Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization.

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103.

Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.

Specific inhibition of coxsackievirus B3 translation and replication by phosphorothioate antisense oligodeoxynucleotides.

The 5' and 3' untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA form highly ordered secondary structures that have been confirmed to play important regulatory roles in viral cap-independent internal translation initiation and RNA replication. We previously demonstrated that deletions in different regions of the 5' UTR significantly reduced viral RNA translation and infectivity. Such observations suggested strongly that viral RNA translation and replication could be blocked if highly specific antisense oligodeoxynucleotides (AS-ODNs) were applied to target crucial sites within the 5' and 3' UTRs. In this study, seven phosphorothioate AS-ODNs were synthesized, and the antiviral activity was evaluated by Lipofectin transfection of HeLa cells with AS-ODNs followed by infection of CVB3. Analysis by Western blotting, reverse transcription-PCR, and viral plaque assay demonstrated that viral protein synthesis, genome replication, and infectivity of CVB3 were strongly inhibited by the AS-ODNs complementary to different regions of the 5' and 3' UTRs. The most effective sites are located at the proximate terminus of the 5' UTR (AS-1), the proximate terminus of the 3' UTR (AS-7), the core sequence of the internal ribosome entry site (AS-2), and the translation initiation codon region (AS-4). These AS-ODNs showed highly sequence-specific and dose-dependent inhibitory effects on both viral protein synthesis and RNA replication. It is noteworthy that the highest inhibitory activities were obtained with AS-1 and AS-7 targeting the termini of the 5' and 3' UTRs. The percent inhibition values of AS-1 and AS-7 for CVB3 protein VP1 synthesis and RNA replication were 70.6 and 79.6 for AS-1 and 73.7 and 79.7 for AS-7, respectively. These data suggest that CVB3 infectivity can be inhibited effectively by AS-ODNs.

Apoptosis associated with esophageal adenocarcinoma: influence of photodynamic therapy.

Tumor cell death in vitro by photodynamic therapy (PDT) has been related to the induction of apoptosis. We measured and compared changes in apoptosis and caspase 3 activity, an effector of apoptosis, in normal and neoplastic esophageal tissues during PDT. Apoptosis index, caspase 3 cleavage activity, pro-caspase 3, p53, and bcl-2 levels were measured in normal and neoplastic tissues of patients with esophageal adenocarcinoma before, during, and after PDT with Photofrin. The apoptotic index was greater in carcinoma tissue compared to adjacent normal tissues. In concert, pro-caspase 3 immunoreactivity was absent and caspase 3-like cleavage activity was over 30-fold greater in carcinoma tissue compared to normal esophageal tissues. These parameters were unaffected by PDT. Variable changes in bcl-2 and p53 immunoreactivity were noted in normal and carcinoma tissues during PDT. Greater levels of apoptosis and caspase 3 activity are hallmarks of esophageal adenocarcinoma compared to normal esophageal tissue. These differences were unaffected by PDT. This may be due to the fact that tissues were obtained 72 h post-PDT therapy. Changes in these parameters may have occurred early after PDT therapy. An assessment of apoptosis and caspase 3 activity prior to 72 h post-PDT may provide further insight into the mechanism involved, although no sustained effects on these parameters by PDT were noted.

Endotoxin infusion in rats induces apoptotic and survival pathways in hearts.

Inflammatory mediators of sepsis induce apoptosis in many cell lines. We tested the hypothesis that lipopolysaccharide (LPS) injection in vivo results in induction of early apoptotic and survival pathways as well as evidence of late-stage apoptosis in the heart. Hearts were collected from control rats and at 6, 12, and 24 h after LPS injection (4 mg/kg). Activation of an apoptotic pathway was identified by a 1,000-fold increase in caspase-3 activity at 24 h (P < 0.05). Confirmation of these results occurred when terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining identified myocardial cells undergoing DNA fragmentation with significant levels at 24 h post-LPS injection. LPS also caused early proapoptotic mRNA (Bax) to increase (16% at 24 h, P < 0.05), whereas the Bax protein initially decreased (35% at 6 h, P < 0.05) and then returned to baseline values by 24 h. Six hours after LPS injection, Bcl-2 (early prosurvival) mRNA levels increased, whereas its protein levels decreased (70%, P < 0.05) and then returned to baseline levels by 24 h. Mitochondrial cytochrome c levels decreased, suggestive of mitochondrial involvement. Thus involvement of proapoptotic and prosurvival pathways in the heart occurs during a septic inflammatory response.

