Liver cell dysplasia: a premalignant condition

Liver cell dysplasia is defined as cellular enlargement, nuclear pleomorphism, and multinucleation of liver cells occurring in groups or occupying whole cirrhotic nodules. The prevalence, natural history, and relationship to the Australia or hepatitis-associated antigen (HAA) have been studied in 552 Ugandan African patients with normal, cirrhotic, and cancerous livers. Liver cell dysplasia was found in only two of 200 (1%) patients with normal livers, in three of 43 (6·9%) of patients with normal livers bearing primary liver cell carcinoma, 35 of 175 (20·3%) patients with cirrhosis, and 80 of 124 (64·5%) of patients with cirrhosis and primary liver cell carcinoma. Cirrhotic patients without dysplasia were, on average, ten years younger than those with dysplasia and the latter were on average six years younger than those with cirrhosis and carcinoma. Liver cell dysplasia occurred more frequently in males than in females. It was found in all but one instance in macronodular or mixed forms of cirrhosis only. There was a strong relationship between dysplasia and the presence of HAA in 104 patients that suggests a possible carcinogenic mechanism for the longincubation (serum or B) hepatitis virus in liver cell carcinoma. It is concluded that the presence of liver cell dysplasia identifies a group of patients with a high risk of liver cell carcinoma and that they should be followed up by serial alpha-fetoprotein estimations.     

Association between the degree of thymic dysplasia and the kinetics of thymic NK cell activity during the graft-versus-host reaction

In this study, by employing different cell doses and parent into F1 hybrid combinations, we have investigated the relationship between the severity of thymic medullary dysplasia and the kinetics of thymic natural killer (NK) cell activity after the induction of graft-versus-host (GVH) reactions. GVH reactions were induced by injecting different doses (30, 20, 10 X 10(6] of C57BL/6 (B6) of A parental lymphoid cells (PLC) into non-X-irradiated adult B6xAF1 (B6AF1) mice. On different days after the induction of GVH reactions, the thymuses were examined histologically and thymocyte NK cell activity was tested by using YAC targets. Our results show that, depending upon the genotype and dose of PLC injected, various degrees of thymic medullary dysplasia (mild, moderate, or severe) can be induced. Furthermore, severe to moderate thymic medullary dysplasia is observed only in those groups of GVH-reactive mice in which thymic NK cell activity occurs early and increases rapidly. In contrast, when mild thymic medullary dysplasia or no thymic alterations was observed, thymic NK cell activity peaked later and was of lower intensity than that of the groups with moderate to severe lesions. These results suggest an association between the degree of thymic medullary dysplasia and the kinetics of NK cell activity in the thymus. Furthermore, the different degrees of thymic medullary dysplasia as described here may serve as a powerful tool to study the role of thymic medullary dysplasia in determining the duration of T-cell immunodeficiency associated with the GVH reactions.     

Large-scale production of functional recombinant CAR in a baculovirus- insect cell system

Coxsackievirus group B and adenovirus receptor (CAR) is a major receptor for the adenovirus groups that has drawn overall attention over the past decade. Although this protein could potentially be used as an agent for the blocking of adenovirus infection, large-scale production of highly purified human CAR in eukaryotic expression system has not been reported. In the present study, we showed the construction of recombinant baculovirus highly-expressing the extracellular domain of human coxsackievirus-adenovirus receptor (exCAR) in High Five insect cells. The recombinant exCAR was recovered from the cell culture medium as a secreted soluble protein and purified by Ni-NTA affinity chromatography. The final yield of recombinant exCAR was about 8-10 mg/l of supernatant with the purity of 96.3%. Binding activity assay showed that the recombinant exCAR exhibited an intact ability of binding to the knob domain of the adenovirus type 5 fiber protein (Ad fiber knob) displayed by T7 phage. These results showed that the recombinant human exCAR produced in insect cells and purified by Ni-NTA chromatography retained its ability to bind to the Ad fiber knob and could potentially be used in therapy of adenovirus infection.     

