Mag3 is a new valuable antigen for the response of T cell proliferation in mite-sensitive patients.     

Similarities between neuronal Lewy bodies in parkinsonism and hepatic Mallory bodies in alcoholism

The aim of the present study was to identify components of the Lewy body, which is a characteristic neuronal lesion in idiopathic Parkinsonism, using histochemical methods that selectively stain the Mallory body, a characteristic lesion of the hepatocyte in alcoholism. Our observation that Lewy bodies stain with phosphotungstic acid hematoxylin, the dye originally used for demonstrating alcoholic hyaline (Mallory bodies), promoted this study. The material consisted of formalin-fixed, brain stem tissue from Parkinsonian subjects, and of similarly preserved liver tissues from alcoholic individuals. The methods selected were Roque's chromotrope 2R-aniline blue, and Liisberg's rhodamine B, which stains Mallory bodies due to its affinity for sites of tissue keratinization. Hence, skin was also included in this study as control tissue. Our results showed that Lewy bodies in the brain, Mallory bodies in the liver and stratum corneum in the skin have identical staining properties with the dyes used, indicating the presence of histochemically similar components. Taking into account the reactions of these dyes with model substances, we suggest that the similar components shared by Lewy bodies and Mallory bodies are arginine-rich proteins and lipids associated with keratinization. Similar findings in both, a toxin-induced lesion of the liver, and a spontaneous lesion of the brain may offer clues for understanding the latter's mode of formation.     

Foci of altered hepatocytes in a fox squirrel (Sciurus niger) with haemochromatosis

Numerous minute milky white foci were distributed throughout the dark brown liver in an adult male fox squirrel. Histologically, the hepatic focal lesions were composed of large eosinophilic granular hepatocytes, which were mostly positive for glutathione S-transferase mu antigen and proliferating cell nuclear antigen. Electron microscopy demonstrated an increased number of mitochondria. These features corresponded to those in the eosinophilic type of foci of altered hepatocytes. Berlin blue stain showed severe haemosiderin deposition in hepatocytes, except in the focal lesions. Since the fox squirrel is known to be liable to develop congenital porphyria, it is suggested that the hepatic anomalies described may be closely associated with the development of porphyria.     

Aggresome formation in liver cells in response to different toxic mechanisms: role of the ubiquitin-proteasome pathway and the frameshift mutant of ubiquitin.

Aggresomes form in cells when intracellular proteins undergo conformational changes, as in so-called conformational diseases. This phenomenon has been observed in the liver and brain and in cell culture in response to abnormal protein formation, such as mutant proteins. In the case of the brain the frameshift mutant ubiquitin (UBB+1) is involved. Mallory body formation in the liver is one example of this phenomenon in vivo. Mallory body formation is common in a variety of liver diseases of diverse pathogenesis. The study of the Mallory body forming model indicated that drug-conditioned hepatocytes form Mallory bodies when mice are given colchicine, ethanol, okadaic acid, or exposure to heat shock. These findings suggest that aggresome formation is a common pathway of liver injury due to diverse mechanisms. To further characterize the role of this common pathway, drug-primed mice were exposed to different types of liver injury, i.e., using such drugs as thioacetamide, galactosamine, tautomycin, and the proteasome inhibitor PS341. Mallory body formation was induced by treatment with all the toxins tested, giving credence to the proposal that aggresome formation in the liver is a common pathway in response to different primary mechanisms of liver injury. The frameshift mutant UBB+1 was invariably found to colocalize with ubiquitin in the Mallory body, indicating its essential involvement in the mechanism of MB formation.     

The role of innate immunity in the pathogenesis of preneoplasia in drug-induced chronic hepatitis based on a mouse model

