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.

[Diagnostic pathway in a case with severe degree of hypertension]. / Iter diagnostico di un caso di ipertensione arteriosa di grado severo.

A 29- year-old male was admitted because of exertion dyspnea and intense headache. These symptoms were associated with severe hypertension, small multiple areas of cerebral ischemia, thrombocytopenia, prolonged aPTT and renal failure. The diagnostic tests performed during hospitalization resulted in a diagnosis of Primary Antiphospholipids Syndrome. The renal biopsy sample suggested histopathological features of uncommon simultaneous occurrence of antiphospholipids nephropathy and a "collapsing variant" of segmental focal glomerulosclerosis. It is fundamental to be aware that this syndrome is very likely to occur, and therefore to perform antiphospholipids antibodies assessment, since only an anticoagulant therapy proves effective; nevertheless, in view of the pathological renal findings, other therapies such as steroids might be added.

[Risks for construction workers: analysis of the upper limb posture]. / Rischi nel comparto edile: analisi della postura degli arti superiori.

Our work focuses on the analysis of risks associated with onset of cumulative trauma disorders (CTD) among construction workers, and the goal is to evaluate the different degree of involvement of articular segments of the upper limbs. A number of workers with different qualification were analyzed using the OCRA Index and check-list protocols, applied to highly or moderate repetitive activities. In order to evaluate the involvement of the various upper limbs segments, we have extracted the information contained in the "posture section" of the protocols, before they are grouped together in the final posture risk value, and analyzed them considering the observed working activities. We obtained an "involvement index" related to any of the upper limb segments, highlighting the information about local involvement during the various phases of work. This "involvement index" may help in analyzing and evaluating the ergonomic risk referring to each articular segment during activity, and can be useful in the analysis and prevention of work related CTDs.

Results of myocardial revascularization by two internal thoracic arteries using a Y-shaped conduit.

MATERIAL AND METHODS: Between August 2002 and September 2004, 276 patients with coronary artery disease underwent surgical treatment using a Y-shaped conduit formed from two internal thoracic arteries (ITA). In 268 (97.1%) patients, myocardial revascularization required exclusively bi-mammary bypass grafting. In creation of the Y-shaped conduit in the capacity of a free transplant, the intercepted right ITA was connected to the left internal thoracic artery (LITA). In the remaining 7 (2.5%) patients, venous bypasses together with the internal thoracic arteries were employed: in one patient (0.36%), the radial artery was used. RESULTS: Three patients (1.08%) died after operation. The survival rate accounted for 98.4%. CONCLUSION: The use of two internal thoracic arteries for complete myocardial revascularization provided beneficial results in the short- and long-term postoperative periods.

Acute effects of rapamycin on glomerular dynamics: a micropuncture study in the rat.

The acute effects of cyclosporin (CsA, 20 mg(kg i.v.) and rapamycin (RAPA, 5 mg(kg i.v.) on glomerular dynamics were separately investigated by renal micropuncture in two groups of intact rats (group CsA and RAPA, respectively) and compared with vehicle-treated rats, used as controls (group CON). Left kidney glomerular filtration rate (GFR) was decreased by CSA (-35% vs. CON, P<0.05), but was not affected by RAPA (-14% vs. CON, NS), whereas the single-nephron GFR (SNGFR) was significantly decreased in both groups (-40% in CsA, P<0.01 and -26% in RAPA, P<0.05 vs. CON). In both groups glomerular plasma flow (GPF) was significantly reduced vs. CON (CsA: -48%, and RAPA: -25%) due to the increase in both afferent (Ra) and efferent (Re) glomerular resistances: group CSA showed a prevalent rise in Re (+98% vs. CON, P<0.001) than in Ra (+66%, P<0.001); in group RAPA the increment was modest and similar in Ra and Re (+33 and +32%, respectively, NS versus CON). A further group of rats was studied in which L-Arginine (ARG), the precursor of nitric oxide (NO), was administered (2.5 mg/Kg/min iv) with RAPA (group ARG). ARG limited the rise in Ra and Re, thereby preserving GPF; nevertheless, SNGFR remained low (-26% vs. CON, P<0.05) due to the decrease in the effective filtration pressure (-26% vs. CON). These data demonstrate that: (1) CsA is nephrotoxic at immunosuppressive doses; (2) RAPA, even at huge doses, has marginal effects on renal and glomerular dynamics; (3) the ARG-NO pathway is only partially involved in the vasoconstriction of superficial nephrons after RAPA administration.

Capsaicin-like effect of resiniferatoxin in the rat stomach.

