POXVIRUS INFECTION AND APOPTOSIS

The following excellent reviews have been published on poxviruses and apoptosis during the last few years: P.C. Turner and R.W. Moyer, Semin. Virology, 8: 453–469, 1998; J.L. Shisler and B. Moss, Semin. Immunol., 13: 67–72, 2001; and H. Everett and G. McFadden, Curr. Opin. Microbiol., 5: 395–402, 2002. These articles dealt with the viral products and the mechanisms by which they interfere with apoptosis. In this review, we summarize new and old information and also introduce a new approach to explore interactions between the host cell and the replicating virus.     

A ROLE FOR APOPTOSIS INDUCED BY ACUTE HERPES SIMPLEX VIRUS INFECTION IN MICE

Acute herpes simplex virus (HSV) infection causes apoptosis in the adrenal cortex and myenteric plexus of the gut, ovary, pituitary gland, and liver of mice. Apoptosis of infected cells is increased in immunosuppressed regions of the adrenal cortex and liver of macrophage-depleted mice. HSV carries the US3 gene which interferes with host cell apoptosis. When the livers of macrophage-depleted mice are infected with a US3-null virus, apoptosis occurs in the narrow areas of inflammatory cell infiltration, restricting viral replication and spread. Thus, these data suggest that apoptosis may function as a primitive immune response to HSV infection in mice.     

CYTOKINES IN EXPERIMENTAL HERPES SIMPLEX VIRUS INFECTION

Herpes simplex virus (HSV) causes productive and latent forms of infection in humans and experimental animals. The primary infection and reactivation of the latent infection evoke an immune response in the host organism, involving activities of macrophages, CD4+ and CD8+ lymphocytes, and B lymphocytes. Strong cytokine responses are associated with the acute and recurrent phases of HSV infection. Also, during the latent phase of HSV infection in the senory ganglia, expression of certain cytokines can be detected. The cytokine response to HSV infection is dominated by proinflammatory and Th1 type cytokines; however, Th2 type cytokines such as interleukin-4 also are expressed in the infected tissue. The use of novel HSV-derived, cytokine-expressing gene therapy vectors necessitates studies on the possible modulation of the host responses by the virus-encoded cytokine transgenes. This review focuses on the roles of certain Th1 and Th2 type cytokines in different phases of the experimental HSV infections.     

VIRUS INFECTION AND APOPTOSIS (ISSUE I): AN INTRODUCTION

Apoptosis is a highly regulated programmed cell death process, which is activated during normal development and by various stimuli that disturb cellular metabolism and physiology. It is a fundamental cellular process that has become a major focus of basic biomedical research. Due to the cell's innate ability to self-destruct, apoptosis is also an important mechanism of host response to viral infection. While there is an enormous amount of literature on the molecular aspects of cellular apoptosis, the field of apoptosis during viral infection is still in its infancy. The goal of this series of review articles is to critically evaluate the existing knowledge of viral infection and apoptosis in important fields of virology. The next several years should be very fruitful in this nascent research area.     

PHYSIOLOGICAL SIGNIFICANCE OF APOPTOSIS DURING ANIMAL VIRUS INFECTION

Apoptosis has been considered to be a host defense mechanism against viral infection in multicellular organisms. This is based on the findings that apoptogenic mutants of insect viruses cannot grow because infected host cells die by apoptosis. This suggests that the apoptotic response of host cells has a deleterious effect on virus infection. Thus, apoptosis is an important host defense mechanism that is capable of inhibiting viral replication during infection. However, in vitro studies indicated that apoptosis alone does not provide the same protection against viral infection in animal cells as it does in the insect cells. Still, most animal viruses have acquired a strategy to overcome host cell apoptosis. In addition, a varying degree of necrosis usually accompanies apoptosis, suggesting a possible contribution of necrosis to the host reactions against virus. To understand the physiological significance of apoptosis during animal virus infection, we have characterized viral growth and the cellular responses against virus infection in a wide variety of virus-cell interaction systems. Mainly based on our own works, we discuss the nature of apoptosis in the animal virus infection and verify its role as a host defense mechanism against virus infection.     

