Polyethylenimine-mediated in vivo gene transfer of a transmembrane superantigen fusion construct inhibits B16 murine melanoma growth.

Immunotherapy has been proposed as a therapeutic strategy in advanced-stage melanomas in which other therapeutic options have little effect. The Staphylococcus enterotoxin A (SEA) has been used to stimulate an antitumoral immune response but its use is hampered by severe systemic side effects. Here, we show that SEA can be targeted to melanoma cells to limit these side effects. More specifically, we used a nonviral vector, the cationic polymer, polyethylenimine (PEI), to express a transmembrane SEA fusion construct (pSEA-TM) in B16F10-induced subcutaneous melanoma in mice. The efficacy of this in vivo transfection was enhanced by concomitant infusion of epinephrine to induce local vasoconstriction. In these conditions, repeated injections of pSEA-TM/PEI complexes elicited a significant response, as evidenced by tumor growth inhibition, without systemic adverse effects. T cell infiltration of the tumors, together with positive lymphocyte proliferation tests, suggested local and systemic immune responses. Altogether, PEI-mediated targeting of SEA to melanoma tumor cells in vivo efficiently stimulates the antitumor immune response without inducing the side effects observed with systemic administration of SEA.

Attractor selection in chaotic dynamics.

For different settings of a control parameter, a chaotic system can go from a region with two separate stable attractors (generalized bistability) to a crisis where a chaotic attractor expands, colliding with an unstable orbit. In the bistable regime jumps between independent attractors are mediated by external perturbations; above the crisis, the dynamics includes visits to regions formerly belonging to the unstable orbits and this appears as random bursts of amplitude jumps. We introduce a control method which suppresses the jumps in both cases by filtering the specific frequency content of one of the two dynamical objects. The method is tested both in a model and in a real experiment with a CO2 laser.

Childhood psychopathological antecedents in early onset schizophrenia.

OBJECTIVE: To describe the premorbid state of early onset schizophrenia (EOS). METHODS: Twenty-three adolescents with EOS were compared to a healthy control group (CG) and to a group of anorexic patients (AG). The premorbid state was studied through the CBCL and the data obtained were analyzed using ANOVA's and t-test. RESULTS: During the premorbid period EOS showed significantly higher scores on all scales, relative to the CG, and only on some scales (social, thought and attention problems, and school competencies) relative to the AG. CONCLUSIONS: Children who develop first episode psychosis during adolescence differ from children with normal development. The premorbid internalizing state is common to AG but social competencies and school problems are the most affected areas in EOS when compared to the AG. It is hypothesized that both EOS and AG can be considered as the expression of a previous vulnerability.

Dopamine agonists and analogues have an antiproliferative effect on CHO-K1 cells.

Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since nearly all parkinsonian patients with clinical impairment are treated with L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA)ergic agonists, a possibility exists that these therapeutic agents can influence the risk of cancer. We studied the antiproliferative effect of these therapeutic agents (and substances structurally correlated) on Chinese hamster ovary (CHO)-K1 cell growth. Among the compounds tested, apomorphine proved to be the most potent inhibitor of CHO-K1 cell growth, with an EC(50) of 3.35 +/- 0.12 micro M. The apomorphine analogues, apocodeine and hydroxyethylnorapomorphine, were less active as inhibitors of CHO-K1 cell growth. The activity of DA, 6-hydroxydopamine (6-OHDA), phenylethylamine (PEA), L-DOPA and bromocriptine as antiproliferative was one order of magnitude lower than that of apomorphine while pergolide was ineffective. To test whether or not the oxidative potential of these compounds was important for their antiproliferative effect, several antioxidants were assayed. Among them glutathione (GSH) and dithiothreitol (DTT) were effective in reversing the anti-proliferative effect of apomorphine, DA, 6-OHDA and PEA, conversely they did not work with bromocriptine. GSH and DTT are sulphydryl-reducing agents; while their effect could explain the efficacy against apomorphine, DA and 6-OHDA, it is difficult to understand why they should have any effect on PEA as this substance does not react with sulphydryl groups. The oxidative potential as a mechanism of action was also questioned by the results obtained with dihydrorhodamine 123, a probe that changes its fluorescent emission wave when oxidized. None of the compounds, with the exception of 6-OHDA, had any effect on the fluorescent emission wave of the probe at the maximal concentrations used to inhibit CHO-K1 cell growth. At concentrations five times higher, apomorphine and DA generated reactive oxygen species but PEA and bromocriptine did not. These data demonstrate that the antiproliferative effect of these compounds is not due to their oxidative potential, but another mechanism must be postulated.

G protein-linked receptors: pharmacological evidence for the formation of heterodimers.

By means of the expression of two chimeric receptors, alpha(2)/M(3) and M(3)/alpha(2), in which the carboxy-terminal receptor portions, containing transmembrane domains VI and VII, were exchanged between the alpha(2C)-adrenergic and the M(3) muscarinic receptor, it has been shown that G protein-coupled receptors are able to interact functionally with each other at the molecular level to form (hetero)dimers. In the present study, we tested the hypothesis that interaction between two different muscarinic receptor subtypes can lead to the formation of a heterodimeric muscarinic receptor with a new pharmacological profile. Initially, muscarinic M(2) or M(3) wild-type receptors were expressed together with gene fragments originating from M(3) or M(2) receptors, respectively. Antagonist binding, performed with pirenzepine and tripitramine, revealed the presence of two populations of binding sites: one represents the wild-type M(2) or M(3) receptors, the other the heterodimeric M(2)/M(3) receptor. In another set of experiments, we constructed a point mutant M(2) receptor M(2) (Asn404-->Ser), in which asparagine 404 was replaced by serine. Although this receptor alone did not show any binding for N-[(3)H]methylscopolamine (up to 2 nM), when cotransfected with M(3), it resulted in the rescue of a high-affinity binding for tripitramine. These findings demonstrate that M(2) and M(3) muscarinic receptor subtypes can cross-interact with each other and form a new pharmacological heterodimeric receptor.