Neurochemical and functional studies were performed to investigate and to compare the effects of resiniferatoxin and capsaicin in the rat stomach. Neonatal administration of resiniferatoxin (0.6-1.6 mumol/kg subcutaneously (s.c.)) produced a marked decrease in gastric calcitonin gene-related peptide-like immunoreactivity in both secretory and non-secretory region of the stomach. Almost complete depletion of the peptide was determined by neonatal administration of capsaicin (164 mumol/kg s.c.). Vasoactive intestinal polypeptide-like immunoreactivity was concomitantly unaffected by resiniferatoxin or capsaicin, thus showing the selectivity of action of the neurotoxins on gastric afferent fibers. Oral administration of an equimolar dose (0.3 nmol/kg) of resiniferatoxin or capsaicin together with 50% ethanol reduced at a similar extent gastric haemorrhagic lesions produced by the mucosal barrier-breaker agent. These findings provide evidence that resiniferatoxin and capsaicin may act on a common neuronal target in the rat stomach and that the acute exciting (protective) effect is of the same magnitude.

Transfection of a retroviral construct carrying a non producer HIV-1 variant induces HIV-1 resistance in CD4+ CEMss cells.

The phenotype of Human Immunodeficiency Virus-1 (HIV)-infected HUT-78 cell clone (F12) has been described (Federico et al, AIDS Res Hum Retrov 1989; 5: 365-96). Briefly, F12 cells are: i) CD4 down-regulated, ii) non producer and iii) fully resistant to homologous superinfection. We tested whether this phenotype was dependent upon the expression of the HIV-1 genome integrated therein. The SstI/SstI F12 provirus was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo)-Geneticine resistance gene. CD4+ HIV-susceptible CEMss cells were transfected with this construct in the sense orientation. Neo-resistant clones exhibited an integrated viral DNA, low viral mRNA expression and (as in F12 cells) the presence of uncleaved gp160, no gp41 and a small amount of p55 gag precursor. Superinfection of the F12/HIV-DNA-transfected CEMss clones showed that these CD4+ cells had acquired a significant (0.7-1.5 logs) resistance towards superinfection with HIV-1. This was observed in all four transfected clones where the F12/HIV DNA was expressed, but not in the control clone that was transfected with the pLj vector alone. These results confirm those that were obtained with human CD4+ CEMss cells infected with a recombinant retrovirus bearing the same SstI/SstI F12/HIV genome (Federico et al, J Gen Virol, 1993, in press). Both sets of results indicate that the expression of this genome in bio-engineered CD4+ human cells results in their intracellular immunization against HIV-1.

An outbreak of Salmonella enteritidis food poisoning from a commercially produced cheese.

Nine suspected cases of food poisoning were reported from three hospitals to the epidemiology and prevention service (Servizio di Epidemiologia e Prevenzione - SEP) of the local health authority in Naples district (Azienda Sanitaria Locale, ASL NA 4) betw

Microvascular leakage induced by substance P in rat urinary bladder: involvement of cyclo-oxygenase metabolites of arachidonic acid.

1. Intravenous administration of substance P (SP) or of the NK1 selective agonist [beta-Ala4, Sar9, Met (O2)11] SP-(4-11) increased vascular permeability in the urinary bladder of urethane-anaesthetized rats, providing evidence for an NK1 receptor-mediated inflammatory response. 2. BW 755C, a dual inhibitor of arachidonate cyclo-oxygenase and lipoxygenase, significantly reduced the plasma extravasation induced by SP, but did not modify the effect of [beta-Ala4, Sar9, Met (O2)11] SP-(4-11). 3. SP-induced microvascular leakage was also inhibited by systemic pretreatment with indomethacin or with the prostaglandin receptor antagonist SC-19220, while it was unaffected by the selective 5-lipoxygenase inhibitor BW A4C or the leukotriene antagonist FPL 55712. 4. Pretreatment of rats with the mast cell degranulating agent compound 48/80 significantly attenuated the inflammatory effect of SP. Indomethacin administration to 48/80-pretreated animals failed to produce further inhibition. 5. These findings indicate that intravascular SP promotes plasma exudation in rat urinary bladder through an NK1-mediated effect on venular permeability and the release of cyclo-oxygenase metabolites of arachidonic acid. The latter effect largely derives from the interaction of the neuropeptide with mast cells.

Full expression of transfected nonproducer interfering HIV-1 proviral DNA abrogates susceptibility of human He-La CD4+ cells to HIV.

An Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus-1 (HIV) variant, and showing a complete resistance to HIV-1 or HIV-2 superinfection, was previously characterized. We demonstrated that the replication of the superinfecting HIVs is blocked at the retrotranscription step, despite the CD4 down-regulation, since HIVs are able to cross the Hut-78/F12 cell membrane. In order to establish if the expression of the HIV-1 variant (F12/HIV) could be per se sufficient to induce the homologous viral interference shown in the F12 cells, the whole F12/HIV provirus was cloned and transfected in He-La CD4+ cells. In F12/HIV expressing He-La CD4+ clones, both the viral proteins expressed and the HIV nonproducer phenotype remain unmodified compared to F12 cells. Furthermore, despite the full expression of CD4 HIV receptors, the life cycle of the superinfecting HIV could be either strongly inhibited or totally abolished, depending on the cell clone considered. The inhibition of the superinfecting HIV was also reproduced when an HIV infectious molecular clone was transfected in F12/HIV He-La CD4+ clones, thus indicating that a post-cDNA synthesis block may operate against the superinfecting HIV. These data demonstrate that HIV susceptibility could be abrogated in cells expressing the F12/HIV genome, even in absence of any CD4 down-regulation.

A nonproducer, interfering human immunodeficiency virus (HIV) type 1 provirus can be transduced through a murine leukemia virus-based retroviral vector: recovery of an anti-HIV mouse/human pseudotype retrovirus.

The expression of a human immunodeficiency virus (HIV) type 1 provirus (F12-HIV) cloned from a nonproducer, chronically infected CD4 down-regulated Hut-78 cell clone (F12) does not lead to the formation of viral particles and, upon transfection in HeLa CD4+ cells, confers resistance to HIV superinfection without affecting the CD4 receptor exposure. In an attempt to transfer the anti-HIV properties of F12-HIV into human primary cell, we constructed a Moloney murine leukemia virus-based retroviral vector containing an F12-HIV genome lacking the 3' long terminal repeat and part of the nef gene, which was expressed under the control of its 5' long terminal repeat. The F12-HIV genome was inserted in the orientation opposite to that of the murine leukemia virus transcriptional unit and was designated the N2/F12-HIV nef-antisense vector. Lymphoblastoid CEMss cells, as well as human peripheral blood lymphocytes, were successfully transduced by the recombinant retrovirus emerging from the producer PA317 clones. CEMss clones expressing the F12-HIV nef-antisense vector became resistant to HIV superinfection even at the highest utilized multiplicity of infection (10(5) 50% tissue culture infective doses per 10(6) cells). In transduced CEMss cells the viral interference induced by the F12-HIV expression is not due to CD4 HIV receptor down-regulation. Nonproducer, interfering HIV proviruses transduced into retroviral vectors may, therefore, provide an alternative strategy for the protection of CD4+ human primary cells from HIV infection, which strategy may be used in designating a safe and efficient gene therapy protocol for patients with AIDS.

Effect of mequitamium iodide (LG 30435) on airway microvascular leakage in the guinea-pig.

Intravenous administration of mequitamium iodide (LG 30435) prevented the increase of tracheobronchial vascular permeability induced either by antigen challenge or by exogenous histamine in the guinea-pig, while it was ineffective against PAF, serotonin or capsaicin. These findings indicate that mequitamium iodide selectively interferes with the effect of histamine on airway microvascular leakage, mediated by histamine H1 receptors, and is more potent than diphenhydramine, mequitazine or astemizole. Histamine receptor antagonism is likely to be major determinant of the antiallergic activity of the compound, although additional mechanisms may be involved.

A recombinant retrovirus carrying a non-producer human immunodeficiency virus (HIV) type 1 variant induces resistance to superinfecting HIV.

A human immunodeficiency virus (HIV) type 1-infected Hut-78 cell clone (F12) shows a peculiar phenotype: it exhibits an altered viral protein pattern, is a nonproducer and is resistant to homologous superinfection. To determine whether this phenotype is dependent upon the expression of the HIV-1 genome integrated therein, the SstI/SstI F12 provirus [deprived of HIV long terminal repeats (LTRs)] was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo) and Geneticin resistance gene. CD4+ HIV-susceptible CEMss cells (a CEM clone able to form large syncytia 2 to 3 days post-HIV infection) were infected with the recombinant retroviruses rescued from the F12/HIV-pLj-transfected (in either sense or antisense orientation) amphotropic packaging cells PA 317. Neo sense resistant gene clones showed approximately 10 copies of viral DNA/cell (without detectable major deletions) only in episomal form, low viral RNA expression and a viral protein pattern characterized by an uncleaved gp160, no gp41 and little, if any, p55 gag precursor (as in F12 cells). Superinfection of these F12/HIV DNA-engineered clones with HIV-1 resulted in a significant reduction in the yield of superinfecting HIV. This effect (more pronounced when the clones were maintained under neo selective pressure) was observed in all five retrovirus-infected clones exhibiting the presence and expression of sense episomal F12/HIV DNA but not in two clones bearing an antisense F12/HIV DNA or in one clone bearing only the pLj vector. These results indicate that bio-engineered human CD4+ cells expressing the F12/HIV genome exhibit a significant resistance to HIV superinfection.