INSECT DEFENSES AGAINST VIRUS INFECTION: THE ROLE OF APOPTOSIS

Insects, with their lack of an adaptive immune response, provide a unique animal model to examine the effects of apoptosis on viral infection. Several members of the baculovirus family of insect viruses have been shown to induce apoptosis during infection of cultured insect cells, and depending on the virus-host combination this apoptotic response can severely limit viral replication. In response to this evolutionary pressure, all baculoviruses studied to date carry antiapoptotic genes, including members of the p35 and IAP (inhibitor of apoptosis) gene families. Recent work has characterized the apoptotic response during infection of the host insect, and the results directly demonstrate the power of apoptosis as an antiviral response.     

62例胰腺癌组织中细胞凋亡的特征

目的研究拟阐明凋亡与胰腺癌分型、分期、分化程度、术后生存期的关系。方法62例胰腺癌标本取自1999年1月至2002年1月中国医科大学第二临床学院普外科及辽宁省人民医院普外科，术后病理证实。22例正常胰腺组织为上述医院的解剖标本。TUNEL法检测正常胰腺组织、胰腺癌组织内的凋亡细胞。结果在22例正常胰腺组织中细胞凋亡率为4／22，程度为（＋）；在62例胰腺癌组织中细胞凋亡阳性率为72．6％（45／62），程度为（＋）～（＋＋＋）。凋亡阳性率在胰腺癌组织分型、分期之间无统计学意义；高分化胰腺癌凋亡阳性率高于低分化胰腺癌（P〈0．05），中分化胰腺癌凋亡阳性率高于低分化胰腺癌（P〈0．01）；高、中分化胰腺癌之间差异无统计学意义；在中、高分化胰腺癌中，细胞凋亡程度（＋＋）～（＋＋＋）与（-）～（＋）的病例术后生存时间比较，前者显著长于后者（P〈0．05）。结论凋亡主要影响胰腺癌的发展、转移及预后；细胞凋亡在一定程度上阻止胰腺癌进展，最终延长胰腺癌患者生存时间。因此采用不同方法诱导胰腺癌组织中细胞凋亡可能为胰腺癌综合治疗提供新思路。     

A MOLECULAR METHOD FOR TYPING HERPES SIMPLEX VIRUS ISOLATES AS AN ALTERNATIVE TO IMMUNOFLUORESCENCE METHODS

Background: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. Study design: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase (pol) gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.     

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.     

胶质瘤细胞增殖与凋亡的研究

对未经其他治疗的手术摘除的胶质瘤标本,采用免疫组化和原位标记方法研究了胶质瘤及其瘤旁组织中细胞增殖以及凋亡的状况。结果表明,增殖细胞核抗原在世界卫生组织（ＷＨＯ）制定的不同级别的胶质瘤中其阳性率具有显著性差异（Ｐ＝ ０．０００５）,而在胶质瘤与其瘤旁组织之间则无显著性差异。恶性程度不同的胶质瘤中细胞发生凋亡的比率具有显著性的差异（Ｐ＝ ０．０１７０）,而在胶质瘤与其瘤旁组织间无显著性差异。增殖细胞核抗原是划分胶质瘤恶性程度的一个较好的指标,与Ｗ ＨＯ 胶质瘤恶性程度分级具有较好的相关性（ｒ＝ ０．４０８９）。Ｗ ＨＯ Ⅱ级星形胶质瘤细胞凋亡明显增多,可能是保留了恶性程度较高的胶质瘤细胞而清除了恶性程度相对较低的胶质瘤细胞,从而可能促进了胶质瘤的恶性进展。胶质瘤邻近的瘤旁组织可能已具有了胶质瘤的某些特性,胶质瘤呈浸润性生长可能与此有密切的关系。     

Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells

A major innate immune response to inhaled conidia of the opportunistic pathogen Aspergillus fumigatus (Af) is the synthesis of pro-inflammatory cytokines, which include tumour necrosis factor (TNF)-alpha, a known inducer of apoptosis. Modulation of host cell apoptosis has been reported to be one of the mechanisms whereby pathogens overcome host cell defences. Our study was designed to investigate whether or not Af conidia could modulate apoptosis induced by TNF-alpha or staurosporine (STS). Exposure of epithelial cells treated by these inducers and exposed to Af conidia decreased the number of apoptotic cells detected by Annexin V staining, analysis of nuclear morphology, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling reaction and immunoblotting. Inhibition of apoptosis by Af conidia was seen in cells of the A549 pneumocyte II line, human tracheal epithelial 16HBE and primary human respiratory cells. Inhibition of apoptosis by Af conidia was also observed when apoptosis was induced by co-cultivating A549 cells with activated human alveolar macrophages. Unlike Af conidia, conidia of Cladosporium cladosporioides as well as latex beads or killed Af conidia have no inhibitory effect on TNF-alpha or STS-induced apoptosis. For TNF-induced apoptosis, the observed anti-apoptotic effect of Af conidia was found to be associated with a significant reduction of caspase-3.     