Innate immunity factors such as conversion of the 26S proteasome to form the immunoproteasome and the Toll-like receptor signaling pathways are activated in chronic hepatitis induced by the carcinogenic drug DDC. Over time, preneoplastic hepatocyte phenotypes appear in the liver parenchyma. These changed hepatocytes expand in number because they have a growth advantage over normal hepatocytes when responding to chronic liver injury. The changed hepatocytes can be identified using immunofluorescent antibodies to preneoplastic cells e.g. FAT10/UbD, A2 macroglobulin, glutathione transpeptidase, alpha fetoprotein, glycipan 3, FAS, and gamma glutamyl transpeptidase. The formation of the preneoplastic cells occurs concomitant with activation of the Toll-like receptor signaling pathways and the transformation of the 26S proteasome to form the immunoproteasome. This transformation is in response to interferon stimulating response element on the promoter of the FAT10/UbD gene. NFκB, Erk, p38 and Jnk are also up regulated. Specific inhibitors block these responses in vitro in a mouse tumor cell line exposed to interferon gamma. Mallory–Denk bodies form in these preneoplastic cells, because of the depletion of the 26S proteasome due to formation of the immunoproteasome. Thus, MDB forming cells are also markers of the preneoplastic hepatocytes. The UbD positive preneoplastic cells regress when the liver injury induced chronic hepatitis subsides. When the drug DDC is refed to mice and chronic hepatitis is activated, the preneoplastic cell population expands and Mallory–Denk bodies rapidly reform. This response is remembered by the preneoplastic cells for at least four months indicating that an epigenetic cellular memory has formed in the preneoplastic cells. This proliferative response is prevented by feeding methyl donors such as S-adenosylmethionine or betaine. Drug feeding reduces the methylation of H₃ K4, 9, and 27 and this response is prevented by feeding the methyl donors. After 8 to 15 months of drug withdrawal in mice the preneoplastic liver cells persist as single or small clusters of cells in the liver lobules. Multiple liver tumors form, some of which are hepatocellular carcinomas. The tumors immunostain positively for the same preneoplastic markers as the preneoplastic cells. Similar cells are identified in human cirrhosis and hepatocellular carcinoma indicating the relevance of the drug model described here to the preneoplastic changes associated with human chronic hepatitis and hepatocellular carcinoma.     

The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.     

Isolation of Mallory bodies and an attempt to demonstrate cell mediated immunity to Mallory body isolate in patients with alcoholic liver disease

Mallory bodies were isolated from necropsy livers from patients with alcoholic hepatitis with and without cirrhosis with a Ficoll viscosity barrier. The purity of Mallory bodies in the isolate varied between 70 and 90%, estimated by counting Mallory bodies and non-Mallory body structures in haematoxylin-eosin stained smears. Electron microscopy confirmed the presence of Mallory bodies in the isolates. The Mallory body isolate was used as antigen in the agarose leucocyte migration inhibition test in order to test the cell-mediated immunity. No significant difference in leucocyte migration was found between controls and patients with alcoholic hepatitis, alcoholic steatosis, alcoholic cirrhosis and miscellaneous liver diseases.     

重组汉滩病毒核蛋白的纯化及鉴定

目的：纯化重组表达的汉滩病毒核蛋白。方法：重组菌经IPTG诱导后，表达的目的蛋白为带有谷胱甘肽转硫酶（GST）标签的融合蛋白，并以包涵体形式存在。将包涵体变性、复性后，采用Glutathione Sepharose 4B亲和色谱对核蛋白进行纯化，并用夹心ELISA和Western blot检测纯化蛋白。结果：表达产物第一次过柱亲和层析后可去除杂蛋白，获得纯化的核蛋白与谷胱甘肽转硫酶的融合蛋白，再经凝血酶酶切，第二次过柱亲和层析后获得的穿过峰为核蛋白，洗脱峰为GST。纯化的融合蛋白和纯化的核蛋白均为SDS－PAGE单点纯，并具有良好的抗原活性。结论：Glutathione Sepharose 4B亲和色谱纯化重组汉滩病毒核蛋白是一种较为有效的方法。     

肝细胞代偿性增生对二甲基亚硝胺致癌作用的影响

目的探讨肝细胞代偿性增生对二甲基亚硝胺（ＮＤＭＡ）致大鼠肿瘤作用的影响。方法对照组用ＮＤＭＡ处理,实验组首次ＮＤＭＡ处理前２４小时行部分肝切除术。观察肿瘤发生情况及γ谷氨酰转肽酶（ＧＧＴ）、胎盘型谷胱甘肽Ｓ转移酶（ＧＳＴＰ）、增殖细胞核抗原（ＰＣＮＡ）和癌基因的表达。结果实验组肝脏ＧＧＴ和ＧＳＴＰ灶的数量和面积高于对照组,ＧＳＴＰ的表达高于ＧＧＴ,肾癌变过程中见ＧＳＴＰ的表达。实验组第５６周肿瘤总发生率和第７１周肿瘤总发生率及肝、肾肿瘤发生率高于对照组。ＰＣＮＡ阳性细胞数量与肝病变组织的增殖情况一致。胰岛素样生长因子Ⅱ（ＩＧＦⅡ）与ｃｍｙｃ、ＨｒａｓｍＲＮＡ的表达在变异肝细胞灶和结节较高,在肝细胞癌和腺瘤较弱,肝组织未见ｃｊｕｎｍＲＮＡ表达。结论肝细胞代偿性增生促进多次低剂量ＮＤＭＡ的致癌作用。ＩＧＦⅡ与ｃｍｙｃ、Ｈｒａｓ基因产物的过量表达在肝癌发生、发展中可能具有协同作用。     

Iron-negative foci in siderotic macroregenerative nodules in human cirrhotic liver. A marker of incipient neoplastic lesions