Histopatholgical spectrum of thymic neoplasms: twelve-year experience at a referral hospital in north India

This study was undertaken to determine the histopathological spectrum and clinical profile of thymic neoplasms at a tertiary referral care centre. A total of 96 thymectomy specimens were received during the study period (1992-2004), which consisted of 54 neoplasms and 42 benign lesions. Among the neoplasms there were 48 thymic epithelial tumors, 3 thymolipomas and 3 thymic carcinoids. The former comprised of 36 male (75%) and 12 female patients (25%) ranging in age from 2-70 years (mean 37 years). Among paraneoplastic syndromes in thymic epithelial tumours, 27 out of 48 (56.25%) cases were associated with myasthenia gravis and one case was associated with pure red cell aplasia. The most frequent histological subtype was cortical thymoma (43.24%) followed by predominantly cortical (24.32%) and well-differentiated thymic carcinoma (18.92%). On staging, all cases of mixed and predominantly cortical subtype were stage 1 whereas one medullary and 2 cortical thymomas and 4 well differentiated thymic carcinoma (WDTC) showed pleural and pericardial invasion (stage III). This study has revealed that half of thymic epithelial tumours presented as myasthenia gravis. The cortical thymoma was the most frequently encountered histologic subtype and most commonly associated with myasthenia gravis.     

In vivo transduction of thymic dendritic cells with adenovirus and its potential use in acute inflammatory diseases

Dendritic cells (DC) represent a potential target for gene therapy. In their ability to process antigens and present them to T cells, DC have been allocated a unique role as initiators of the immune response in both the innate and acquired immunity. Recent in vitro studies have showed the feasibility of DC transduction with adenoviral recombinants. In cancer therapy, targeting of DC with adenovirus has been proved to be effective in inhibiting tumour growth, as well as in reducing the number of tumour metastases. The aim of our study is to evaluate the feasibility of in vivo transduction of DC in a murine lymphocyte-rich compartment (thymus) as a potential treatment for acute inflammatory diseases. Nearly 50% of the total thymic DC were transduced with a first-generation adenoviral construct following intrathymic injection, and post-transductional inflammation was neglectable. Transduction of thymic cells with adenoviral recombinants was able to induce the expression of an intracellular protein (beta-galactosidase, green fluorescent protein), as well as the secretion of human interleukin-10, within the local compartment. Furthermore, this induction of the latter significantly decreased thymic apoptosis in the applied model of acute bacterial peritonitis (cecal ligation and puncture).     

Infection of mouse liver by human adenovirus type 5.

CBA mice, inoculated intravenously with large doses of adenovirus type 5, showed raised levels of serum aspartate aminotransferase (SAAT; EC 2.6.I.I) and died within a few days from histologically demonstrable hepatic necrosis. After inoculation of I LD50, virus was rapidly taken up by the tissues where infectivity then declined greatly. Organ titres then increased about 100-fold by 48 h p.i. but, in the liver, which showed intranuclear inclusion bodies, and by electron microscopy, scattered intranuclear and intracytoplasmic adenovirions, the increase was 10000- to 100000-fold. P antigen was detected by single radial diffusion in liver extracts, and by immunofluorescence in 80% of liver cells at 36 h p.i. Hexon, penton base and fibre antigens appeared later and in fewer cells. The maximum amount of hexon, of demonstrable type 5 specificity, was shown by radioimmunoassay to be equivalent to up to 5 x 1011 whole adenovirions/g liver. It is concluded that human adenovirus type 5 undergoes an abortive but lytic infection in most liver cells but that replication may proceed to completion in a few.     

Thymic involution in murine graft-versus-host reaction. Epithelial injury mimicking human thymic dysplasia.

