Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide

The onset of vascular leakage and hemorrhagic diathesis is one of the life-threatening complications occurring in dengue patients, yet the pathogenic mechanisms are not well understood. In this study, we demonstrated that Abs against dengue virus nonstructural protein 1 (NS1) generated in mice cross-reacted with human endothelial cells and mouse vessel endothelium. After binding, mouse anti-NS1 Abs induced endothelial cell apoptosis in a caspase-dependent manner. Inducible NO synthase expression could be observed; it showed a time- and dose-dependent correlation with NO production. Endothelial cell apoptosis, characterized by exposure of phosphatidylserine on the cell surface and nuclear DNA fragmentation, was blocked by treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Further studies demonstrated that the expression of Bcl-2 and Bcl-x(L) decreased in both mRNA and protein levels, whereas p53 and Bax increased after anti-NS1 treatment. Cytochrome c release was also observed. All of these effects could be inhibited by N(omega)-nitro-L-arginine methyl ester. Taken together, anti-NS1 Abs act as autoantibodies that cross-react with noninfected endothelial cells and trigger the intracellular signaling leading to the production of NO and to apoptosis. Endothelial cell damage may cause vascular leakage that contributes to the pathogenesis of dengue disease.     

Hydrogen peroxide activation of ERK5 confers resistance to Jurkat cells against apoptosis induced by the extrinsic pathway

Reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂) exhibit both pro-survival and pro-death signaling in leukemic cells. We examined the effect of exogenous H₂O₂ on Fas ligand (FasL) -induced apoptosis in Jurkat cells. H₂O₂ applied prior to (pre-conditioning) and during (post-conditioning) FasL stimulation attenuated early apoptosis through activation of EKR5. H₂O₂ increased the activated caspase-8 sequestered in the mitochondria thereby decreasing cell death through the extrinsic apoptotic pathway. In addition, inhibition of a protein tyrosine phosphatase likely explains the post-conditioning requirement for H₂O₂. Given that chemotherapeutic agents used for the treatment of acute lymphoblastic leukemia are thought to work partly through production of ROS, a simultaneous inhibition of the ERK5 pathway may abrogate the ROS-initiated pro-survival signaling for an enhanced cell kill.     

Effects of OPA1 mutations on mitochondrial morphology and apoptosis: relevance to ADOA pathogenesis

To characterize the molecular links between type-1 autosomal dominant optic atrophy (ADOA) and OPA1 dysfunctions, the effects of pathogenic alleles of this dynamin on mitochondrial morphology and apoptosis were analyzed, either in fibroblasts from affected individuals, or in HeLa cells transfected with similar mutants. The alleles were missense substitutions in the GTPase domain (OPA1(G300E) and OPA1(R290Q)) or deletion of the GTPase effector domain (OPA1(Delta58)). Fragmentation of mitochondria and apoptosis increased in OPA1(R290Q) fibroblasts and in OPA1(G300E) transfected HeLa cells. OPA1(Delta58) did not influence mitochondrial morphology, but increased the sensitivity to staurosporine of fibroblasts. In these cells, the amount of OPA1 protein was half of that in control fibroblasts. We conclude that GTPase mutants exert a dominant negative effect by competing with wild-type alleles to integrate into fusion-competent complexes, whereas C-terminal truncated alleles act by haplo-insufficiency. We present a model where antagonistic fusion and fission forces maintain the mitochondrial network, within morphological limits that are compatible with cellular functions. In the retinal ganglion cells (RGCs) of patients suffering from type-1 ADOA, OPA1-driven fusion cannot adequately oppose fission, thereby rendering them more sensitive to apoptotic stimuli and eventually leading to optic nerve degeneration.     

Direct demonstration of the human suppressor inducer subset by anti-T cell antibodies

Prior studies indicated that sera of patients with active juvenile rheumatoid arthritis (JRA) contain anti-T cell antibodies reactive with the T4+ inducer population. More important, depletion of this T cell subset with JRA anti-T cell antibodies (JRA+ T cells) and C abrogated T5/T8+ suppressor T cell function. In the present study, we utilized Ig-coated plate techniques and JRA anti-T cell antibodies to fractionate the T4+ population into T4+JRA+ and T4+JRA- subsets and characterize the individual T4+ inducer subset. It was shown that whereas only the T4+JRA- population responded maximally to the soluble antigens, TT and mumps, both T4+JRA+ and T4+JRA- subsets proliferated equally well to mitogens and alloantigens. Furthermore, B cell immunoglobulin production induced by T4+JRA- T cells was approximately twice that induced by the reciprocal T4+JRA+ subset. In contrast, the T4+JRA+ subset alone activated T8+ T cells to become suppressor effector cells. These results suggest that the T4+JRA+ subset is the inducer of suppressor subpopulation whereas the T4+JRA- subset functions maximally as the inducer of B cells. It is believed that the suppressor inducer population may have a central role in the immunoregulatory network in man.     