Cell death in the developing vertebrate limb: A locally regulated mechanism contributing to musculoskeletal tissue morphogenesis and differentiation

Our aim is to critically review current knowledge of the function and regulation of cell death in the developing limb. We provide a detailed, but short, overview of the areas of cell death observed in the developing limb, establishing their function in morphogenesis and structural development of limb tissues. We will examine the functions of this process in the formation and growth of the limb primordia, formation of cartilaginous skeleton, formation of synovial joints, and establishment of muscle bellies, tendons, and entheses. We will analyze the plasticity of the cell death program by focusing on the developmental potential of progenitors prior to death. Considering the prolonged plasticity of progenitors to escape from the death process, we will discuss a new biological perspective that explains cell death: this process, rather than secondary to a specific genetic program, is a consequence of the tissue building strategy employed by the embryo based on the formation of scaffolds that disintegrate once their associated neighboring structures differentiate.     

T-CELL AND NEURONAL APOPTOSIS IN HIV INFECTION: IMPLICATIONS FOR THERAPEUTIC INTERVENTION

The pathogenesis of HIV infection involves the selective loss of CD4 + T cells contributing to immune deficiency. Although loss of T cells leading to immune dysfunction in HIV infection is mediated in part by viral infection, there is a much larger effect on noninfected T cells undergoing apoptosis in response to activation stimuli. In the subset of patients with HIV dementia complex, neuronal injury, loss, and apoptosis are observed. Viral proteins, gp120 and Tat, exhibit proapoptotic activities when applied to T cell and neuronal cultures by direct and indirect mechanisms. The pathways leading to cell death involve the activation of one or more death receptor pathways (i.e., TNF-α, Fas, and TRAIL receptors), chemokine receptor signaling, cytokine dysregulation, caspase activation, calcium mobilization, and loss of mitochondrial membrane potential. In this review, the mechanisms involved in T-cell and neuronal apoptosis, as well as antiapoptotic pathways potentially amenable to therapeutic application, are discussed.     

ALPHAVIRUSES AND APOPTOSIS

Many reports have indicated that infection with SV or SFV induces apoptosis both in cultured cells and in the CNS of mice. In general, the ability of virus strains to induce apoptosis correlates with their neurovirulence, although both apoptosis and neurovirulence are age dependent, i.e., resistance increases with age. SV can induce apoptosis simply by the process of membrane fusion and entry, by the expression of the envelope proteins, or by the expression of the nonstructural protein, nsP2. However, viral particles are not necessary to activate apoptosis, since transfection with viral RNA or even viral RNA expressing only the nonstructural proteins will result in apoptosis.The cellular pathways involved in alphavirus-induced apoptosis are complex, and much remains poorly understood. Experimental results point to the involvement of both the mitochondrial and the death receptor pathways. To date, there are no reports implicating the ER stress pathway.     

APOPTOSIS IN AUTOIMMUNE AND NON-AUTOIMMUNE THYROID DISEASE

The elimination of autoreactive T cells in the thymus involves the process of programmed cell death. Animal model studies, using the lpr and gld strains of mice, have identified FAS receptor (FAS) and FAS ligand (FAS-L) as important components of this mechanism. Whether FAS and FAS-L are also implicated in the autoimmune destruction of a target organ, such as the thyroid, remain hypothetical. An accompanying paper in this issue has addressed the question by FACS and immunocytochemical analysis of FAS expression and apoptosis in thyrocytes grown in culture and in intact thyroid tissues obtained from Hashimoto's thyroiditis, multinodular goitre and Graves' disease. The overall results suggest that the degree of FAS expression on target cells may determine their sensitivity to T-cell mediated cytotoxicity in the absence of perforin or granzyme directed apoptosis mechanisms. © 1997 John Wiley & Sons, Ltd.     

