Implementation of GOLD consensus report in real life: results from the Velletri-Lariano (VELA) cohort.

BACKGROUND: COPD is one of the leading causes of morbidity and mortality. Pharmacotherapy improves quality of life and reduces exacerbations although low adherence with prescribed treatments may represent a barrier to optimal disease management. The first objective of this paper is to report the distribution of COPD patients according to GOLD categories, in a sample of patients from a cohort study in an area of the Latium region in Italy. The second objective is to evaluate the agreement between the distributions of severity obtained from the HCPs and the experts included in the study board (Board). METHODS: COPD patients were given a card to collect demographic and clinical data at baseline. Information in those cards was independently evaluated by HCPs and Board to include each patient into one of the four GOLD categories. RESULTS: In a sample of 187 stable COPD patients, 59% male, mean age 70 year, the distribution of GOLD categories according to the Board was: 6% A, 34% B, 2% C, and 58% D. A discrepancy in GOLD classification was observed between the study board and field-based HCPs, regarding more than 50% of the patients, with a clear trend to underestimate the frequency of patients in D level (21%) and to overestimate the frequency in C level (21%). CONCLUSIONS: These results describe for the first time the distribution of COPD patients in an Italian cohort according to the GOLD categories, with the highest frequencies in levels B and D. The misclassification from HCPs may impact the therapeutic approach and the clinical outcomes.

Insertion torque of dental implants after microvascular fibular grafting.

We have measured the necessary torque to the initial stabilisation of dental implants in revascularised bony transplants for reconstruction of the maxilla and mandible in edentulous patients. We installed 28 dental implants in 7 patients who had had reconstructions of the maxilla and mandible by microsurgical flaps. At the time of the installation of the implants, we measured the torque for final stabilisation. The minimum torque was 20 Newton centimetres (Ncm) in 11 implants, and the maximum 45Ncm in 8. The measure of torque was not influenced by sex, age group, or time between transplant and implant.

Bradykinin B2 receptor antagonist off label use in short-term prophylaxis in hereditary angioedema.

Hereditary angioedema type I (HAE-C1-INH) is an inherited disorder characterized by repeated severe angioedema attacks mostly triggered by traumas, emotional stress, increased estrogen levels or surgical procedures, in particular, odontostomatological interventions. Icatibant, a bradykinin B2 receptor antagonist, has been approved for treatment of HAE attacks. In this paper we describe the “off label” administration of icatibant as short-term prophylaxis of dental extraction in a patient with HAE with the aim of preventing perioperative angioedema attacks. The drug showed an effective and safe profile. Thus, a short-term prophylaxis of angioedema attacks in patients with HAE may be arranged on a multidisciplinary basis, according to the clinical history of each single patients.

Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix.

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.

Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts.

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.

Tyrosine kinase-dependent release of an adenovirus preterminal protein complex from the nuclear matrix.

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) through the interaction of Ad preterminal protein (pTP). The NM is a highly salt-resistant fibrillar network which is known to anchor transcription, mRNA splicing, and DNA replication complexes. Incubation of rATP with NM to which pTP was bound caused the release of pTP as a pTP-NM complex with a size of 220 to 230 kDa; incubation with 5' adenylylimidodiphosphate (rAMP-PNP) showed no significant release, indicating that rATP hydrolysis was required. With NM extracts, it was shown that a pTP-NM complex which was capable of binding Ad origin DNA could be reconstituted in vitro. A number of high-molecular-weight NM proteins ranging in size from 120 to 200 kDa were identified on Far Western blots for their ability to bind pTP. rATP-dependent release of pTP from the NM was inhibited in a dose-dependent fashion by the addition of tyrosine kinase inhibitors, such as quercetin, methyl-2,5-dihydroxycinnamate, or genistein. NM-mediated phosphorylation of a poly(Glu, Tyr) substrate was also significantly abrogated by the addition of these compounds. rATP-dependent release of Ad DNA termini bound to the NM via pTP was also blocked by the addition of these inhibitors. These results indicate that a tyrosine kinase mechanism controls the release of pTP from its binding sites on the NM. These data support the concept that phosphorylation may play a key role in the modulation of pTP binding sites on the NM.

Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells.

Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.

Recombinant interleukin 1 and tumor necrosis factor acting in synergy to release thromboxane, 6-KETO-PGF1 alpha and PGE2 by human neutrophils.

The cyclooxygenase pathway promotes formation of an endoperoxide that is the precursor of prostaglandins (PG), thromboxanes (Tx) and prostacyclins (PGI2), all of which have important biologic activities. In this study, we examined the ability of human polymorphonuclears (PMN) to synthesize TRxA2, 6-KETO-PGF1 alpha and PGE2 in response to human recombinant interleukin 1 (IL1) and tumor necrosis factor (TNF) alone and in combination. Blood was obtained from healthy donors and whole blood was centrifuged over Ficoll-Hypaque in 2% dextran for 30 min. PMNs were resuspended in Gey's buffer, exposed to the IL1 and TNF at 300 ng/ml and 0.5 ng/ml concentrations, and incubate for 30 min. at 10(6) cell/ml. Results indicate that IL1 and TNF alone have little or no effect on human neutrophils to synthesize TxA2, 6-KETO-PGF1 alpha and PGE2 production. This effect was completely inhibited by two non-steroidal anti-inflammatory drugs (i.e. indomethacin and proglumetacin).

Restoration of anti-interleukin-1 depressed natural killer activity by human recombinant interferon alpha or gamma, human recombinant interleukin-2 and indomethacin.

The lymphokines interleukin-2 and interferon alpha or gamma are synthesized and secreted by activated mononuclear cells (MC) and play a critical role in the proliferative expansion of T-lymphocyte effector cells during the immune response. The pretreatment of human peripheral blood mononuclear cells (PBMC) with antibody IgG against human interleukin-1 (IL-1) from normal rabbit serum, inhibited their natural killer (NK) activity against both myeloid (K562) and lymphoid (MOLT-4) cell lines. Percent specific lysis of tumor cells decreased by increasing the antibody anti-IL-1 dose in an almost linear fashion. When the effector cells were pretreated with human recombinant interleukin-2, human interferon alpha or gamma and indomethacin alone or in combination, the inhibitory effect of antibody against human IL-1 was almost totally reversed.

Lipoxin A augments release of thromboxane from human polymorphonuclear leukocyte suspensions.

Lipoxin A (LXA) is a novel eicosanoid, generated by the interactions of lipoxygenases, which has a variety of biological actions. When added to human polymorphonuclear leukocytes, LXA stimulated thromboxane formation which was monitored as TxB2 by radioimmunoassay. The compound augmented the formation of TxA2 stimulated by the ionophore of divalent cations (A23187). Formation of thromboxane was inhibited by two non-steroidal anti-inflammatory drugs (i.e. indomethacin and proglumetacin). Results of the present study indicate that LXA can provoke the release and transformation of endogenous arachidonic acid to thromboxane. Moreover, they suggest a relationship between lipoxin A and the formation of cyclooxygenase pathway products.

Mitogen dose-dependent effect of weak pulsed electromagnetic field on lymphocyte blastogenesis.

The effects of pulsed extremely low frequency electromagnetic fields on human peripheral blood lymphocyte mitogenesis induced by phytohaemoagglutinin, concanavalin A or calcium ionophore A23187 were studied. The dependence of the field effect on mitogen concentrations was investigated. Field exposure produced strong inhibition of DNA synthesis when optimal doses of mitogens were used, confirming our previous findings. Opposite effects were observed at suboptimal concentration of mitogens. Experiments performed by exposing cell cultures to the field for short periods indicated that a field application of at least 6 h is needed to influence irreversibly lymphocyte blastogenesis.

Augmentation of thromboxane production in vitro by polymorphonuclears and macrophages exposed to IL1/LP.

