Death receptor ligation triggers membrane scrambling between Golgi and mitochondria.

Subcellular organelles such as mitochondria, endoplasmic reticulum (ER) and the Golgi complex are involved in the progression of the cell death programme. We report here that soon after ligation of Fas (CD95/Apo1) in type II cells, elements of the Golgi complex intermix with mitochondria. This mixing follows centrifugal dispersal of secretory membranes and reflects a global alteration of membrane traffic. Activation of apical caspases is instrumental for promoting the dispersal of secretory organelles, since caspase inhibition blocks the outward movement of Golgi-related endomembranes and reduces their mixing with mitochondria. Caspase inhibition also blocks the FasL-induced secretion of intracellular proteases from lysosomal compartments, outlining a novel aspect of death receptor signalling via apical caspases. Thus, our work unveils that Fas ligand-mediated apoptosis induces scrambling of mitochondrial and secretory organelles via a global alteration of membrane traffic that is modulated by apical caspases.

Primary effusion lymphoma cells undergoing human herpesvirus type 8 productive infection produce C-type retroviral particles.

Primary effusion lymphomas (PELs) are invariably infected by the human herpesvirus 8 (HHV8)that is present in most PEL cells as latent virus but replicates in a subset of permissive cells to produce infectious progeny. Here we show that productively infected PEL cells release C-type retrovirus-like particles encoding an Mn++-dependent RT activity, which is typical of endogenous retroviruses. Strikingly, C-type particles are produced only in cells showing advanced HHV8 morphogenesis. Phorbol esters, which induce productive HHV8 replication and morphogenesis in PEL cells, increase RLP production. Phosphonoacetic acid, a blocker of HHV8 late gene expression, inhibits the production of C-type particles, whereas neutralizing anti-alphaIFN antibodies, which are known to increase HHV8 assembly, increases C-type particle production. These data suggest that factors expressed in advanced stages of HHV8 reactivation support endogenous C-type particle morphogenesis in PEL cells.

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

Apoptotic death of Listeria monocytogenes-infected human macrophages induced by lactoferricin B, a bovine lactoferrin-derived peptide.

Listeria monocytogenes, an intracellular facultative food-borne pathogen, was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of murine macrophages. These cells represent the predominant compartment of bacterial multiplication and die as a result of necrosis. In this study we showed that human non-activated and IFN-gamma-activated macrophagic-like (THP-1) cells infected with L. monocytogenes, mainly die by necrosis rather than by an apoptotic process. Two natural products derived from bovine milk, lactoferrin and its derivative peptide lactoferricin B, are capable of regulating the fate of infected human macrophages. Bovine lactoferrin treatment of macrophages protects them from L. monocytogenes-induced death whereas lactoferricin B, its derivative peptide, determines a shifting of the equilibrium from necrosis to apoptosis.

The prognostic significance of acetowhitening of the vulva and HPV-DNA test. A multicentre study.

Acetowhitening of the vulva has been related to a subclinical human papillomavirus (HPV) infection. No consense has been reached about undertaking -or not- any therapy for these acetowhite changes. We have observed from our clinical experience and in a 10 years observational follow-up, that acetowhitening of the vulva regarding high risk (16-18) and low risk (6-11) HPV groups (as assessed by PCR analysis) significantly decreased; and acetowhitening areas negative to polymerase chain reaction (PCR), significantly increased from 53% (202/382) to 85% (276/325) (P<0.001). Our findings suggest that independently from HPV type and in the absence of cofactors, there is a statistically significant spontaneous remission of these areas.

In vitro effect of synthetic flavanoids on astrovirus infection.

In this study we investigated the activity of halogeno-, cyano- and amidino-isoflavenes, isoflavans and flavans on the multiplication of human astroviruses. These are naked small round viruses which have been recognized as causative agents of human gastroenteritis, and whose capsid proteins are similar to those of picornaviruses. Although all drugs tested caused a dose-dependent reduction of viral antigen synthesis as monitored by immunofluorescence, the chloro derivatives were the most effective.

