Biomedical subjects

A Fontana

Publications and source records attributed to A Fontana.

At least 19 recordsLinked to original sources

PubMed1995

T cell apoptosis induced by interleukin-2 deprivation or transforming growth factor-beta 2: modulation by the phosphatase inhibitors okadaic acid and calyculin A.

The phosphatase inhibitors okadaic acid and calyculin A were used to examine the role of phosphorylation processes in T cell apoptosis induced by interleukin-2 (IL-2) deprivation or transforming growth factor-beta 2 (TGF-beta 2). Okadaic acid and calyculin A inhibited IL-2-driven T cell proliferation and induced apoptosis at concentrations known to inhibit protein phosphatase 1. High concentrations of both agents caused toxic changes of prominent cellular swelling and dilatation of rough endoplasmic reticular profiles. When the T cells were induced to undergo apoptosis by IL-2 deprivation, okadaic acid and calyculin A delayed loss of membrane integrity, nucleosomal size DNA fragmentation, and loss of bcl-2 mRNA. However, T cells deprived of IL-2 in the presence of okadaic acid or calyculin A revealed DNA breaks by in situ DNA end labeling and apoptotic morphology by electron microscopy and failed to show enhanced survival after reexposure to IL-2. Although TGF-beta-mediated signaling is thought to involve the dephosphorylation of specific substrates, okadaic acid and calyculin A not only failed to inhibit, but actually augmented, TGF-beta 2-induced inhibition of T cell proliferation and induction of apoptosis. Exposure to either TGF-beta 2 or the phosphatase inhibitors prevented phosphorylation of the retinoblastoma protein RB. In summary, okadaic acid and calyculin A: (i) induce T cell apoptosis in the presence of IL-2, (ii) allow us to distinguish essential from epiphenomenal features of T cell apoptosis after IL-2 deprivation, and (iii) cooperate with TGF-beta 2 in inducing growth arrest and apoptosis of murine T cells via intracellular cascades that converge in the prevention of RB phosphorylation.

Animals↗

PubMed1993

Oral challenge with alternative nonsteroidal antiinflammatory drugs (NSAIDs) and paracetamol in patients intolerant to these agents.

The reliability and safety of the oral challenge procedure were evaluated in the diagnosis and prevention of intolerance to nonsteroidal anti-inflammatory drugs (NSAIDs). 112 NSAID-intolerant patients were submitted to oral challenge with aspirin (acetylsalicylic acid), dipyrone, paracetamol, imidazole-hydroxybenzoate or nimesulide to confirm historic intolerance and to evaluate tolerance to other NSAIDs. A significant correlation was demonstrated between history of intolerance and the results of oral challenge in aspirin-intolerant patients (p < 0.001). Of 237 challenges with various NSAIDs and paracetamol in 101 patients, 19 challenges were positive and 2 patients developed anaphylactic shock. The ratio of positive: total challenges with alternative NSAIDs and paracetamol were as follows: 7 of 83 for paracetamol, 2 of 49 for imidazole-hydroxybenzoate and 0 of 30 for nimesulide. On the basis of these results, nimesulide was evaluated as an alternative NSAID in a second group of 284 NSAID-intolerant patients. Challenge with nimesulide elicited a positive response in 14 patients (in 6 patients the response was delayed), although adverse reactions were mild in all instances. In conclusion, a positive history of intolerance to a given NSAID is a sufficient reason to contraindicate its use even for diagnostic purposes, e.g. oral challenge. Oral challenge should therefore be restricted to the assessment of the tolerability of alternative NSAIDs. In the present study nimesulide appeared to be the safest currently available alternative in patients with NSAID intolerance.

Acetaminophen↗

PubMed1992

Human small cell lung cancer expresses the octamer DNA-binding and nervous system-specific transcription factor N-Oct 3 (brain-2).

Small cell lung cancer (SCLC) cells express several characteristics of neuronal cells, including synthesis of neuropeptides and expression of the respective receptors. Establishment and maintenance of the neuronal phenotype of SCLC may depend on expression of gene transcription factors inherent to the central nervous system. The present study shows the nervous system-specific transcription factor N-Oct 3 (brain-2) to be expressed in all 13 SCLC cell lines investigated. Furthermore, N-Oct 3 (brain-2) was also found in SCLC-derived skin metastasis. In contrast, in extracts and RNA of non-SCLC cell lines and non-SCLC tumor tissues, such as lung squamous, large cell, and adenocarcinoma, expression of N-Oct 3 (brain-2) was not detectable. These data support the concept that SCLC cells derive from the neuroectodermal cell lineage since expression of N-Oct 3 (brain-2) protein is highly abundant at the neural tube stage and in the adult restricted to the neuroectodermal cell lineage.