Host gene regulation during coxsackievirus B3 infection in mice: assessment by microarrays.

Host genetic responses that characterize enteroviral myocarditis have not yet been determined. The injurious and inflammatory process in heart muscle may reflect host responses of benefit to the virus and ultimately result in congestive heart failure and dilated cardiomyopathy. On the other hand, host responses within the myocardium may secure the host against acute or protracted damage. To investigate the nature of modified gene expression in comparison with normal tissue, mRNA species were assessed in myocardium using cDNA microarray technology at days 3, 9, and 30 after infection. Of 7000 clones initially screened, 169 known genes had a level of expression significantly different at 1 or more postinfection time points as compared with baseline. The known regulated genes were sorted according to their functional groups and normalized expression patterns and, subsequently, interpreted in the context of viremic, inflammatory, and healing phases of the myocarditic process.

Nuclear factor-kappaB activation by the photochemotherapeutic agent verteporfin.

The nuclear factor-kappa B (NF-kappaB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-kappaB principally resides within the cytoplasm in association with inhibitory kappa (IkappaB) proteins. The status of IkappaB and NF-kappaB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IkappaB proteins may be caspase substrates during apoptosis. However, the level of IkappaBbeta was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IkappaBalpha levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IkappaBalpha levels were transiently depressed after PDT. At these times, p50 and RelA NF-kappaB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a kappaB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-alpha, a well-characterized NF-kappaB activator. Productive NF-kappaB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IkappaBalpha is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

Structural and functional analysis of the 5' untranslated region of coxsackievirus B3 RNA: In vivo translational and infectivity studies of full-length mutants.

The lengthy 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure, which plays an important role in controlling viral transcription and translation. Our previous work has delineated the internal ribosome entry site (IRES) by mutation of mono- and bicistronic plasmids containing the 5'UTR and subsequent cell- free translation in rabbit reticular lysate (D. Yang, J. E. Wilson, D. R. Anderson, L. Bohunek, C. Cordeiro, R. Kandolf, and B. M. McManus. (1997). Virology 228, 63-73). To further identify the sequence elements responsible for viral translation and infectivity in tissue culture cells, >30 full-length mutants of CVB3 were constructed by mutations of the IRES and its flanking regions. Viral RNAs were transcribed from these constructs and transfected into HeLa cells. When the stem-loops G and H in the putative IRES were deleted, viral infectivity was abolished and viral protein translation was also undetectable by immunoblot analysis. However, when stem-loops A and B were deleted or stem-loop E was partially deleted, viral protein translation could be detected although cytopathic effect could not be observed. The data suggest that the crucial sequence of the IRES is located at stem-loops G and H. Further serial deletion mapping up and down stream of the crucial sequence defined more accurately the 5' and 3' boundaries of the IRES, located at nucleotides (nts) 309-432 and 639-670, respectively. These results indicate that the core sequence of the IRES should be located at nts 432-639. This IRES segment is much shorter and located closer to the initiation codon than that of poliovirus. To further define critical nucleotides within the IRES core, site-directed mutagenesis was conducted at the IRES core sequence by PCR. A 46-nt deletion in the pyrimidine-rich tract of stem-loop G abolished viral translation and infectivity. Interestingly, five single-nt substitutions in the pyrimidine-rich tract aimed at destabilizing the base pairing between the viral IRES and host 18S rRNA did not abolish CVB3 infectivity although viral protein translation was significantly reduced. This finding suggests that ribosomal internal initiation of translation and viral infectivity not only may require RNA secondary structure but also may need tertiary structure and perhaps the assistance of host protein factors.