Mild, moderate, and severe graft-versus-host (GVH) reactions were induced in four series of experiments in 71 CBA X A and C57BL/6 X A F1 hybrid mice. At regular intervals post-GVH reaction induction (Days 4-42), the animals were sacrificed, autopsied, and histologically studied. Visceral alterations of GVH reaction were recorded in the spleen, lymph nodes, liver, kidney, gut, and thymus. A spectrum of thymic changes was documented, ranging from obliteration of a definable cortex and medulla with loss of Hassall's corpuscles to marked involution with complete disappearance of the gland. Ultrastructural studies revealed damage to both lymphocytes and epithelial cells along with lymphocyte emperipolesis of epithelial cells, lymphocytolysis within epithelial cells, and accumulation of numerous autophagic vacuoles containing fragments of cellular debris within epithelial cells and histiocytes. The resemblance of these alterations to human thymic dysplasia as observed in primary immunodeficient conditions was striking. The theoretical implications of these studies for the pathogenesis of human congenital immunodeficiency states are considered.     

人胸腺基质淋巴生成素基因重组腺病毒载体的构建及表达

目的: 构建含人胸腺基质淋巴生成素基因(TSLP)的重组腺病毒载体并表达, 以研究其免疫学功能。方法: 将由人胚肺细胞扩增得到的TSLP基因, 克隆于真核表达载体pcDNA3. 1中, 再亚克隆至穿梭质粒pShuttle中, 并与腺病毒骨架载体pAdEasy -1共同转化大肠杆菌。以获得的重组质粒线性化后转染HEK293细胞, 并包装成病毒颗粒。采用PCR法对重组腺病毒基因进行鉴定, 并以Westernblot检测TSLP蛋白的表达。结果: 通过细菌内同源重组, 成功构建带有人胸腺基质淋巴生成素基因的重组腺病毒质粒, 转染 293细胞后, 包装的重组病毒经PCR检测表明, 基因组含有目的基因,病毒的滴度可达 1×1011pfu/L。Westernblot证实, 感染的肿瘤细胞中有相应基因产物的表达。结论: 通过菌内重组可高效制备带有特定基因的重组病毒。所制备的Ad -TSLP可成功表达相应基因产物, 为进一步研究这一新型细胞因子的功能奠定了基础。     

Mice Engrafted with Human Fetal Thymic Tissue and Hematopoietic Stem Cells Develop Pathology Resembling Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3⁺ thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells.     

Disseminated Adenovirus Disease in Immunocompromised Patient Successfully Treated with Oral Ribavirin: A Case Report

In patients with immunological disorders, adenovirus infections are associated with significant rates of morbidity and mortality. Only few hematological units use molecular virological methods, such as polymerase chain reaction, for surveillance of adenovirus infection, and treatment strategies have never been evaluated in multicenter clinical trials. This report describes the detection and treatment of human adenovirus (HAdVs) disseminated disease in the case of a 46-year-old immunocompromised female having myelodysplastic syndrome with refractory cytopenia with multilineage dysplasia: International Prognostic Scoring System 1. Serum and urine samples were tested for the presence of adenoviral DNA using the quantitative real-time polymerase chain reaction (PCR) assay. For additional confirmation, sequencing of PCR products was also performed. With real-time PCR, we detected HAdV DNA in both serum and urine samples. The viral level constantly decreased with applied oral ribavirin therapy. As the result of sequencing, HAdVs type 11 was determined. Surveillance of adenovirus by real-time PCR is useful in detecting and monitoring disseminated HAdV infection; it is a potential standard diagnostic approach that could assist clinicians to decide whether antiviral therapy ought to be administered.     

Expression of early activation antigen (CD69) during human thymic development.