Carcinoma, Small Cell↗

PubMed1992

Interleukin-8 is produced in neoplastic and infectious diseases of the human central nervous system.

The presence of interleukin-8 (IL-8), a leukocyte chemotactic factor, was examined in primary and metastatic central nervous system tumors and in nonneoplastic acute meningoencephalitides. In vitro: (a) 11 of 12 glioblastoma cell lines constitutively expressed IL-8 mRNA; (b) 5 of 6 of these cell lines secreted IL-8 protein as detected by enzyme-linked immunosorbent assay and a glucosaminidase release bioassay; and (c) IL-1 beta or tumor necrosis factor was able to augment both IL-8 mRNA steady state levels and protein secretion of all cell lines tested except IN-319. IL-8 was also found in vivo. (a) IL-8 poly A+ mRNA was detected in 2 of 2 low grade astrocytomas, 1 of 2 anaplastic astrocytomas, and 6 of 6 glioblastomas. (b) IL-8 protein was present in the cyst fluid of 1 of 4 low grade astrocytomas, 1 anaplastic astrocytoma, 2 of 2 glioblastomas, 1 oligodendroglioma grade III, and one central nervous system cervical carcinoma metastasis. (c) The cerebrospinal fluid of 3 of 4 metastatic lymphomas, 2 of 16 glioblastomas, 1 of 2 low grade astrocytomas, but none of 3 anaplastic astrocytomas and none of 9 meningiomas contained IL-8. The presence of IL-8 was not restricted to central nervous system tumors as 2 of 2 bacterial meningitis and 5 of 5 acute viral meningitis patients contained considerable IL-8 levels in the cerebrospinal fluid. (d) Immunohistochemical analysis showed IL-8 immunoreactivity in perivascular tumor cells in 11 of 15 glioblastoma sections. These data suggest that IL-8 secretion could be a key factor involved in the determination of the lymphoid infiltrates observed in brain tumors and the development of cerebrospinal fluid pleocytosis in meningoencephalitides.

Astrocytoma↗

PubMed1992

Transforming growth factor beta 2 inhibits cerebrovascular changes and brain edema formation in the tumor necrosis factor alpha-independent early phase of experimental pneumococcal meningitis.

Macrophages and granulocytes seem to play a key role in the pathogenesis of bacterial meningitis. Transforming growth factor beta (TGF-beta) leads to macrophage deactivation, as well as to inhibition of cytokine production and of endothelial granulocyte adhesion. We have investigated the influence of TGF-beta on regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain edema formation during the early phase of experimental meningitis. Rats which were inoculated intracisternally with live pneumococci or with pneumococcal cell wall hydrolyzed by the M1 muramidase (PCW-M) developed an increase of rCBF and ICP within 4 h postintracisternal challenge. A single intraperitoneal injection of TGF-beta 2 but not of TGF-beta 2 vehicle-control prevented the changes of rCBF. Furthermore, TGF-beta 2 significantly reduced the increase of ICP in rats inoculated with PCW-M. Likewise, the elevation of brain water content after intracisternal injection of pneumococci or PCW-M was blocked by pretreatment of rats with TGF-beta 2. TGF-beta 1 exhibited similar inhibitory effects in PCW-M-injected rats. The beneficial effects of TGF-beta 2 on the initial phase after pneumococcal inoculation seem to be tumor necrosis factor alpha- (TNF-alpha) independent since (a) intracisternal or intraperitoneal injection of neutralizing anti-TNF-alpha antibodies did not significantly influence rCBF, ICP, and brain water content in PCW-M-induced meningitis; and (b) TNF-alpha was only occasionally detected at low levels in cerebrospinal fluid at 4 h after PCW-M application.

Animals↗

PubMed1992

Granulocyte-macrophage colony-stimulating factor (GM-CSF) production by glioblastoma cells. Despite the presence of inducing signals GM-CSF is not expressed in vivo.