Simultaneous activation of apoptosis and inflammation in pathogenesis of septic shock: a hypothesis

Sepsis, a widely prevalent disease with increasing morbidity and mortality, is thought to result from uncontrolled inflammatory responses to microbial infection and/or components. However, failure of several experimental anti-inflammatory therapies has necessitated re-evaluation of the paradigm underlying the pathogenesis of this complex disorder. Apoptotic cell death forms a second dominant feature of septic shock in patients and animal models. Anti-apoptotic strategies may protect animals from septic death. However, simultaneous occurrence of apoptosis and inflammation is necessary for septic death. At the cellular level, apoptosis plays a central role in the development of the lymphoid system and regulation of immune responses. Immune activation renders cells refractory to apoptosis while apoptosis of activated lymphocytes is an important immunoregulatory mechanism. Factors such as complement factor 5a, caspase-1 and mitogen-activated protein kinase, which participate in apoptosis as well as pro-inflammatory pathways, may be responsible for simultaneous activation of apoptosis and inflammation in sepsis. Further identification of other similar biochemical events capable of co-activating inflammation and apoptosis may provide new targets for therapy of this hitherto untreatable disease.     

Characteristics of MNNG induced repair synthesis and DNA synthesis induced by human cytomegalovirus

DNA synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse embryo and human embryo cells was compared with DNA synthesis induced in these cells by human cytomegalovirus. In virus infected human embryo cells grown in the medium depleted of arginine DNA synthesis showed resistance to hydroxyurea and arabinofuranosylcytosine, similarly as repair synthesis induced by MNNG. DNA synthesis induced by the virus in mouse embryo cells was partially sensitive to both inhibitors.     

Cutaneous changes induced by a dose of DEC in rodents with skin microfilaria: relevance of these phenomena for understanding the Mazzotti reaction and the pathogenesis of human onchocerciasis

A single dose of DEC was given to Lemniscomys striatus parasitized by Monanema martini. The histopathological analysis of ear-lobe skin sections shows: a massive escape of microfilariae from the lymph capillaries, a major vascular exudative reaction with edema and diapedesis of blood cells. This inflammatory process leads to the destruction of the extralymphatic microfilariae and it induces a subacute and chronic dermatitis at the 7th day. These findings explain the pathogenesis of Mazzotti reaction during treatment of human onchocerciasis. The use of DEC produces precisely the factor that is responsible for the severity of the human disease: inflammatory reactions around the extralymphatic microfilariae.     

Antioxidants protect against increased risk of atherosclerosis induced by exposure to cigarette smoke: Histological and biochemical study

Background and objectivesThis study aimed to assess the dose-dependent effect of antioxidants in protection against cardiovascular changes induced by exposure to cigarette smoke.Design and settingThis was an experimental study, conducted at King Fahd Medical Research Center, King Abdulaziz University.Materials and methodsThis study was carried out on 57 male albino rats divided into nine groups. Rats of experimental groups were exposed to cigarette smoke from a total of 100 cigarettes per week for four weeks in a specially designed chamber. The antioxidants used (vitamin C, E, and B-carotene) were administrated at low (9, 7.2, and 0.27 mg/day) and high doses (18, 14.4, and 0.54 mg/day), respectively, through gastric feeding tubes. The lipid profile was estimated, and the carotids and heart were removed, weighed, and then processed, and the carotid intima-media thickness was measured. Statistical analysis was performed using the Statistical Package for Social Sciences.ResultsThe lipid profile was significantly improved in all groups treated with low or high doses of antioxidants after or during the exposure to cigarette smoke. Improvement was marked in the group treated with a high dose of antioxidants.The histological changes, as well as the intima-medial thickness of the carotid artery induced by exposure to cigarette smoke, have been improved by treatment with antioxidants (at either low or high doses), either after or during exposure to cigarette smoke. Improvement was marked in the group treated with a low dose of antioxidant. Treatment with antioxidants could not improve degenerated cardiac muscle fibers, while they could reduce the thickness of the branches of the coronary vessels.ConclusionThese results indicated that antioxidants ameliorated the cigarette smoke contribution to atherosclerosis, but they could not completely reverse the changes induced by cigarette smoke. Simultaneous intake of antioxidants could ameliorate the cigarette-smoke-induced changes apart from those of the heart.     

Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation

The inhibitory effects of Chinonin, a natural antioxidant extracted from a Chinese medicine, on apoptotic and necrotic cell death of cardiomyocytes in hypoxia-reoxygenation process were observed in this study. The possible mechanisms of Chinonin on scavenging reactive oxygen species and regulating apoptotic related genes bcl-2 and p53 were also investigated. Neonatal rat cardiomyocytes were subjected to 24-h hypoxia and 4-h reoxygenation. Cell death was evaluated by DNA electrophoresis on agarose gel, cell death ELISA and annexin-V-FLUOS/propidium iodide (PI) double staining cytometry. Hypoxia caused the increase of apoptotic rates and the release of lactate dehydrogenase (LDH), while reoxygenation not only further increased the apoptotic rates and leakage of LDH, but also induced necrosis of cardiomyocytes. In addition, hypoxia increased the levels of NO(2)(-)/NO(3)(-) and thiobarbituric acid reacted substances (TBARS), while reoxygenation decreased NO(2)(-)/NO(3)(-), but further increased TBARS in the cultured media. Moreover, hypoxia up-regulated the expression levels of bcl-2 and p53 proteins, while reoxygenation down-regulated bcl-2 and further up-regulated p53. Chinonin significantly decreased the rates of apoptotic and necrotic cardiomyocytes, and inhibited the leakage of LDH. It also diminished NO(2)(-)/NO(3)(-) and TBARS, down-regulated the expression level of p53 protein, and up-regulated bcl-2 protein, respectively. The results suggest that Chinonin has preventive effects against apoptotic and necrotic cell death and its protective mechanisms are related to the antioxidant properties of scavenging nitric oxide and oxygen free radicals, and the modulating effects on the expression levels of bcl-2 and p53 proteins.     

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine

There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 muM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.     

Bile acid activation of the gadd153 promoter and of p53-independent apoptosis: relevance to colon cancer

Bile acids are strongly implicated in the etiology of colon cancer. Bile acids also induce apoptosis, and this action may be a key to understanding their role in colon cancer. However the mechanism of bile acid induction of apoptosis is not known. We present evidence of bile acid activation of the gadd153 promoter (a promoter activated by DNA damaging agents). We also show that bile acid induction of apoptosis is p53-independent. In addition, bile salts were found to induce blebbing preceding the actual morphological onset of apoptosis, which indicates early cytoskeletal alterations.     

The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics

Viruses have coevolved with their hosts, acquiring strategies to subvert host cellular pathways for effective viral replication and spread. Human cytomegalovirus (HCMV), a widely-spread β-herpesvirus, is a major cause of birth defects and opportunistic infections in HIV-1/AIDS patients. HCMV displays an intricate system-wide modulation of the human cell proteome. An impressive array of virus–host protein interactions occurs throughout the infection. To investigate the virus life cycle, proteomics has recently become a significant component of virology studies. Here, we review the mass spectrometry-based proteomics approaches used in HCMV studies, as well as their contribution to understanding the HCMV life cycle and the virus-induced changes to host cells. The importance of the biological insights gained from these studies clearly demonstrate the impact that proteomics has had and can continue to have on understanding HCMV biology and identifying new therapeutic targets.     

Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis

Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.     

MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma.     

Modulation of cell proliferation and gene expression by alpha-tocopheryl phosphates: relevance to atherosclerosis and inflammation

The effect of a mixture of alpha-tocopheryl phosphate and di-alpha-tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP-1 monocytic leukaemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which alpha-tocopherol was equally inhibitory. TPm led in non-stimulated THP-1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low density lipoprotein surface binding and oxLDL uptake. In non-stimulated THP-1 cells, alpha-tocopherol had only very weak effects on these events. Contrary to alpha-tocopherol, TPm was cytotoxic to THP-1 cells at high concentrations. Thus, TPm is able to inhibit the major aggravating elements involved in the progression of atherosclerosis. The higher potency of TPm may be due to a better uptake of the molecule and to its intracellular hydrolysis, providing more alpha-tocopherol to sensitive sites. Alternatively, a direct effect of the phosphate ester on specific cell targets may be considered.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

Regulation by anti-CD2 monoclonal antibody of the activation of a human T cell clone induced by anti-CD3 or anti-T cell receptor antibodies

In this study the effect of anti-cluster designation (CD) 2 monoclonal antibodies (mAb) on the activation of a cloned human T cell line, HY837, after triggering the CD3/T cell receptor (TcR) complex by anti-CD3 or anti-TcR mAb is described. HY837, which reacts with a series of mAb directed at different epitopes on the TcR, could be induced to proliferation and interleukin 2 (IL-2) production by soluble mAb directed at the CD3/TcR complex in the absence of accessory cells. mAb directed at the CD2 epitope T11-1 were shown to block the IL-2 production by HY837, as well as the expression of the IL-2 receptor, induced by anti-CD3 mAb, resulting in the inhibition of the proliferative response. The effect of anti-CD2 mAb on the proliferative response of HY837, induced by anti-CD3 mAb, was not due to a competition for Fc binding sites. In contrast, the proliferative responses and IL-2 production of HY837, induced by mAb directed at the TcR, were shown to be enhanced by the action of the anti-CD2 mAb. These results indicate that effects mediated by anti-CD3/TcR mAb cannot always be extrapolated to antigen-mediated effects and show that anti-CD2 mAb may regulate the T cell response, induced by mAb directed at the CD3/TcR complex, depending on which part of this complex is triggered during activation.