The novel early activation antigen, EA1, has been shown to be induced by mitogens, antigens and the tumour promoter, phorbol myristate acetate (PMA), on human lymphocytes. This antigen has been designated to be CD69. EA1 has also been shown to be expressed on thymocytes without exogenous activation stimuli. In order to characterize further the expression of EA1 on thymocytes, the ontogeny of its expression was studied. EA1 appeared between 7 and 9.5 weeks of gestation, after colonization of the thymic rudiment with CD7+ T cell precursors, but before the onset of compartmentalization of the thymus into cortical and medullary zones. After cortico-medullary differentiation, the majority of medullary thymocytes expressed EA1 while only a fraction of the cortical thymocytes expressed this antigen. In the fetal and post-natal cortex, EA1 expression appeared to cluster in the subcapsular cortex. EA1+ cells were also scattered throughout the inner cortex. By two-colour fluorocytometric analysis of post-natal thymocytes, it was shown that EA1 was expressed on 30 to 65% of thymocytes. EA1 was expressed on CD4+ CD8+ as well as on the more immature CD4- CD8- thymocytes. In contrast to circulating T cells, thymocytes were much less responsive to PMA stimulation for the expression of EA1. Molecular characterization showed that EA1 on thymocytes had the same structure as that of activated peripheral T cells. In addition, thymic EA1 was constitutively phosphorylated. Thus, EA1 expression is acquired early during thymic development after colonization of the thymic rudiment by CD7+ T cell precursors. However, the specific role that EA1 may play in the activation and function of developing thymocytes remains to be determined.     

Subtypes of bovine adenovirus type 2 exhibit major differences in region E3

The genomes of two adenovirus type 2 strains which were isolated from different hosts have been investigated. One of these strains designated ORT-111 was originally isolated from a lamb in Hungary during an outbreak of pneumoenteritis. This isolate was typed as bovine adenovirus type 2 (Ad bos 2) in a neutralization assay. The genome of ORT-111 was compared to that of the prototype strain of Ad bos 2, a virus which exclusively has been isolated from cattle. Electron microscopic heteroduplex analysis showed that 95% of the genomes were well matched, forming stable duplexes at Tm -6 degrees. Two distinct substitution loops were, however, seen which were approximately 0.5 and 1.0 kbp long. The centers of the two loops were located 5.3 and 7.7 kbp from one end of the Ad bos 2 genome. In order to map these regions relative to the gene map of human adenovirus type 2 (Ad2), restriction enzyme cleavage fragments of the two bovine viruses were cloned and hybridized to different sets of restriction fragments of human Ad2. From these results it was apparent that the centers of the two substitution loops were located at coordinates 76 and 83, respectively; thus at positions which fall within region E3 and the adjacent gene for polypeptide VIII of human Ad2. The observed differences between the genomes of the two Ad bos 2 strains are in sharp contrast to those previously observed when the genomes of different human adenovirus serotypes were compared. In the latter case the hexon and the fiber genes showed the most pronounced variation.     

Viral pathogenesis and immunity within the thymus

Replication of viruses within the thymic microenvironment may have a unique impact on viral persistence and pathology. The author’s laboratory has studied thymic infection by both human and murine retroviruses. For human lentiviruses, such as HIV-1, the consequences of persistent thymic replication are frequently a severe disruption of the normal processes of thymopoiesis and potentially of progression to AIDS. Murine retroviruses, such as Gross murine leukemia virus, establish persistent infection with less cytopathic, but no less devastating effects. These include the alteration of immune recognition to retroviral antigens by the peripheral immune response, the thymic persistence of virus, and the establishment of viral-induced thymic leukemia. This article summarizes the analysis of both the common and distinctive means of pathology induced by these two retroviral families with particular attention on the influence and impact of the thymus as a unique site of virus replication.     

Thymic involution mimicking thymic dysplasia: a consequence of transfusion-induced graft versus host disease in a premature infant

A premature infant with ostensibly normal immunologic function received two exchange blood transfusions for hyperbilirubinemia and several blood and platelet transfusions for pancytopenia. A fatal graft vs host reaction (GvHR) ensued that was diagnosed by skin biopsy one day prior to death at age 40 days. Autopsy revealed characteristic alterations of GvHR in the spleen, lymph nodes, gut, and liver. In addition the thymus was small (3.5 g), lymphoid-depleted, lacked corticomedullary demarcation and Hassall's corpuscles, and demonstrated lymphocyte invasion and injury of epithelial cells. The thymic cytomorphology acquired added meaning when it was discovered that a chest roentgenogram obtained at one day of age demonstrated a normal thymic shadow. It is concluded that the GvHR induced a direct injury to the thymic epithelium, resulting in an acquired form of thymic dysplasia. The theoretical implications of this observation are considered.     