One of the morphologic hallmarks of human gliomas are inflammatory infiltrates with accumulation of macrophages in the tumor site. The signals leading to the macrophage response are only at the beginning of being understood. Novel chemotactic factors that have recently been characterized as secretory products of glioblastoma cells may attract mononuclear cells from the blood. Within the tumor tissue blood-derived monocytes and macrophages of the brain tissue, the microglial cells, may increase in cell numbers due to tumor-derived growth factors. Both astrocytoma cell lines and cultured astrocytes have been shown recently to produce granulocyte-macrophage (GM)-CSF. We show that in vitro not only astrocytoma but also glioblastoma cell lines secrete GM-CSF when stimulated with TNF-alpha or IL-1. However, there is no evidence for GM-CSF production by glioblastoma cells in vivo: fresh tumor samples lack the mRNA for GM-CSF and the protein is not detectable in the tumor cyst fluids or the cerebrospinal fluids of glioblastoma patients. This contrasts IL-1 and IL-6 that are detectable in the tumor cyst fluids and IL-6 also in the cerebrospinal fluids of the patients. Unlike GM-CSF, transforming growth factor-beta 2 mRNA is expressed in ex vivo tested glioblastoma tissues. Absence of GM-CSF in vivo may be explained by the presence of tumor-derived inhibitory factors, such as transforming growth factor-beta 2 and PGE which suppress GM-CSF production by glioblastoma cells in vitro. The accumulation of macrophages at the tumor site may be due to local elaboration of chemoattractants and/or not yet defined growth factors rather than due to GM-CSF production.

Chemotactic Factors↗

PubMed1992

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.

Animals↗

PubMed1992

Protease inhibitors interfere with the transforming growth factor-beta-dependent but not the transforming growth factor-beta-independent pathway of tumor cell-mediated immunosuppression.

Tumor cells have been reported to exert inhibitory effects on the activation of T lymphocytes in vitro. We show that the IL-2-stimulated proliferation of a Th cell line is suppressed when the T cells are cocultured with human glioblastoma and melanoma cell lines. The use of two Th cell clones that differ in their responsiveness to growth-inhibition by transforming growth factor-beta (TGF-beta) and the analysis of tumor cell-derived supernatants as well as of TGF-beta 1/TGF-beta 2 gene expression allowed to distinguish two pathways of tumor-induced immunosuppression. Glioblastoma cells exert their immunosuppressive effects by producing biologically active TGF-beta 2, whereas the immunosuppressive state induced by melanoma cells is TGF-beta-independent and requires direct contact between tumor cell and T cell. The TGF-beta-dependent immunosuppression is down-regulated by various protease inhibitors and up-regulated by estradiol via modulation of the production of biologically active TGF-beta 2 by glioblastoma cells leaving total activatable TGF-beta 2 unaffected. No such modulation is functional for the TGF-beta-independent pathway of immunosuppression. We conclude that the production of active TGF-beta by tumor cells is regulated at a posttranslational level by the coordinated action of several proteolytic enzymes.

Animals↗

PubMed1992

Macrophage-induced cytotoxicity of N-methyl-D-aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines.

The co-localization of activated macrophages and damaged neurons observed in brain injury and degenerative brain diseases may hint to macrophage-induced neuronal cytotoxicity. Recently, macrophages have been found to secrete neurotoxic molecules such as radical oxygen intermediates and glutamate, the latter interacting with N-methyl-D-aspartate (NMDA) receptors. As shown in the present study, brain macrophages termed microglial cells co-cultured with differentiated cerebellar neurons excert potent neurotoxic effects. Neurotoxicity is unlikely to be due to cytokines since tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6 and interferon (IFN)-alpha/IFN-beta/IFN-gamma had no such effects. In contrast, when treating neurons with H2O2 or oxygen radical-generating systems cytotoxicity was induced. Furthermore, microglia were found to produce O2- and H2O2 when triggered with phorbol 12-myristate 13-acetate. However, in co-cultures of neurons and microglia, oxygen-radical scavengers catalase and superoxide dismutase, failed to protect neurons from microglia-induced killing. Moreover, when using undifferentiated neurons which are susceptible to H2O2 but not to NMDA receptor-dependent killing, microglia did not destroy the neurons. Thus, the amount of reactive oxygen intermediates produced by microglia in co-culture do not reach the critical concentrations required for neurotoxicity. As dibenzocyclohepteneimide, an antagonist to NMDA receptors neutralized neurotoxicity in microglia-neuronal co-cultures, excitatory amino acids released by microglia are suggested to compose the major determinant of neurotoxicity.

Animals↗

PubMed1992

Neurons and neuroblastoma as a source of macrophage colony-stimulating factor.

