Platelet aggregation and aspirin non-responsiveness increase when an acute coronary syndrome is complicated by an infection.

BACKGROUND: Epidemiologic studies have shown that there is an association between acute respiratory infection and acute coronary syndrome. The aim of this study was to analyze the thrombotic risk, assessed by platelet aggregation and aspirin non-responsiveness, in patients with an acute coronary syndrome complicated by an infection. METHODS: Patients with an acute coronary syndrome who were admitted to the intensive care unit and hospitalized for at least 3 days in 2002 and 2003 were eligible for the study. Three hundred and fifty-eight patients were included, of whom 66 had an infection during their hospital stay. Platelet aggregation was analyzed by an aggregometer using laser light (PA-200, laser light scattering). Aspirin non-responsiveness was defined as a closure time of

Ribavirin up-regulates IL-12 p40 gene expression and restores IL-12 levels in Leishmania-treated PBMCs.

Ribavirin, a nucleoside analogue that interferes with viral mRNA synthesis and inhibits the replication of RNA and DNA viruses, has been recently proposed as an effective immune response modulator. In the present report, we studied the effect of ribavirin on IL-12 p40 gene expression in peripheral blood mononuclear cells (PBMCs) of healthy subjects. We also studied ribavirin effects on PBMCs activated with lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) and treated with Leishmania donovani antigens. We provide evidence that ribavirin was able to up-regulate IL-12 p40 gene expression and to restore levels of IL-12 p40 gene expression and IL-12 secretion in fully activated PBMCs that were strongly inhibited by L. donovani antigens. Because effective management of leishmanial disease is usually associated with a prevalent T-helper 1 immune response with elevated production of IL-12,our preliminary results may be of particular interest, provided that they will be confirmed by further in vitro and in vivo studies, when considering a possible use of ribavirin as adjuvant in severe leishmanial disease.

Effects of fish farming on microbial enzyme activities and densities: comparison between three Mediterranean sites.

AIM: The effects of fish farming on microbial enzyme activities and heterotrophic bacterial density were investigated in three Mediterranean sites before and after the start of mariculture. METHODS AND RESULTS: Microbial activities were measured on water and sediment samples by using fluorogenic substrates specific for leucine aminopeptidase, beta-glucosidase and alkaline phosphatase (AP); bacterial counts were determined by Marine agar plates. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of activity and abundance values obtained before and after the experiment showed that fish farming mainly affected the levels of microbial activities; they were significantly enhanced both in water and sediments, reaching an increase of 183.66 times for AP in Castellammare Gulf. After mariculture, no significant variations were recorded in heterotrophic bacterial density in the waters, while significant changes were observed in the sediments. Effects induced appeared to be extended not only to stations in which cages were located, but also to control sites far from the direct influence of fish farming.

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain.

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.

Thymectomy and multiple sclerosis: ultrastructural study of an experimental model.

Transmission electron microscopy was performed on specimens of the thymus of rats induced for acute experimental allergic encephalomyelitis (EAE). The ultrastructural alterations of the thymus were progressive and correlated with EAE development. The thymic disorganization was due to a progressive degeneration of both epithelial cells and thymocytes. These data suggest a direct involvement of the epithelial thymic cells and thymocytes in EAE pathogenesis and may suggest the intriguing therapeutic concept of thymectomy in the management of multiple sclerosis.

Group B streptococci persist inside macrophages.

Group B streptococci (GBS) are an important cause of neonatal sepsis, pneumonia and meningitis. In the early phase of infection, macrophages and polymorphonuclear cells (PMN) are the first immune cells that interact with GBS. In this in vitro study, to gain insight into GBS-macrophage interaction in the absence of type-specific antibodies, we examined the features of GBS survival in thioglycollate-elicited murine peritoneal macrophages and the effect of GBS on the protein kinase C (PKC)-dependent transduction pathway. Our results demonstrate that type Ia GBS, strain 090 (GBS-Ia) and type III GBS strain COH 31r/s (GBS-III), after in vitro phagocytosis survive and persist intracellularly in macrophages for up to 24 and 48 hr, respectively. However, macrophage activation by interferon-gamma (IFN-gamma) and lipopolysaccharide from Escherichia coli (LPS) caused a significant reduction in the time of intracellular persistence. Macrophage activation by IFN-gamma and LPS seems to be a multifactorial event involving multiple intracellular signal pathways also including PKC. Since PKC is one of the components in the signal network leading to macrophage activation and an important target for several intracellular micro-organisms, we wondered whether PKC could have a role in intracellular GBS survival. Both PKC depletion by treatment with phorbol 12-myristate 13-acetate (PMA) for 18 hr and PKC inhibition by Calphostin C rendered macrophages more permissive for the intracellular GBS survival. Furthermore, GBS-infected macrophages were unable to respond to PMA and LPS, activators of PKC, by inducing antimicrobial activity. The ability of GBS to impair PKC-dependent cell signalling was also demonstrated by the reduced c-fos gene expression in GBS-infected macrophages with respect to control macrophages, after LPS stimulation. In conclusion, our results indicate that GBS survive in macrophages and impairment of PKC signal transduction contributes to their intracellular survival.