Release of cytochrome c, Bax migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis.

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.

Early release of mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 with a porphyrin-derived photosensitizer: Bcl-2 and Bcl-xL overexpression do not prevent early mitochondrial events but still depress caspase activity.

Certain nonmetallic porphyrins have potent antitumor activity upon visible light irradiation. Treatment of HeLa cells with nanomolar amounts of the photochemo therapeutic agent verteporfin and red light mobilized caspases 2, 3, 6, 7, 8, and 9, caused degradation of specific caspase substrates, and resulted in morphologic changes consistent with apoptosis. Caspase processing was detectable by 1 hour after light irradiation. The mitochondrial 7A6 epitope, recognized by monoclonal antibody APO2.7, became accessible, and cytochrome c was detectable within the cytosolic fraction of cells treated with verteporfin immediately after light irradiation. The general caspase inhibitor benzyloxycarboyl-Val-Ala-Asp-fluoromethylketone did not prevent 7A6 expression produced by photosensitization at peptide concentrations which completely prevented caspase activation and cleavage of caspase-specific substrates. Enforced overexpression of Bcl-2 or Bcl-xL prevented cytochrome c release and 7A6 expression produced by ultraviolet B light treatment, but did not prevent cytochrome c release or 7A6 expression elicited by verteporfin photosensitization. Bcl-2 or Bcl-xL overexpression delayed morphologic changes, depressed caspase activation, and limited substrate degradation, but did not protect against loss of viability after verteporfin photosensitization. This observation indicates that cells overexpressing Bcl-2 or Bcl-xL exhibit resistance to caspase activation even after the appearance of cytochrome c in the cytosol. Porphyrin photosensitizers are effective chemotherapeutic agents that elicit primary proapoptotic mitochondrial events even in the setting of heightened Bcl-2 or Bcl-xL expression.

Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication.

Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks.

Viral myocarditis: identification of five differentially expressed genes in coxsackievirus B3-infected mouse heart.

Differences in host susceptibility to viral myocarditis caused by a given strain of coxsackievirus B3 (CVB3) are known to be largely related to host genetic factors. Little is known, however, about the key genes that encode determinants (mediators) of myocarditis development or the nature of injury. To identify these genes and further understand the molecular mechanisms of the disease process, we have used a murine model and the differential display technique to fingerprint mRNAs from CVB3-infected mouse hearts. Total RNA was extracted from hearts of 4- and 10-week-old A/J(H-2(a)) mice at day 4 after CVB3 infection, and mRNAs were detected by reverse transcriptase-polymerase chain reaction and subsequently analyzed on polyacrylamide DNA sequencing gels. The differentially displayed bands were confirmed by Northern hybridization using the bands as cDNA probes. Twenty-eight upregulated or downregulated bands were selected from the sequencing gels; among these, 2 upregulated and 3 downregulated cDNA fragments were confirmed by Northern hybridization. DNA sequence analysis and GenBank searching have determined that 4 of the 5 candidate genes are homologous to genes encoding Mus musculus inducible GTPase, mouse mitochondrial hydrophobic peptide (a subunit of NADH dehydrogenase), mouse beta-globin, and Homo sapiens cAMP-regulated response element binding protein (CREB) binding protein (CBP), respectively. The remaining candidate gene matches an unpublished cDNA clone, M musculus Nip21 mRNA (GenBank accession number, AF035207), which is homologous to human Nip2, a Bcl-2 binding protein. Our data suggest preliminarily that both structural and nonstructural genes are involved in myocarditis development. For the structural gene, beta-globin, we further confirmed its downregulation at the protein level by measuring the mean cell volume of red blood cells and found it was marginally reduced in the CVB3-infected group (P<0.06), with no change in hemoglobin concentration. Cardiac myoglobin concentration was also measured and found to be decreased (P<0.005), with a parallel decrease in total soluble protein in the CVB3-infected mouse myocardium (P<0.01). We also noted that the ratio of myoglobin to total protein was not significantly changed; this may be due to the downregulation of additional genes in the host heart, a number being observed on the differential display gels. The significant downregulation of beta-globin major gene expression in the heart may be relevant to impaired cardiac function in both the early and late postinfection period. The other identified nonstructural genes are known to be involved in regulation of gene expression, signal transduction pathways, and apoptotic cell death. The altered expression of structural and nonstructural genes may play important roles in the mediation of myocarditis development and perhaps other pathological processes in the heart.