Limited expression of R5-tropic HIV-1 in CCR5-positive type 1-polarized T cells explained by their ability to produce RANTES, MIP-1alpha, and MIP-1beta.

Human T helper (Th) cells (Th1- or Th2-oriented memory T cells as well as Th1- or Th2-polarized naive T cells) were infected in vitro with an R5-tropic HIV-1 strain (BaL) and assessed for their profile of cytokine production, CCR5 receptor expression, and HIV-1 p24 antigen (p24 Ag) production. Higher p24 Ag production was found in CCR5-negative Th2-like memory T cells than in CCR5-positive Th1-like memory T cells. By contrast, p24 Ag production was higher in Th1-polarized activated naive T cells in the first 4 days after infection. However, p24 Ag production in Th1-polarized T cells became comparable or even lower than the production in Th2-polarized populations later in infection or when the cells were infected with HIV-1BaL after secondary stimulation. The higher levels of p24 Ag production by Th1-polarized naive T cells soon after infection reflected a higher virus entry, as assessed by the single round infection assay using the HIV-chloramphenicol acetyl transferase (HIV-CAT) R5-tropic virus that contains the envelope protein of HIV-1 YU2 strain. The limitation of viral spread in the Th1-polarized populations, despite the initial higher level of T-cell entry of R5-tropic strains, was due to the ability of Th1 cells to produce greater amounts of beta-chemokines than Th2 cells. In fact, an inverse correlation was observed between Th1-polarized naive T cells and Th1-like memory-activated T cells in regards to p24 Ag production and the release of the following CCR5-binding chemokines: regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta. Moreover, infection with the HIV-1BaL strain of Th1-polarized T cells in the presence of a mixture of anti-RANTES, anti-MIP-1alpha, and anti-MIP-1beta neutralizing antibodies resulted in a significant increase of HIV-1 expression. These findings suggest that Th1-type responses may favor CD4(+) T-cell infection by R5-tropic HIV-1 strains, but HIV-1 spread in Th1 cells is limited by their ability to produce CCR5-binding chemokines. (Blood. 2000;95:1167-1174)

Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap.

Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from Xenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.

Characterization of HIV-1 Tat proteins mutated in the transactivation domain for prophylactic and therapeutic application.

Previous work from our group showed that genetic immunization of mice with HIV-1 tat genes (tat22 and tat22/37), encoding Tat proteins mutated in the transactivation domain and lacking Tat-transactivating activity, evoke an immune response to wild-type Tat, both humoral and cellular. In the present work we report that the mutated Tat proteins localize within the cells, are released and taken up by the cells in a fashion similar to wild-type Tat. Moreover, the exogenous mutated Tat proteins interfere with the transactivating function of extracellular wild-type Tat. These results support the notion that tat22 and tat22/37 genes may represent good candidates for the development of an anti-HIV-1 vaccine, especially for HIV-1 infected patients.

gag, vif, and nef genes contribute to the homologous viral interference induced by a nonproducer human immunodeficiency virus type 1 (HIV-1) variant: identification of novel HIV-1-inhibiting viral protein mutants.

We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr, pol, or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.

Anti-HIV viral interference induced by retroviral vectors expressing a nonproducer HIV-1 variant.

A Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus (HIV-1) variant shows complete resistance to HIV-1 or HIV-2 superinfection. The F12-HIV provirus produces an altered HIV-1 protein pattern and cannot generate even immature viral particles. We demonstrated that HeLa CD4+ cells transfected with the F12-HIV genome resist HIV superinfection through a CD4-independent mechanism. As F12-HIV appears to be a useful system to induce anti-HIV intracellular immunization, we constructed various retroviral vectors containing the F12-HIV genome, modified by elimination of the F12 3'LTR and part of its nef gene, inserted 'antisense' with respect to the Moloney murine leukemia virus 5' LTR. Here we show that recombinant retroviral particles carrying the N2/F12-HIV nef- (as) construct can stably transduce both CEMss human cells and primary human peripheral blood lymphocytes, inducing the expression of the F12-HIV genome. These results could open the way to an anti-AIDS gene therapy strategy based on F12-HIV-induced intracellular immunization.