Resistance to iron accumulation is known as a phenotypic marker of neoplasia and preneoplasia in experimental hepatocarcinogenesis in rodents overloaded by iron. This study was aimed to evaluate whether such iron-free foci are present and valuable for identification of preneoplastic and incipient neoplastic lesions in human cirrhotic livers, especially within macroregenerative nodules in which hepatocellular carcinoma is known to arise. Iron-free foci were found in siderotic macroregenerative nodules in liver cirrhosis. These foci were classic and overt carcinoma or borderline lesions showing an expansive growth pattern. Borderline lesions were composed of hyperplastic or small basophilic hepatocytes with hyperchromasia and distinct nuclear membrane showing pseudoglandular, trabecular, compact, and scirrhous patterns. These data suggest that iron stain is valuable for identification of neoplastic or borderline lesions representing a transition from hyperplastic nodule to carcinoma in human liver cirrhosis.     

Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver

In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.     

Alcoholic hepatitis: granulocyte chemotactic factor from Mallory body-stimulated human peripheral blood mononuclear cells

The characteristic histopathological features of acute alcoholic hepatitis include hyaline degeneration of hepatic parenchymal cells (Mallory bodies), hepatocellular necrosis, and granulocyte infiltration of the liver. The chemotactic response of neutrophils to highly purified Mallory bodies was studied. Mallory bodies, per se, were not chemotactic for granulocytes, nor did they generate chemotactic factors when incubated with serum. However, a factor(s) chemotactic for both granulocytes and mononuclear cells was generated when Mallory bodies were incubated with mononuclear cells, both from patients with alcoholic hepatitis or from normal controls. It was concluded that Mallory bodies stimulate peripheral blood mononuclear cells to release a factor chemotactic for granulocytes and mononuclear cells. This factor may be important in the etiology of the cellular infiltration in the livers of patients with alcoholic hepatitis.     

Glutathione S-transferase π-class as a tumour marker in lingual preneoplastic and neoplastic lesions of rats and humans

Immunocytochemical expression of the π class glutathione S-transferase (GST) was investigated in preneoplastic and neoplastic lingual lesions in a 4-nitroquinoline 1-oxide (4NQO)-induced rat genetic model [Wistar/Furth rats (WF) and Dark-Agouti rats (DA)] and in human surgical material [fibrous polyp, mild to moderate dysplasia, severe dysplasia, carcinoma in situ (CIS), squamous cell carcinoma (SCC)]. Two polyclonal antibodies raised against rat (GST-P) and human (GST-π) antigens were used. In the rat model, DA and WF rats showed contrasting susceptibility to 4NQO, DA rats having a much higher tumour incidence and a significantly shorter survival time than WF rats. While the established lingual SCC in DA and WF rats all expressed GST-P, the number of GST-P⁺ foci in the preneoplastic lingual epithelium was significantly higher in DA (14.5 ± 6.5) than in WF rats (5.5 ± 2.6; P < 0.0001). In contrast, GST-π epithelial staining in human specimens was more variable and the results overlapped in different groups. More frequent nuclear and/or basal cell staining was detected in severe dysplasia, CIS and SCC than in benign and mild to moderate dysplastic lesions. Although the π class GST may be a useful marker for rat lingual carcinogenesis, its value in clinical applications is unclear. GST-π staining patterns and their distribution may be helpful in identifying high-risk lingual lesions in humans.     

Glutathione S-transferase in bone marrow metastases of disseminated neuroblastoma.

Antisera to each of the three main cytosolic forms of glutathione S-transferase (GST; alpha, mu, and pi) has been used to characterise GST expression by metastatic neuroblastoma in bone marrow trephine biopsies taken from 15 patients at presentation and from five of this group at relapse. There was no correlation between expression of extra-nuclear alpha or mu GST and outcome, and no consistent pattern at relapse. Seven of eight expressing nuclear pi GST at presentation died of resistant disease. Three of five cases with no detectable nuclear pi class GST remain alive and disease free. The results provide no encouragement for further investigation of alpha or mu GST in this disease but larger studies of uniformly treated patients may show whether nuclear pi GST expression at presentation indicates likely relapse.     

Human hepatic preneoplasia: phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia

 Foci of altered hepatocytes (FAH) represent preneoplastic lesions, as shown in various animal models of hepatocarcinogenesis, but their significance in the human liver has not been established. The cellular composition, size distribution and proliferation kinetics of FAH in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC) and their possible association with small-cell change of hepatocytes (SCC) were therefore studied. FAH, including glycogen-storing foci (GSF), mixed cell foci (MCF) and basophilic cell foci, were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with (29/32) than in those without HCC (55/79). FAH were observed more frequently in HCC-free cirrhosis associated with hepatitis B or C virus or chronic alcoholic abuse (high-risk group) (37/47) than in that due to other causes (low-risk group) (12/21). MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of nodules of altered hepatocytes (39.3%) than were GSF (8.5%). The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSF. Oncocytic and amphophilic cell foci were also observed, but their significance remains to be clarified. Two types of SCC, namely diffuse and intrafocal SCC, were identified, but only intrafocal SCC was found to be related to increased proliferative activity and more frequent nodular transformation of the FAH involved, suggesting a close association with progression from FAH to HCC. Received: 11 March 1997 / Accepted: 2 June 1997     