Accumulation of macrophages in brain tissue as observed in nervous system injury may be due to local production of hematopoietic colony-stimulating factors (CSF). The present work shows human neuroblastoma cells and murine neurons, namely granule cells of the cerebellum, to produce macrophage (M)-CSF which guides expansion and differentiation of macrophage lineage cells. The mRNA-encoding M-CSF but not the respective protein is present in mouse brain including cerebellum. Neither granulocyte M-CSF nor IL-3 is produced by cerebellar neurons or neuroblastoma. By their production of M-CSF, neurons may regulate the macrophage response and lead to local expansion and enhanced function of macrophages in inflammatory diseases of the central nervous system.

Animals↗

PubMed1992

Production of macrophage colony-stimulating factor by astrocytes and brain macrophages.

Morphological hallmarks of inflammatory and degenerative diseases of the brain are hypertrophy of astrocytes and accumulation of macrophages recruited from circulating blood monocytes and/or from resident macrophages, the so-called microglial cells. Recently, production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by astrocytes has been suggested to contribute to the macrophage response. Here we report that in addition to GM-CSF, murine astrocytes also produce macrophage (M)-CSF upon stimulation with tumor necrosis factor alpha, interleukin-1 and lipopolysaccharides. The bioactivity detected in supernatant of astrocytes was characterized using the M-CSF-dependent cell line M-NFS-60 and neutralizing anti-M-CSF antibodies. RNase protection analysis showed M-CSF mRNA already in unstimulated astrocytes without striking up-regulation by the stimuli. Thus, in astrocytes the expression of the M-CSF gene is predominantly regulated at the posttranscriptional level.

Animals↗

PubMed1992

[Isolation and characterization of a flocculating mutant of Saccharomyces diastaticus].

A flocculating strain of Saccharomyces diastaticus NRRL2416 was isolated after ethidium bromide mutagenesis and density gradient separation. Flocculation did not change the general characteristics of the strain. Flocculation was better on maltodextrin-containing media than on others. It was found to be calcium-dependent and sensitive to growth conditions. The deflocculating effects of some sugars suggested that mannose may be involved in the mechanism of flocculation, as for strains of the FLO1 phenotypic group.

Culture Media↗

PubMed1992

Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease.

Oligonucleotide-directed mutagenesis has been used to replace glycine residues by alanine in neutral protease from Bacillus subtilis. One Gly to Ala substitution (G147A) was located in a helical region of the protein, while the other (G189A) was in a loop. The effects of mutational substitutions on the functional, conformational and stability properties of the enzyme have been investigated using enzymatic assays and spectroscopic measurements. Single substitutions of both Gly147 and Gly189 with Ala residues affect the enzyme kinetic properties using synthetic peptides as substrates. When Gly replacements were concurrently introduced at both positions, the kinetic characteristics of the double mutant were roughly intermediate between those of the two single mutants, and similar to those of the wild-type protease. Both mutants G147A and G189A were found to be more stable towards irreversible thermal inactivation/unfolding than the wild-type species. Moreover, the stabilizing effect of the Gly to Ala substitution was roughly additive in the double mutant G147A/G189A, which shows a 3.2 degrees C increase in Tm with respect to the wild-type protein. These findings indicate that the Gly to Ala substitution can be used as a strategy to stabilize globular proteins. The possible mechanisms of protein stabilization are also discussed.

Alanine↗

PubMed1992

War zone traumas and posttraumatic stress disorder symptomatology.

The diagnosis and clinical understanding of posttraumatic stress disorder (PTSD) rests upon the explicit identification of traumatic experiences that give rise to a well-defined constellation of symptoms. Most efforts to investigate the characteristics of these experiences have attempted to specify war zone stressors as objectively as possible. In this study, we add specification of the psychological meaning of war zone stressors to their objective specification. Eleven traumas are organized in terms of four roles that veterans played in the initiation of death and injury; namely, target, observer, agent, and failure. These roles can be ordered in terms of the degree of personal responsibility involved in the initiation of death and injury. The relationships of these roles to current symptomatology were examined in combination with a set of objective measures of war zone stressors. The sample consisted of the first 1709 Vietnam theater veterans who were assessed in a national evaluation of the PTSD Clinical Teams initiative of the Department of Veterans Affairs. Results show that having been a target of others' attempts to kill or injure is related more uniquely than any other role to symptoms that are diagnostic criteria for PTSD. On the other hand, having been an agent of killing and having been a failure at preventing death and injury are related more strongly than other roles to general psychiatric distress and suicide attempts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

BAL modifications after antiblastic and immunomodulant therapy in lung cancer.