Death of bystander cells by a novel pathway involving early mitochondrial damage in human immunodeficiency virus-related lymphadenopathy.

Destruction of immune cells in peripheral lymphoid tissues plays presumably a pivotal role in acquired immune deficiency syndrome pathogenesis. We found that cell suspensions obtained from lymph nodes of eight human immunodeficiency virus (HIV)-infected individuals contained variable proportions (2.1% to 18.3%, median 11.2%) of dead lymphocytes permeable to supravital dyes, represented by CD4+, CD8+, and B cells. The frequency of dead cells correlated directly (R = 0.847) with the amount of HIV provirus in the cell populations, and HIV provirus was enriched in the dead cell fractions. Similar proportions of dead cells were observed in cell suspensions from lymphadenopathic lymph nodes of HIV- donors, but not from small resting HIV- lymph nodes. Electron microscopic and flow cytometric analyses revealed that most dead cells from HIV+ lymph nodes lacked internucleosomal DNA fragmentation but displayed combined features of apoptosis and necrosis, eg, chromatin condensation and mitochondrial swelling. Cells with similar morphology were readily identified in lymph node tissue sections, and marked mitochondrial swelling could be occasionally observed in cells with otherwise normal morphology. Our findings have two major implications. One is that the in vivo cell death in HIV-infected lymph nodes occurs predominantly through a novel pathway, related to but distinct from classical apoptosis and characterised by early and severe mitochondrial damage. The second implication is that HIV-related lymphadenopathy is accompanied in vivo by massive destruction of uninfected lymph node cells. Comparable levels of cell death were observed in other inflammatory lymphadenopathies not related to HIV; however, the uniquely endless and generalized nature of HIV lymphadenopathy might render this "inflammatory" cell destruction a powerful pathogenetic mechanism, accounting for the progressive disruption and depletion of lymphoid tissues seen in HIV infection.

[Prognostic significance of desmoplasia in breast carcinoma. A preliminary clinical study]. / Significato prognostico della desmoplasia nel carcinoma della mammella. Studio clinico preliminare.

This study carried on 61 patients with breast cancer confirmed the biological assumption of a higher malignancy in tumors with strong desmoplastic reaction. Patients in stage II A, II B and III presenting a marked desmoplastic reaction have a shorter DFS (disease free survival). Therefore, desmoplastic reaction may be considered a marker of local malignancy and metastatic process.

[Bone marrow changes in HIV-positive patients (clinical, cytological, histological, and ultrastructural study of 57 cases)]. / Alterazioni osteomidollari in pazienti HIV positivi (studio clinico, citologico, istologico ed ultrastrutturale su 57 casi).

The authors take into consideration clinical, cytological, histological and ultrastructural pattern of 57 HIV+ patients. They want to quantify bone marrow alterations and research their relation with haematological pattern of these patients. They think that peripheral haematological deficit is related with cellular and stromal alterations of the bone marrow. In fact there are many morphological cellular alterations. The most characteristic are that of megakaryocytes. The alterations of these cells are, probably, responsible for bone marrow early sclerosis of these patients. The plasma cells are also numerous and activated. They respect an immunological response.

Aberrant, noninfectious HIV-1 particles are released by chronically infected human T cells transduced with a retroviral vector expressing an interfering HIV-1 variant.