Infection of human enterocyte-like cells with rotavirus enhances invasiveness of Yersinia enterocolitica and Y. pseudotuberculosis.

Mixed infection with rotavirus and either Yersinia enterocolitica or Y. pseudotuberculosis was analysed in Caco-2 cells, an enterocyte-like cell line highly susceptible to these pathogens. Results showed an increase of bacterial adhesion and internalisation in rotavirus-infected cells. Increased internalisation was also seen with Escherichia coli strain HB101 (pRI203), harbouring the inv gene from Y. pseudotuberculosis, which is involved in the invasion process of host cells. In contrast, the superinfection with bacteria of Caco-2 cells pre-infected with rotavirus resulted in decreased viral antigen synthesis. Transmission electron microscopy confirmed the dual infection of enterocytes. These data suggest that rotavirus infection enhances the early interaction between host cell surfaces and enteroinvasive Yersinia spp.

Effect of HSV-2 infection on the expression of HPV 16 genes in CaSki cells.

Human papillomaviruses (HPVs) have been proposed to be the most important etiological factors for cervical cancer although different agents may act in conjunction. Herpes simplex virus type 2 (HSV-2) infection is considered as a possible cofactor to malignant transformation. To examine the influence of HSV-2 infection on the HPV genes expression, CaSki cells bearing 60 to 600 copies of HPV-16 DNA per cell were used as a model system. Twenty hours post HSV-2 infection the mRNA transcripts for HPV-16 early (E1, E2 and E6) and late (L1) genes were analysed by RT-PCR assay. Results indicated that the level of transcription of E1, E2 and E6 genes was up to 3-fold enhanced in HSV-2 infected CaSki cells suggesting that HSV-2 infection could increase the risk of cervical cancer by overexpression of both HPV regulatory and oncogenic genes.

Effect of polyions on the infectivity of SA-11 rotavirus in LCC-MK2 cells.

We investigated the effect of different polyions on the early phases of SA-11 rotavirus infection in susceptible LLC-MK2 cells in order to clarify the influence of electrostatic interactions in rotavirus binding to cell membranes and to select antiviral compounds able to prevent viral attachment. When added during the viral attachment step, polymers having positive charge (protamine, protamine sulphate, DEAE-dextran, histone and poly-L-lysine hydrobromide) enhanced virus infection whereas those having negative charge (mucin, heparin, heparan sulphate, alpha-1-acid glycoprotein and dextran sulphate) inhibited the viral replication. The effect of polyanions on SA-11 rotavirus and on cell membrane receptors has also been examined. Results obtained indicated that while mucin and alpha-1-acid glycoprotein act directly on virus particles, the target of heparin, heparan sulphate and dextran sulphate is the host cell membrane.

Vulvar Paget's disease: review of the literature, considerations about histogenetic hypothesis and surgical approaches.

Paget's disease of the vulva is a rare neoplasm that occurs on the apocrine glands. It predominantly is an intraepithelial lesion, but has the potential for dermal invasion and on occasion has been associated with an underlying adenocarcinoma.

HPV and intraepithelial neoplasia recurrent lesions of the lower genital tract: assessment of the immune system.

OBJECTIVE: To evaluate the immune state in patients with genital relapse HPV and intraepithelial lesions of the lower genital tract. METHOD: Forty-three patients were selected. Twenty-one were affected by recurrent HPV infection either alone or combined with intraepithelial neoplasia treated by laser surgery, and 22 had been previously-treated and clinically cured without recurrence during a follow-up from 18 to 24 months. The diagnostic protocol included colposcopy with eso- and endocervical cytology histologically confirmed by directed biopsy. Afterwards patients underwent a systemic immunogenic evaluation. RESULTS: NK cell reduction was strictly related to HPV infection associated with intraepithelial lesions; B-lymphocyte reduction was percentually greater in patients affected by HPV alone; activation of R-IL2 increased in a percentage overlapping in the two groups indicating patient reaction to the virus. CONCLUSION: Our study supports the theory that immune response directed against viral antigens is one of the most important effectors in the control of HPV infections and that HPV is the cause of a systemic rather than local lesion.