The bronchoalveolar compartment can be easily investigated with BAL (bronchoalveolar lavage) before and after antiblastic therapy. We studied 50 patients affected by primary lung cancer, of whom 31 served as a control group and 19 were submitted to BAL after chemo- and/or radiotherapy. Data from BAL performed in an unaffected lung area show that antiblastic therapy can produce alterations in the terminal airways without clinical evidence. Chemotherapy causes a significant impairment of the alveolo-capillary barrier. Radiotherapy is able to affect lymphocytes, with a CD4/CD8 reduction. The concomitance of both therapies produces synergistic effects. Immunomodulant therapy with thymostimulin in otherwise untreated lung cancer patients seems able to modify alveolar lymphocyte number and subsets, but these are preliminary data which need further substantiation.

Bronchoalveolar Lavage Fluid↗

PubMed1992

Modulation of the immune response by transforming growth factor beta.

For the past several years immunologists have been fascinated by a series of experiments showing that transforming growth factor beta (TGF beta) suppresses T- and B-lymphocyte growth as well as IgM and IgG production by B cells. Moreover, while exerting chemotactic activity on monocytes and inducing expression of interleukin-1 and interleukin-6 by these cells, TGF beta interferes with bacterially induced tumor necrosis factor alpha production, oxygen radical formation and the adhesiveness of granulocytes to endothelial cells. These mechanisms may provide the basis for the effect of TGF beta to prevent the microvascular changes associated with brain edema formation in bacterial meningitis. Given the potential of lymphocytes as well as macrophages to produce TGF beta 1, this cytokine may exert negative feedback signals on the immune response, provided the cytokine is processed from its latent form to the bioactive homodimer. Potent effects of TGF beta have been observed in experimental animals including the inhibition of the generation of virus-specific cytotoxic T cells and antiviral antibodies as well as the diminution of cellular infiltrates with decreased major histocompatibility complex class-II expression and CD8+ T cells in the tissue of virally infected animals. TGF beta may also be of importance in tumor immunology. By the production of bioactive TGF beta as detected in glioblastoma and acute T-cell leukemia, tumor cells may induce an immunodeficiency state and escape immune surveillance. In inflammation, monitoring of TGF beta in the tissue will bring light on the immune regulation in acute and chronic inflammatory diseases.

Animals↗

PubMed1992

Dissociation and posttraumatic stress disorder in Vietnam combat veterans.

OBJECTIVE: This study compared current dissociative symptoms and dissociation at the time of specific traumatic events in Vietnam combat veterans with posttraumatic stress disorder (PTSD) and Vietnam combat veterans without PTSD. METHOD: Vietnam combat veterans who sought treatment for PTSD (N = 53) were compared to Vietnam combat veterans without PTSD (N = 32) who sought treatment for medical problems. Dissociative symptoms were evaluated with the Dissociative Experiences Scale. Dissociation at the time of a combat-related traumatic event was evaluated retrospectively with the modified Dissociative Experiences Questionnaire. The Combat Exposure Scale was used to measure level of combat exposure. RESULTS: There was a significantly higher level of dissociative symptoms, as measured by the Dissociative Experiences Scale, in patients with PTSD (mean = 27.0, SD = 18.0) than in patients without PTSD (mean = 13.7, SD = 16.0). This difference persisted when the difference in level of combat exposure was controlled with analysis of covariance. PTSD patients also reported more dissociative symptoms at the time of combat trauma, as measured retrospectively by the Dissociative Experiences Questionnaire (mean = 11.5, SD = 1.6) than non-PTSD patients (mean = 1.8, SD = 2.1). CONCLUSIONS: Dissociative symptoms are an important element of the long-term psychopathological response to trauma.

Adult↗

PubMed1992

In vitro production of HIV-1 specific antibodies from "at risk" seronegative subjects.

We investigated a group of well characterized seronegative subjects "at risk" for HIV-1 infection including heterosexual partners of HIV-1 infected subjects and intravenous drug abusers. Pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMCs) were studied for their ability to produce antibodies against HIV-1 structural proteins in vitro. Viral activity by means of HIV-1 isolation from PBMCs and presence of serum p24 antigen were also tested. In 7/42 cases (16.6%) HIV-1 immunoreactive specific antibodies were found, mostly directed against the envelope proteins (gp 120/160). Remarkably, none of these in vitro antibody producers yielded HIV-1 isolation in cell cultures or had detectable serum levels of p24 antigen.

Blotting, Western↗