Adenovirus hepatitis in two successive liver transplants in a child

Adenoviruses can produce severe disease, especially in patients who are immunosuppressed. We present a unique case in which adenovirus type 5 was demonstrated retrospectively, by using immunohistochemical methods, in the appendix of a liver transplant donor. The donor had intussusception, which has been associated with adenovirus infection. Both the initial and a second liver transplant in the recipient were severely damaged by adenovirus type 5. These findings were demonstrated immunohistochemically, as well as on electron microscopy. The recipient died due to the infection and ensuing complications.     

Demonstration of phenotypic abnormalities of thymic epithelium in thymoma including two cases with abundant Langerhans cells.

A panel of monoclonal antibodies that phenotypically define stages of normal human thymic epithelial (TE) cell maturation was used to compare thymic epithelium of nine thymomas with hyperplastic thymic epithelium in myasthenia gravis (MG) and thymic epithelium of normal thymuses. It has been shown previously that normal thymic epithelial cells express antigens of early TE cell maturation (A2B5, TE-4) throughout thymic ontogeny and acquire antigens 12/1-2, TE8, and TE-15 at 14 to 16 weeks of fetal gestation. Hyperplastic MG thymic epithelial cells expressed TE antigens in phenotypic patterns similar to that seen in normal postnatal thymus, ie, TE in subcapsular cortex and medulla was TE4+, A2B5+, and 12/1 - 2+ and Hassall's bodies were reactive with antibodies TE8 and TE15. In contrast, thymic epithelium in primary mediastinal thymomas was TE4+, A2B5+, TE8-, and greater than 75% of thymoma epithelium was 12/1 - 2-, a thymic epithelial phenotype similar to that seen on normal fetal thymic epithelium at 14 to 16 weeks fetal gestation. In one subject with a mature epithelial histologic pattern, thymoma epithelium was found to be strongly TE8+, a phenotype suggestive of a later stage of TE maturation. Lymphocytes in five of seven thymomas with immature thymic epithelial cells predominantly expressed immature thymocyte phenotype while two thymomas with immature epithelial phenotype showed a predominance of Langerhans cells and surrounding lymphocytes expressing a mature phenotype. Lymphocytes in the thymoma with differentiated epithelial cells expressed a mature thymocyte phenotype. Thus, in thymomas of varying histologic types, phenotypic abnormalities of thymic epithelium are present; these phenotypic abnormalities may reflect abnormal thymic epithelial maturation.     

Potential role of Homer-2a on cutaneous vascular anomaly

Homer protein was identified based on its rapid induction in rat hippocampal granule cell neurons following excitatory synaptic activity. Although the presence of the Homer gene in the peripheral tissues has been observed in previous reports, the physiological function of the Homer protein in these tissues has not been noted. In this experiment, a Homer-2a cDNA fragment was successfully amplified by RTPCR in the involuting phase of human hemangioma but not in the human vascular malformation and normal vessel. After isolation of full Homer cDNA in a mouse liver cDNA library, E1-deleted recombinant adenovirus expressing the Homer protein (Adv.CMV.mHomer-2a) was constructed to determine its physiological function in peripheral tissues. Adv.CMV.mHomer2a, but not Adv.CMV.LacZ (recombinant adenovirus expressing Beta-galactosidase), strongly inhibited the growth rate of HUVECs (human umbilical vein endothelial cells) probably via inducing apoptosis determined by acridine orange/ethidium bromide (AO/EB) staining methods. This study suggests that the Homer gene is present in human specimens in the involuting phase of hemangioma, and it might be involved in the growth control.     