Viral childhood diarrhoea in Rome: a diagnostic and epidemiological study.

During the period May 1987-January 1989, faecal samples from 417 paediatric inpatients admitted to the main paediatric hospital in Rome were screened by direct electron microscopy and rotavirus enzyme-linked immunosorbent assay. Rotaviruses were detected in 18.2% of cases and adenoviruses in 7%, whereas astroviruses were found in 1% of cases. Different percentages of rotavirus excretors were revealed by enzyme-linked immunosorbent assay and electron microscopy. This discrepancy seems to be due to false positive results introduced by enzyme-linked immunosorbent assay. Analysis of electron microscopy-positive samples by rotaviral RNA polyacrylamide gel electrophoresis showed different electropherotypes of rotavirus among which a single, largely predominant long electropherotype (55.4%) was revealed. Short electropherotype subgroup I rotaviruses were demonstrated in about 10.7% of samples.

An epidemiological study on viral infantile diarrhoea in Tirana.

During the period May 1993-April 1994, an epidemiological survey was conducted on enteric viruses which cause gastroenteritis in infants and young children in Tirana, Albania. Specimens from 321 cases were screened by direct electron microscopy and by an enzyme-linked immunosorbent assay specific for rotavirus group A antigen. By ultrastructural analysis, rotaviruses were detected in 10.3% of cases and adenoviruses in 0.6%, whereas small round structured viruses and small round viruses were found in 2.8% and 2.2% of cases, respectively. Different percentages of rotavirus excretors were revealed by enzyme-linked immunosorbent assay (12.15%) and electron microscopy. Samples rotavirus-positive in at least one of these assays were also analyzed by agglutination of latex particles and electron microscopy results were confirmed. Analysis of electron microscopy-positive samples by rotaviral RNA polyacrylamide gel electrophoresis showed five different long electropherotypes of rotavirus among which a single, largely predominant electropherotype (65.5%) was observed.

[Preliminary diagnostic evaluation of submucosal myoma using operative hysteroscopy]. / Valutazione diagnostica preliminare al trattamento dei miomi sottomucosi con isteroscopia operativa.

BACKGROUND AND AIM: The authors aim to underline the importance of preliminary diagnostic evaluation in the treatment of submucous leiomyoma using hysteroscopy. MATERIALS AND METHODS: The study examined 18 patients monitored at the Institute of Obstetrics and Gynecology of "La Sapienza" University of Rome between January and December 1995 in whom it was possible to make a correct definition of the lesion to be treated (number, site, size, etc.) using 3 different diagnostic methods: hysteroscopy (HS), transvaginal scan (TSV) and transvaginal echohysterography (TVHS) The authors focused attention on three different parameters: leiomyoma size, extension (intracavity/intramural portion) and evaluation of the residual leiomyoma, which are essential for optimal endoscopic resection. RESULTS: HS enabled the number, size, site, origin, base, submucous portion and relations with tube operings to be evaluated, but did not allow the myometrial part of the lesion to be examined. CONCLUSIONS: Integration with TSV, even if this does not allow a precise definition of the extension, highlights the size, site, involvement of myometrial structure and relations with the perimetrium, thus allowing the possibility of evaluating the residual myometrium. Compared to the above methods, TVHS highlights the effective extension (namely the submucous/intramural portion) and localization of the neoformation.

Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes.

It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.

killerFLIP: a novel lytic peptide specifically inducing cancer cell death.

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIPL as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIPL motif indeed induced similar cell death, suggesting the importance of the c-FLIPL residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.