APOPTOSIS OF CRYPT EPITHELIAL CELLS IN ULCERATIVE COLITIS

In the colon of ulcerative colitis (UC) patients, apoptotic bodies have been recognized in routine histopathological preparations. To investigate the extent of the apoptosis, colonic biopsies were examined from involved and uninvolved areas of untreated active UC and from normal areas in patients with colonic polyps, utilizing various markers of apoptosis. The markers included DNA breaks detected by TUNEL, Fas (CD95/APO-1) and Fas ligand (Fas-L) localized by immunohistochemistry, electron microscopic features of apoptosis, and laddering of extracted DNA. Apoptosis marker positive cells were found mainly on the luminal epithelium of the normal colon and were present in active UC in crypts of involved and uninvolved areas of the colon, in addition to the luminal epithelium. The DNA extracted from active UC colon electrophoresed as a ladder. These findings suggest that the loss of epithelial cells in active UC occurs mainly by apoptosis in crypts of involved and adjacent uninvolved areas and that the Fas/Fas-L interaction is a mediator of the apoptosis.     

遗传性视网膜变性rd小鼠及其感光细胞凋亡研究

目的 研究遗传性视网膜变性rd小鼠感光细胞层的发育变化及细胞凋亡。方法 对出生后5d到40d的rd小鼠及对照小鼠视网膜感光细胞层进行光镜及超微结构观察、TUNEL法检测及形态计量学分析。结果 与同龄对照鼠相比,rd小鼠出生后第10d视网膜开始变性,尔后1周内感光细胞迅速减少,第18d时只残留一层视椎细胞。rd小鼠出生后第10d感光细胞层开始出现TUNEL染色阳性细胞,第14d及16d达到高峰。电镜下变性高峰期rd小鼠视网膜感光细胞层可见大量浓缩核、染色质边聚及凋亡小体。结论 rd小鼠视网膜感光细胞在发育过程中变性,并通过凋亡的方式死亡。     

IN THE ABSENCE OF EPSTEIN-BARR VIRUS INFECTION, PHORBOL ESTER MODULATES APOPTOSIS IN CYCLOHEXIMIDE-TREATED BURKITT'S LYMPHOMA (BJA-B) CELLS

We have shown previously that cycloheximide (CHX), a potent protein synthesis inhibitor, induces high levels of apoptosis in Epstein-Barr virus free (EBV(−)) Burkitt's lymphoma (BJA-B) cells, with comparably reduced levels of apoptosis in the EBV positive (EBV(+)) cells. Modulation of CHX-induced apoptosis in EBV(−) and (+) B cells is reported here using concurrent treatment with phorbol ester (phorbol 12,13-dibutyrate, PdBu). Cells were collected at 0, 3, 6, 12, 24 and 48 hours after treatment with (i) 1 μg/ml CHX, (ii) 0.1 μg/ml PdBu (1 hour pretreatment before 0 h), or (iii) CHX + PdBu (CHX added at 0 h, 1 hour after PdBu). Control cultures were untreated. Apoptotic, necrotic or viable cells were quantified using histological, ultrastructural and biochemical parameters. Protein synthesis was assessed using ³⁵S-methionine incorporation. Intracellular calcium concentrations were measured using flow cytometry. PdBu alone had little effect on cell death. High levels of CHX-induced apoptosis in EBV(−) cells were significantly reduced by concurrent addition of PdBu ( P  < 0.005). In contrast, low levels of CHX-induced apoptosis in EBV(+) cells were not significantly altered by PdBu treatment. In EBV(−) cells, a negative relationship was observed between levels of apoptosis and calcium concentrations, whereas in EBV(+) cells, there was negligible correlation between these parameters. Thus high levels of CHX-induced apoptosis in EBV(−) cells occur via a PKC-dependent pathway, whereas CHX treatment of EBV(+) cells induces comparatively low levels of apoptosis that occur via a PKC-independent mechanism. The results application in the therapeutic intervention for cancers developing in association with EBV infection.     

用单纯疱疹病毒I型跨神经元输送对大鼠小脑纤维联系的研究

实验选用ＳＤ大鼠２３只，分别在小脑前、后叶定位定量注射单纯疱疹病毒Ｉ型存涤２－５天或１０天后灌杀取脑切片，经抗ＨＳＶ１血清行组织化学ＡＢＣ法反应。结果显示，免疫阳性神经元呈类似Ｇｏｌｇｉ样染色，胞体和树突标记清晰。注射部位不同或术后动物存活时间不同，其标细胞出现的数量和部位同。１．后叶注射后１天可出现小脑Ｐｕｒｋｉｎｊｅ细胞及缝核群，网状结构，室周灰质，丘脑腹外侧核发现标记细胞。２．前叶注射后３天