Relevance of hepatic preneoplasia for human hepatocarcinogenesis

Different lesions have been suggested to represent preneoplastic conditions in human liver. They include liver cell dysplasia, separated in large-cell change (LCC) and small-cell change (SCC), adenomatoid hyperplasia, and the more recently identified foci of altered hepatocytes (FAH) and nodules of altered hepatocytes (NAH). FAH have been demonstrated to represent preneoplastic lesions in various animal models of hepatocarcinogenesis. To demonstrate prevalence and significance of FAH in the human liver, the cellular composition, size distribution, and proliferation kinetics of these lesions were studied in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC). FAH including glycogen-storing foci (GSF), mixed cell foci (MCF), and basophilic cell foci were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with than without HCC. MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of NAH than GSF. The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSP. We also investigated the relationship of FAH to liver cell dysplasia. Occurrence of SCC, rather than that of LCC, confers FAH an increased proliferation activity and higher risk to nodular transformation, and, hence, should be considered a precancerous condition. Histological detection of FAH and SCC through needle-aspiration liver biopsy can be used for monitoring HCC development in high-risk populations, such as HBV carriers with chronic hepatitis and/or cirrhosis.     

Thickened area of external granular layer and Ki-67 positive focus are early events of medulloblastoma in Ptch1+/− mice

Patched1 (Ptch1) encodes a receptor for Sonic hedgehog (Shh) and is major gene related to human medulloblastoma (MB) in the Shh subgroup. MB is thought to arise from residual granule cell precursors (GCPs) located in the external granular layer (EGL) of the developing cerebellum. As the detailed preneoplastic changes of MB remain obscure, we immunohistochemically clarified the derived cell, early events of MBs, and the cerebellar developmental processes of Ptch1^+/? (Ptch1) mice, an animal model of human MB of the Shh subgroup. In Ptch1 mice, the earliest proliferative lesions were detected at PND10 as focal thickened areas of outer layer of the EGL. This area was composed of GCP-like cells with atypia and nuclei disarrangement. In the latter cerebellar developmental period, GCP-like cell foci were detected at high incidence in the outermost area of the cerebellum. Their localization and morphological similarities indicated that the foci were derived from GCPs in the EGL. There were two types of the foci. A Ki-67-positive focus was found in Ptch1 mice only. This type resembled the GCPs in the outer layer of EGL characterized by having proliferating activity and a lack of neuronal differentiation. Another type of focus, Ki-67-negative, was observed in both genotypes and exhibited many of the same features of mature internal granule cells, suggesting that the focus had no preneoplastic potential. Due to morphological, immunohistochemical characteristics, our results indicate that the focal thickened area of EGL and Ki-67-positive foci are preneoplastic lesions of MB.     

Malignant progression of adenomyoepithelial adenosis of the breast

A case of breast tumor is described, which consisted of dense and uniform proliferation of ducts and lobules composed of both epithelial and myoepithelial cells and in which multiple foci of adenocarcinoma were observed. The tumor surrounding the carcinoma foci was identified as 'adenomyoepithelial adenosis'. Adenomyoepithelial adenosis was not monoclonal by clonal analysis, but revealed a relatively high labeling index for proliferating cell nuclear antigen by immunohistochemistry. Although it was still undetermined whether adenomyoepithelial adenosis is a non-clonal nonneoplastic lesion or a biclonal neoplastic one, the lesion was shown to reveal high proliferative activity in both glandular epithelial and myoepithelial cells and was considered to be prone to progress to obvious carcinoma.     

Phenotypic characterization and functional activity of human milk macrophages

A large population (about 80%) of the cells obtained from colostrum and early human milk were considered to be macrophages by the following criteria: nonspecific esterase stain, adherence, phagocytosis and IgG-Fc receptor expression. The majority of freshly isolated human milk macrophages (HMM phi) stain for the monocyte antigen OKM1. Another monocyte antigen, 61D3, was expressed only by 30% of HMM phi. Class II antigens were expressed by HMM phi. About 85% of the cells were DR-positive whereas 50% were DS-positive as assessed with a panel of monoclonal antibodies directed against class II antigens. Monocyte and class II antigens were gradually lost during in vitro culture. HMM phi can support proliferative response to antigens and mitogens when cocultured with autologous peripheral T cells. The proliferative response was significantly reduced when monoclonal antibodies to DR or DS were added to the assay. These results indicate that HMM phi have the phenotype and functional characteristics of antigen presenting cells.