Increased severity of viral myocarditis in mice lacking lymphocyte maturation.

The role of lymphocytes in the pathogenesis of viral myocarditis is controversial. To better understand how lymphocyte maturation controls a virus-induced myocarditic process, a murine model of viral myocarditis was utilized. Encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene (RAG)-2 knockout 129/SV mice. The RAG2 participate in the maturity of T and B lymphocytes. Survival rate, heart weight (HW), HW to body weight (BW) ratio, viral genome, cardiac inflammation and myocardial necrosis were evaluated after EMCV (500 plaque forming unit/mouse) inoculation. On post-inoculation day 10, the survival rate of C57BL/6, 129/SV and RAG2 knockout mice were 42, 90 and 0%, respectively. Myocardial viral titer was significantly (P<0.05) higher in C57BL/6 and RAG2 knockout mice than in 129/SV mice. In situ hybridization demonstrated the EMCV genome in the myocardium of RAG2 knockout and C57BL/6 mice, but not in 129/SV mice. At day 8, HW and HW/BW ratios were elevated (P<0.05) in RAG2 knockout mice as well as C57BL/6 mice compared with 129/SV mice. Myocardial necroses were more severe in RAG2 knockout mice than in wild-type 129/SV mice. In conclusion, matured lymphocytes protect the development of viral myocarditis which includes viral replication and myocardial apoptosis.

Attenuated acute cardiac rejection in NOS2 -/- recipients correlates with reduced apoptosis.

BACKGROUND: The mechanisms through which NOS2-mediated pathways regulate graft failure in acute cardiac rejection are ill defined. To determine whether apoptosis promoted by NOS2 may contribute, we used a heterotopic transplant model to study mouse cardiac allografts placed in recipients with targeted gene deletion of NOS2. METHODS AND RESULTS: Using 5 different indexes of apoptosis, we showed that mouse cardiac allografts placed in NOS2 -/- recipients (n=7) had reduced apoptotic activity compared with those in NOS2 +/+ controls (n=8). There were significantly fewer TUNEL-positive nuclei per high-powered field (P<0.01), less DNA fragmentation (antinucleosome ELISA; P<0.05), lower corrected transcript levels for caspase-1 and -3 (32P reverse transcriptase-polymerase chain reaction; P<0.01), and reduced caspase-3 activity (cleavage of DEVD-pNA [P<0.001] and poly [ADP-ribose] polymerase) in grafts from NOS2 -/- recipients. This concordant reduction in apoptotic indexes paralleled the improved histological outcome of grafts transplanted into NOS2 -/- recipients (assessed as rejection scores; P=0.012). To identify pathways controlled by NOS2, we compared intragraft transcript levels of potential triggers and regulators. Whereas Fas ligand/Fas and tumor necrosis factor (TNF)-alpha/TNF receptor-1 levels were not altered by NOS2 deficiency, transcript levels for p53 were significantly lower in grafts from NOS2 -/- recipients, coinciding with a significant increase in the antiapoptotic Bcl-2/Bax balance and decrease in Bcl-Xl levels. CONCLUSIONS: Using NOS2 knockout mice, we demonstrated that NOS2-mediated pathways can promote acute rejection, at least in part, by inducing apoptotic cell death. When NOS2 is present, p53 might control NOS2-mediated apoptosis by stimulating Bax and repressing Bcl-2 and Bcl-Xl expression, which may activate the cell death program in the rejecting heart.

Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy.

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.

Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells.

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.

Interaction of viral proteins with host cell death machinery.

In recent years, intense research has been directed towards understanding molecular mechanisms involved in viral pathogenesis. It is now known that many viruses manipulate host defense mechanisms to prevent apoptosis in order to maximize viral replication. Towards the end of their replication cycle, certain viruses direct the synthesis of proteins that induce apoptosis or cell lysis thereby facilitating viral release from the cell. The present review summarizes the current understanding of interactions between viral proteins and the host cell death machinery.

Myocarditis as systemic disease: new perspectives on pathogenesis.

1. Myocarditis may be an early indicator of or may subsequently lead to dilated cardiomyopathy in humans. This hypothesis has evolved from research on viruses that induce myocarditis, wherein the coxsackie B group viruses (CVB) in the family Picornaviridae are the most common known viral infectants of heart muscle. 2. Many competing hypotheses exist as to the pathogenesis of CVB3-induced myocarditis, including direct virus-induced myocyte damage and immunopathological disease with autoimmune sequelae. Evidence to support the direct-damage and viral RNA-persistence hypothesis is derived from in situ hybridization and gene amplification studies. 3. Recent use of terminal deoxynucleotidyl transferase-mediated nick-end labelling indicates that this injury in target organs is largely non-apoptotic in nature. Most apoptotic bodies in cardiac tissue are derived from immune cells. 4. Beyond infection of heart muscle, CVB3 can also associate with, infect and persist in cells of immune origin. The CVB3 localizes to follicles in spleens and lymph nodes of the murine host and this particular localization may continue in mice susceptible to more aggressive myocarditis. Whether virus-immune cell association in these compartments is advantageous (or essential) to the host in the evolution of anti-viral immune responses or whether it is more advantageous to the virus in immunosuppression of the host is not known. 5. We suggest that CVB3 can directly perturb or alter the immune response, thereby delaying viral clearance from vulnerable systemic organs. Both host and viral genetic factors can influence susceptibility, persistence and disease progression. 6. Picornaviruses use a unique method for the initiation of translation, involving the internal binding of the ribosome on a sequence element of the 5' untranslated region, termed an internal ribosome entry site (IRES). 7. The IRES of CVB3 is located at approximately stem loops G, H and I, spanning nucloetides 530 and 630. Arrest of host translation is also a feature of picornavirus infection. Such regulation of host cell translation machinery no doubt fosters viral replication at the expense of the host cell. 8. Differences between cell types in the mechanisms, along with those at other key steps in the viral life cycle and in signalling via kinase pathways, may determine viral tropism and cellular destruction and the physiological outcome of neighbouring cells.

Complement component 3 interactions with coxsackievirus B3 capsid proteins: innate immunity and the rapid formation of splenic antiviral germinal centers.

Innate immunity is central to the clearance of pathogens from hosts as well as to the definition of acquired immune responses (D. T. Fearon, and R. M. Locksley, Science 272:50-53, 1996). Coxsackievirus B3 (CVB3), a human cardiopathic virus, was evaluated for the ability to activate the alternative and classical pathway of complement. CVB3 proteins interact with complement component 3 (C3, a soluble protein effector of innate immunity) after either in vitro exposure to mouse serum or in vivo murine infection and activate the alternative pathway of complement. In addition, we demonstrate that viral antigen retention and localization in germinal centers is dependent on C3, while virus antigen retention in extrafollicular regions in the spleen is not. In vivo depletion of native C3 abolished the rapid formation of virus-specific germinal centers (by day 3 post-CVB3 infection) in the absence of serum anti-CVB3 antibodies. These studies demonstrate that innate immune mechanisms, such as C3 interaction with CVB3, are essential for splenic antiviral germinal center formation in naive (antigen nonsensitized) mice resistant (C57BL/6J strain) and susceptible (A/J strain) to CVB3-induced myocarditis.