腺病毒介导入B7—1基因对肝癌细胞免疫原性的影响

目的 研究同时含人ｐ５３、ＧＭ－ＣＳＦ、Ｂ７－１、ＩＬ－２基因的重组腺病毒载体Ａｄ－ｍｕｌｔｉｇｅｎｅｓ,对肝癌细胞免疫原性的影响。方法 应用人外周血淋巴细胞和肿瘤细胞的混合培养、外周血Ｔ淋巴细胞杀伤活性试验,分析导入目的基因的人肝癌细胞免疫原性的变化。结果 导入Ａｄ－ｍｕｌｔｉｇｅｎｅｓ的肝癌细胞系ＨｅｐＧ２、ＢＥＬ－７４０２,体外能刺激正常人外周血Ｔ淋巴细胞的增殖,并能增强Ｔ淋巴细胞的杀瘤细胞活性,加入ＩＬ－１２有协同增强作用。结论 肝癌细胞导入含Ｂ７－１基因的Ａｄ－ｍｕｌｔｉｇｅｎｅｓ后,其免疫原性得到增强。     

New human adenovirus isolated from a renal transplant recipient: description and characterization of candiate adenovirus type 34.

An antigenically distinct adenovirus is described which was isolated in March 1972 from the urine of a 17-year-old Caucasian male who was experiencing fever after receiving a kidney transplant from a cadaver in February. The adenovirus could not be isolated in April from a pharyngeal swab which yielded cytomegalovirus. Complement-fixation, hemagglutination-inhibition, and/or serum-neutralization tests on sequential serum specimens from the patient confirmed that the adenovirus infection occurred during March and showed that infections with cytomegalovirus and respiratory syncytial virus also occurred during late March and April. The patient's persistent fever, for which other causes could not be found, may have been associated with one or more of these infections. Upper respiratory symptoms and lung involvement were not found during this period. Mild liver dysfunction during this time could not be clearly related to adenovirus infection because of the presence of multiple other causes. The adenovirus may have been latent in the donor kidney and become active in the new host as a consequence of immunological impairment. The adenovirus, purified by terminal dilution and plaque procedures, has antigenic, morphological, biophysical, host susceptibility, and hemagglutinating properties characteristic of adenovirus group IA. Buoyant densities in CsCl are 1.340 g/ml for the virion, 1.304 g/ml for the group CF antigen (hexon), 1.295 g/ml for the major soluble complete hemagglutinin (dodecon), and 1.206 g/ml for the minor soluble complete hemagglutinin (tentatively, fiber dimer). The virus does not cross-react in reciprocal hemagglutination-inhibition and serum-neutralization tests with antisera to adenovirus types 1 to 33. We propose this virus as candidate adenovirus type 34 (Compton).     

A novel cell surface molecule on cortical epithelial cells of the human thymus

A novel cell surface molecule, DN4, defined by an mAb raised against human thymic epithelial cells, showed a specificity for epithelial cells of the thymic cortex. This antigen was not expressed at detectable levels on any other types of tissues in the human body except for the thymus and bone marrow. Immunohistochemical analysis revealed that the reactivity of anti-DN4 mAb was restricted to the thymic cortex, and the antigen-expressing cells were arranged in a reticular network with long processes between thymocytes. The cellular nature of DN4-positive cells was identified as cortical epithelial cells, as DN4 was expressed in a subpopulation of freshly prepared thymic stromal cells which contain a large amount of keratin and expression of DN4 was strictly confined to the cortical area within the thymus on immunohistochemical analysis of frozen tissues. Immunofluorescence and flow cytometric analysis revealed that a subpopulation of bone marrow cells was also positive for DN4 (20%). The large blasts of normal bone marrow cells were clearly labeled with anti-DN4 mAb, in contrast to small-sized bone marrow cells. This finding suggests that DN4 seems to be transiently expressed in certain blastic stages during the differentiation of bone marrow cells.

Immunoprecipitation of ¹²⁵I-labeled cell lysates from THP-1 and U937 cell lines with anti-DN4 mAb yielded a single chain glycoprotein with an approximate size of 80–85 kd. There was a reduction in apparent molecular weight of approximately 40 kd in the immunoprecipitation of cell lysates after endoglycosidase F treatment. Thus, DN4 seemed to have a considerable amount of carbohydrate group.

DN4 appears to be a novel cortical epithelial cell antigen of the human thymus, and although the role of this molecule has not been well established experimentally, the possibility can be suggested that the DN4 molecule might be involved in the positive selection of thymocytes which occurs predominantly in the thymic cortical area.