The expression of the nonproducer F12-HIV-1 genome has been previously shown to protect the host cell from HIV superinfection. In order to estimate the efficacy of the F12-HIV genome as an anti-HIV reagent also in cells already infected, an HIV-1 chronically infected Hut-78 cell clone (D10) was superinfected with an amphotropic mouse/human pseudotype retrovirus whose genome expresses both the F12-HIV genome and the selection marker gene (i.e. the c-DNA of a truncated form of the nerve growth factor receptor, NGFr) under the control of F12-HIV 5'LTR. D10 cells homogenously expressing the F12-HIV genome (T-D10) released unaltered amounts of retrovirions whose infectivity was, however, dramatically impaired (from 9 x 10(3) in D10 to < 10(0.5). TCID50/ml in T-D10 supernatants). Electron microscopy showed that the morphology of retrovirions released by T-D10 cells was heavily altered, both in size and shape. Furthermore, no retrotranscription products were detectable in CD4 cells challenged with T-D10 retrovirions. For the first time, the block in the infectivity of HIV released from already infected cells through the expression of an anti-HIV retroviral vector was demonstrated. These data could have important implications both from a perspective of F12-HIV-based anti-HIV gene therapy and, in general, on the role that nonproducer and/or defective HIV could play 'in vivo' in HIV infection and AIDS pathogenesis.

Ultrastructural and immunohistochemical characterization of the tunica albuginea in Peyronie's disease and veno-occlusive dysfunction.

The tunica albuginea and corpus cavernosum from patients with Peyronie's disease (PD), patients with veno-occlusive dysfunction (VOD), and those from normal control subjects were studied by transmission electron microscopy and immunohistochemical staining for type I, III, and V collagens, platelet-derived growth factor (PDGF) AA and BB homodimers, and PDGF alpha and beta receptors. Ultrastructural modifications resembling a fibrotic reaction were detected in the two pathological tunica albuginea, but not in those from control subjects. Ultrastructural data demonstrated a general increase in fibrous and amorphous extracellular matrix material in the pathological tunica albuginea. The amorphous material probably represents glycoproteins and proteoglycans. The fibrous material, representing collagen, appears disorganized in the tissue and does not display the typical and homogeneous diameter, size, and spatial arrangement. Large areas of extracellular and intracytoplasmic, partially degraded, fibers are visible. An increased type I/III collagen ratio was detected by immunohistochemistry in the two pathological tunica albuginea. Moreover, a strong expression of type V collagen, correlated to fibroblasts, was revealed. Fibroblasts from control tissues, on the other hand, were totally negative. Finally, PDGF AA and BB were positive in fibroblasts from pathological tunica albuginea but were negative in control tissues. PDGF beta receptor was positive in pathological and normal tissue fibroblasts. Tunica albuginea from PD and VOD show similar ultrastructural and immunohistochemical alterations, whereas the corpus cavemosum shows no visible modifications.

Role of neutrophils and lymphocytes in inhibition of a mouse mammary adenocarcinoma engineered to release IL-2, IL-4, IL-7, IL-10, IFN-alpha, IFN-gamma, and TNF-alpha.

Impressive inhibition of tumor growth has been observed after transduction of cytokine genes into tumor cells. Secreted cytokines do not affect the proliferation of a tumor directly but activate a host immune reaction strong enough to overcome its oncogenic capacity. However, the reaction mechanisms activated are difficult to interpret; because these mechanisms have been derived from experiments with different tumors, comparisons are hindered. To compare the reactive mechanisms induced by each cytokine, BALB/c mice were challenged with the parental cells of the syngeneic spontaneous mammary adenocarcinoma TSA, or with TSA cells engineered to release IL2, IL4, IL7, IL10, IFN alpha, IFN gamma, and TNF alpha, and the tumor growth area was studied histologically, ultrastructurally, and immunohistochemically. These observations were integrated with data on the growth and rejection patterns of TSA cells in mice depleted of natural killer (NK) cells, granulocytes, CD4+, or CD8+ lymphocytes. The rejection of TSA-IL2 and TSA-TNF alpha cells was associated with the massive presence of neutrophils, that of TSA-IL4 and TSA-IL7 cells with neutrophils and very small areas of colliquative necrosis, and that of TSA-IFN alpha and TSA-IL10 cells with extensive areas of ischemic-coagulative necrosis and some neutrophils. TSA-IFN gamma cells displayed a delay in growth, but were not rejected. Their growth areas comprised necrotic zones of ischemic necrosis devoid of neutrophils. The selective depletion experiments demonstrated that rejection of engineered TSA cells depends on several leukocyte populations. The weight of each population varied with the secreted cytokine, although neutrophils and CD8+ lymphocytes constantly played the major role. Employment of the same tumor line engineered with the genes of different cytokines showed that each cytokine evokes a distinct reaction and that tumor inhibition results from a complex mechanism in which neutrophils and CD8+ lymphocytes and ischemic necrosis are of primary importance.

Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-alpha 1 and herpes simplex thymidine kinase followed by ganciclovir.