Association of fission proteins with mitochondrial raft-like domains.

It was shown that receptor-mediated apoptosis involves a cascade of subcellular events including alterations of mitochondria. Loss of mitochondrial membrane potential that follows death receptor ligation allows the release of apoptogenic factors that result in apoptosis execution. Further important mitochondrial changes have been observed in this regard: mitochondrial remodeling and fission that appear as prerequisites for the occurrence of the cell death program. As it was observed that lipid rafts, glycosphingolipid-enriched structures, can participate in the apoptotic cascade being recruited to the mitochondria under receptor-mediated proapoptotic stimulation, we decided to analyze the possible implication of these microdomains in mitochondrial fission. We found that molecules involved in mitochondrial fission processes are associated with these domains. In particular, although hFis1 was constitutively included in mitochondrial raft-like domains, dynamin-like protein 1 was recruited to these domains on CD95/Fas triggering. Accordingly, the disruption of rafts, for example, by inhibiting ceramide synthase, leads to the impairment of fission molecule recruitment to the mitochondria, reduction of mitochondrial fission and a significant reduction of apoptosis. We hypothesize that under apoptotic stimulation the recruitment of fission-associated molecules to the mitochondrial rafts could have a role in the morphogenetic changes leading to organelle fission.

The adenine nucleotide translocator 1 acts as a type 2 transglutaminase substrate: implications for mitochondrial-dependent apoptosis.

In this study we provide in vitro and in vivo evidence showing that the protein disulphide isomerase (PDI) activity of type 2 transglutaminase (TG2) regulates the correct assembly and function of the mitochondrial ADP/ATP transporter adenine nucleotide translocator 1 (ANT1). We demonstrate, by means of biochemical and morphological analyses, that ANT1 and TG2 physically interact in the mitochondria. Under physiological conditions, TG2's PDI activity regulates the ADP/ATP transporter function by controlling the oligomerization of ANT1. In fact, mitochondria isolated from hearts of TG2(-/-) mice exhibit increased polymerization of ANT1, paralleled by an enhanced ADP/ATP carrier activity, as compared to mitochondria belonging to TG2(+/+) mice. Interestingly, upon cell-death induction, ANT1 becomes a substrate for TG2's cross-linking activity and the lack of TG2 results in a reduction of apoptosis as well as in a marked sensitivity to the ADP/ATP exchange inhibition by atractyloside. These findings suggest a complex TG2-dependent regulation of the ADP/ATP transporter and reveal new important avenues for its potential applications in the treatment of some mitochondrial-dependent diseases, including cardiovascular and neurodegenerative diseases.

Xeno-cannibalism as an exacerbation of self-cannibalism: a possible fruitful survival strategy for cancer cells.

The term self-cannibalism, or autophagy, was coined to describe the ability of the cells to cannibalize their own damaged organelles or proteins. It was morphologically described as the presence of double-membraned autophagic vesicles filled with diverse cellular materials or debris inside the cells. Hence, more recently, the presence of autophagic vacuoles has been associated with cell survival, including cell senescence and cancer and appears to be activated by nutrient deprivation. The occurrence of autophagic processes can also lead, as final event, to the death of the cell. In this review we summarize the results reported in literature on a phagic process that appears to be related to self-cannibalism: the xeno-cannibalism. This was described as the ability of certain cells, e.g. metastatic cells, to cannibalize their siblings as well as cells from the immune system. Interestingly, metastatic tumor cells are also able to engulf and digest living cells, including autologous lymphocytes that should kill them, i.e. CD8(+) cytotoxic lymphocytes. This can represent a formidable opportunity for metastatic cells to survive in adverse conditions such as those they encounter in their "journey" towards the target organ to establish a colony. Altogether these findings seem to suggest a pathogenetic role for cannibalic behavior in human pathology and point at this surprising cellular aggressiveness as an innovative pharmacological target in the clinical management of metastatic disease.