Human peripheral blood polymorphonuclear leukocytes (PMNs) and murine peritoneal macrophages (M phi) were stimulated to generate thromboxane upon treatment with highly purified human interleukin 1/leukocytic pyrogen (IL1/LP) at various concentrations. Thromboxane B2 was measured by radioimmunoassay in the cell-free supernatants of cell suspensions after 1 hour incubation at 37 degrees C. Thromboxane B2 amounts increased in a way which depended on the dose of IL1 added to the cell cultures.

A role for Ca2+ in the effect of very low frequency electromagnetic field on the blastogenesis of human lymphocytes.

The DNA synthesis of lymphocytes triggered by phytohemagglutinin or phorbol-myristate-acetate is strongly reduced by the externally applied electromagnetic field (ELF). Ca2+ uptake by stimulated lymphocytes is also reduced by ELF. The effect appears to be synergistic with that of the well-known calcium blocker agent, verapamil.

Human prostatic carcinoma in tissue culture: correlations between histological diagnosis and in vitro parameters.

Prostatic cell biology is still largely unknown so that even the natural history of prostatic carcinoma is unpredictable. In order to correlate new observations with the prognosis of patients with prostatic carcinoma of various grades, we followed up 24 in vitro samples from surgical specimens of prostatic carcinoma. Fragments from 7 grade-I, 10 grade-II, 6 grade-III; and 1 grade-IV tumors were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10% horse serum and 50 ng/ml each of hydrocortisone and insulin. Epithelial cells grown from the explants continued to grow for a maximum of 120 days and their morphology varied from a fairly regular monolayer of polygonal cells to irregular patterns of overlapping growth with many giant multinucleated cells. Although our data need a longer clinical follow-up time and larger numbers to achieve any statistical significance, the present findings seem to indicate rather clearly that a short life span in culture, a regular growth and a positive secretion activity is typical of low-grade tumors and that a longer life span, an irregular growth and a negative secretion in vitro are characteristics of high-grade tumors. A longer clinical follow-up of these patients will be important in the future to indicate whether these findings can be of any real prognostic value.

Dimethylsulphoxide stimulation of dome formation in cultured rat prostate epithelial cells.

Primary cultures of normal rat prostate produce cells with epithelial characteristics that may be useful in the study of cell differentiation and prostate physiology. These epithelial cells, grown in DME plus 10% FCS, 10% HS, and 50 ng/ml each of hydrocortisone and insulin, can be induced to differentiate into dome ( hemicyst ) forming cells by 250 mM DMSO, a molecule that acts as a differentiation inducer in Friend erythroleukemia cells, Madin-Darby canine kidney cells, and in mouse and rat mammary epithelial cells. Dome formation in cell culture is the consequence of transepithelial transport of water and ions, resulting in fluid accumulation between the culture dish and the cell layer. Dome formation in prostate epithelial cells, besides representing an example of induction of differentiation in a cell culture system, suggests that prostate (ductal?) epithelial cells have fluid reabsorption capabilities.

Reduced mitogenic stimulation of human lymphocytes by extremely low frequency electromagnetic fields.

Blastogenesis of human peripheral blood lymphocytes stimulated in vitro by non-specific mitogens (PHA, ConA, PWM) upon exposure to extremely low frequency EMF has been studied. Different frequencies of square waveforms have been used. PHA-stimulation resulted in strong inhibitions as measured by [3H]thymidine incorporation. A frequency window (3-50 Hz) within which ConA-induced blastogenesis was significantly inhibited has been individuated. The mitogenic effect of PWM was significantly affected only at 3 Hz.

Effect of salicylates on lymphocyte blastogenesis in vitro: association with other non-steroid anti-inflammatory drugs.

High concentrations of the non-steroid anti-inflammatory drugs, alone and in association, reduce the blastogenesis of human lymphocytes in vitro. This effect is probably due to the toxicity of these agents and not to the inhibition of prostaglandin (PG) prosuction. Therefore drugs, such as, salicylates and associations of salicylates with other non-steroid anti-inflammatory drugs, which have a weak action on PG-synthesis, also inhibit proliferation of lymphocytes.