Transduction of the murine interferon-alpha (IFN-alpha) gene into various malignant mouse tumor cells has resulted in the loss of tumorigenicity and an acquired capacity to induce long-lasting antitumor immunity following their injection into immunocompetent syngeneic mice. In the present study, we investigated the effectiveness of IFN-alpha-producing tumor cells in the therapy of mice with established mouse tumors. In DBA/2 mice bearing subcutaneous (s.c.) Friend erythroleukemia cell (FLC) tumors, we found that to achieve some antitumor response (i) it was necessary to inject high numbers of IFN-alpha-producing FLC, which occasionally lead to the formation of slowly growing tumors; and, that (ii) repeated injections of irradiated IFN-alpha-FLC did not result in any antitumor effect. The therapeutic potential of IFN-alpha-producing FLC rendered sensitive to ganciclovir (GCV), by transfer of the herpes simplex virus thymidine kinase (tk) gene, was investigated. Complete tumor rejection and cure was observed in > or = 70% of the animals after injection of high numbers (10(7)) of IFN-alpha-producing tk-expressing tumor cells followed 4 days later by repeated GCV treatments, whereas only a slight increase in survival time was obtained after administration of control tk-expressing tumor cells (not producing IFN) and GCV. Tumor rejection was associated with a dramatic destruction of tumor tissue and with the subsequent development of a potent and long-lasting antitumor immunity. No therapeutic effect was observed in immunosuppressed nude mice. These data indicate that this approach may represent an effective and safe therapeutic strategy for antitumor cytokine gene therapy.

Heavy metal toxicity following apical and basolateral exposure in the human intestinal cell line Caco-2.

Caco-2 is a cell line, derived from a human colon carcinoma, that retains the ability to differentiate in culture into absorptive intestinal cells. Caco-2 cells were used to evaluate the toxicity of three heavy metals-the essential trace elements zinc and copper, and the xenobiotic cadmium. The cells were cultivated on permeable filters until differentiated and were then exposed to the metals either from the apical (luminal) or from the basolateral (serosal) side. Toxicity was measured in dose-effect experiments with reference to cell survival and integrity of the cell monolayer. The metals were more toxic when presented to the basolateral than to the apical cell side. The toxicity ranking was cadmium > > copper > zinc. The cell's ability to transport each metal across the monolayer and the resulting intracellular accumulation could account for the cytotoxic effects. A specific toxic effect observed on a specialized function of these cells was the interference of cadmium in tight-junction integrity as shown by changes in the transepithelial electrical resistance, in the rate of transport of a specific marker across the cell monolayer, and by morphological alterations of the tight junctions.

Transduction of genes coding for a histocompatibility (MHC) antigen and for its physiological inducer interferon-gamma in the same cell: efficient MHC expression and inhibition of tumor and metastasis growth.

The mouse mammary carcinoma TS/A, of BALB/c (H-2d) origin, was transfected with the murine interferon-gamma (IFN-gamma) gene (Int. J. Cancer 55: 320, 1993). We used IFN-gamma transfectants as recipients for a second round of transfections with murine allogeneic class I histocompatibility (H-2b) genes that are modulated by IFN. Transfectants with either gene alone, as well as parent TS/A cells (TS/A-pc), were used as controls. Only double transfectants expressed high levels of the allogeneic H-2b genes, while in H-2b single transfectants the expression was very low (but was induced by treatment with exogenous IFN-gamma). The tumorigenic potential of IFN-gamma or H-2b single transfectants was reduced in comparison to TS/A-pc. IFN-gamma+H-2Kb double transfectants were almost nontumorigenic, while IFN-gamma+H-2Db clones gave rise to tumors in about one-half of mice. The experimental metastatic ability of all IFN-gamma+H-2b double transfectants was very low. IFN-gamma single transfectants were known to induce a strong macrophage response in the host. The expression of allogeneic H-2 antigens added a T-lymphocyte-mediated response that accounted for the lower tumorigenicity of double transfectants. These results show that it is possible to steer the immune response evoked by tumor cells for therapeutic purposes. Moreover, the high H-2 expression obtained in IFN-gamma+H-2b double transfectants suggests that single IFN-gamma transfectants are ideal recipients for all IFN-sensitive genes. This approach can be used also for other general-purpose inducers of gene expression.

[The biological effects of the CO2 laser studied in experimental intestinal resections]. / Studio degli effetti biologici del laser CO2 nelle resezioni intestinali sperimentali.

An histological and E.M. study has been performed on rat's intestinal tract resection to evaluate CO2 laser effects. Necrotic effect of laser is evident up to 120 micron. While the intestinal tissue results normal at 480 micron from resection line. Muscular and fibrous components appear more resistant to damage while E.M. presents a new organization of its components forming a new compact and continue tissue. This new organization may be involved in the protection of the subepithelial spaces, and may have an important role in intestinal anastomosis scar process.

IFN-alpha 1 gene expression into a metastatic murine adenocarcinoma (TS/A) results in CD8+ T cell-mediated tumor rejection and development of antitumor immunity. Comparative studies with IFN-gamma-producing TS/A cells.

Cells from a spontaneous, invasive, and metastasizing mouse mammary adenocarcinoma (TS/A-pc) were transfected with a retroviral vector containing the mouse IFN-alpha 1 gene. TS/A clones secreting varying amounts of IFN-alpha 1 were isolated and their tumorigenicity was evaluated after s.c. or i.v. injection into immunocompetent BALB/c mice. Almost all of the IFN-alpha-secreting TS/A clones failed to grow in a high percentage of mice or formed small tumors after a long latency time, whereas TS/A-pc or transfection control cells always grew into large s.c. tumors. Rejection was mainly mediated by CD8+ T lymphocytes and partially by polymorphonuclear cells, as demonstrated by selective immunosuppression experiments and histologic and ultrastructural data. After rejection, a significant portion of mice displayed an immune resistance to the subsequent challenge with TS/A-pc. When the metastatic ability of IFN-alpha-secreting clones was compared with that of previously characterized IFN-gamma-secreting TS/A clones, it was found that the expression of IFN-alpha into TS/A tumor cells resulted in a potent inhibition of metastases formation, whereas IFN-gamma expression either did not affect or even enhanced the metastatic behavior of TS/A cells. These results provide strong evidence for the usefulness of IFN-alpha-producing tumor cells for the development of gene therapy strategies and vaccines against metastatic tumors.

Defective response to T cell mitogens in mice injected with human immunodeficiency virus type 1-infected U937 cells.

Swiss mice were injected intraperitoneally with uninfected or human immunodeficiency virus type 1 (HIV-1) infected human U937 cells. At 6 days, no residual human cells were detected in mouse tissues as determined by PCR analysis of DNAs from injected mice using primers and probes for the human HLA-DQ alpha gene. At 6 to 12 months, approximately 60% of the HIV-1-infected mice had antibodies to HIV-1 gp 120 and gp41 proteins. Fifteen percent of the animals showed evidence of HIV-1 infection as determined by PCR analyses of DNA from peripheral blood leukocytes and by in situ hybridization for detection of HIV-1 mRNA in peritoneal cells. In this set of experiments, spleen cells from mice sacrificed at different times after injection were cultured for 48 h in the presence or absence of mitogens [i.e.: concanavalin (Con A) or anti-CD3 antibody] and then tested for lymphocyte proliferation. At 10 to 12 months, splenocytes from approximately 80% of Swiss mice injected with HIV-1-infected U937 cells exhibited a marked defect in their proliferative response to Con A or anti-CD3 antibody as compared with spleen cells from both uninjected or U937 cell-injected mice. Similar results were obtained at 12 months in C3H/HeJ mice. Non-responding spleen cells from HIV-1-injected Swiss mice did not proliferate in response to anti-CD3 antibody even in the presence of co-stimulatory molecules such as phorbol myristate acetate or anti-CD28 antibody. Splenocytes from these mice also exhibited an impaired capacity to produce interferon-gamma and interleukin-4 after mitogen stimulation. No T cell defects were observed in control-injected mice. Immunofluorescence analyses revealed a significant decrease in the percentage of both CD4+ and CD8+ spleen cells in HIV-1-injected mice. These data indicate that immunocompetent mice can be used to investigate some HIV-1-related immune dysfunctions in vivo.

[A histological and ultrastructural study of the effects of the argon laser scalpel on the kidney]. / Studio istologico ed ultrastrutturale degli effetti del bisturi laser ad argon sul rene.

The authors carried an histological and ultrastructural study on rat kidney following polar excision by argon laser. The involvement of the remainder portion of the kidney was evaluated analysing the depth of the damage induced by the laser radiation. Restitution ad integrum was found below 360 micron thickness with a different involvement of the various kidney components. Glomeruli showed a deep damage caused by intravascular photocoagulation. On the contrary, tubular and extracellular damage was less serious and the damaged area extended for only 240 micron from the section, probably for a lack of absorbance of the argon radiation by clear and water-rich tissues.