Apomorphine has a potent antiproliferative effect on Chinese hamster ovary cells.

Apomorphine is a potent non selective agonist at the D1 and D2 dopamine receptors acting both pre- and post-synaptically. In this report we describe a novel function of apomorphine, independent from its dopaminergic activity. Apomorphine inhibits Chinese hamster ovary (CHO)-K1 cell proliferation in a dose-dependent manner. The EC50 of apomorphine-induced inhibition of CHO-K1 cell proliferation determined by cell counting was 3.24 +/- 0.07 microM. Remarkably, the dose-response curve obtained by measuring the incorporation of [3H]thymidine was practically identical to the previous one giving an EC50 of 3.52 +/- 0.04 microM. The dopaminergic antagonists SCH23390 and spiperone at a concentration of 10 microM (well beyond their Kd values for the dopamine D1- and D2-like receptors respectively) were not able to antagonize the effect of apomorphine on CHO-K1 cell proliferation. Apomorphine exerts its effect early during incubation; CHO-K1 cells exposed to apomorphine for a period as short as 1 h and then allowed to grow for three days were significantly reduced in number with respect to untreated control cells. After four hours of exposition to apomorphine (10 microM) the antiproliferative effect was similar to that seen when this compound was present in the bath for all three days. Concentrations of apomorphine higher than 10 microM induced cell death, and the colony was completely destroyed at 50 microM. Cytometric analyses showed a significant accumulation of CHO-K1 cells in the G2/M phase.

Structure of MHC class I and class II cDNAs and possible immunodeficiency linked to class II expression in the Mexican axolotl.

Despite the fact that the axolotl (Ambystoma spp. a urodele amphibian) displays a large T-cell repertoire and a reasonable B-cell repertoire, its humoral immune response is slow (60 days), non-anamnestic, with a unique IgM class. The cytotoxic immune response is slow as well (21 days) with poor mixed lymphocyte reaction stimulation. Therefore, this amphibian can be considered as immunodeficient. The reason for this subdued immune response could be an altered antigenic presentation by major histocompatibility complex (MHC) molecules. This article summarizes our work on axolotl MHC genes. Class I genes have been characterized and the cDNA sequences show a good conservation of non-polymorphic peptide binding positions of the alpha chain as well as a high diversity of the variable amino acids positions, suggesting that axolotl class I molecules can present numerous antigenic epitopes. Moreover, class I genes are ubiquitously transcribed at the time of hatching. These class I genes also present an important polylocism and belong to the same linkage group as the class II B gene; they can be reasonably considered as classical class Ia genes. However, only one class II B gene has been characterized so far by Southern blot analysis. As in higher vertebrates, this gene is transcribed in lymphoid organs when they start to be functional. The sequence analysis shows that the peptide binding region of this class II beta chain is relatively well conserved, but most of all does not present any variability in the beta 1 domain in inbred as well as in wild axolotls, presuming a limited antigenic presentation of few antigenic epitopes. The immunodeficiency of the axolotl could then be explained by an altered class II presentation of antigenic peptides, putting into question the existence of cellular co-operation in this lower vertebrate. It will be interesting to analyze the situation in other urodele species and to determine whether our observations in axolotl represent a normal feature in urodele amphibians. But already two different models in amphibians, Xenopus and axolotl, must be considered in our search for understanding immune system and MHC evolution.

Activation by mitogens and superantigens of axolotl lymphocytes: functional characterization and ontogenic study.

Urodele amphibians have weak and slow immune responses compared to mammals and anuran amphibians. Using new culture conditions, we tested the ability of lymphocytes of a well-studied salamander, the Mexican axolotl (Ambystoma mexicanum) to proliferate in vitro with diverse mitogenic agents. We demonstrated that the axolotl has a population of B lymphocytes that proliferate specifically and with a high stimulation index to the lipopolysaccharide (LPS) known as a B-cell mitogen in mammals. This proliferative capacity is observed without significant changes throughout ontogenesis. In the presence of LPS, axolotl B lymphocytes are able to synthesize and secrete both isotopes of immunoglobulin described in this species, IgM and IgY. Moreover, a distinct lymphocyte subpopulation is able to poliferate significantly in response to the mitogens usually known as T-cell specific in mammals, phytohaemagglutinin (PHA) and concanavalin A (Con A). The activated cells are T lymphocytes, as shown by depletion experiments performed in vitro with monoclonal antibodies, and in vivo by thymectomy. Splenic T lymphocytes of young axolotls (before 10 months) do not have this functional ability, which suggests maturation and/or migration phenomena during T-cell ontogenesis in this species. Axolotl lymphocytes are able to proliferate in vitro with a significant stimulation index to staphylococcal enterotoxins A and B (SEA and SEB). These products act on mammalian lymphocytes as superantigens: in combination with products of the major histocompatibility complex (MHC), they bind T-cell receptors with particular V beta elements. The fact that these superantigens are able to activate lymphocytes of a primitive vertebrate suggests a striking conservation of molecular structures implied in superantigen presentation and recognition.

Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS.

The duration of human immunodeficiency virus (HIV-1) infection prior to the development of AIDS is variable, and for most patients the exact time of infection is not known. A group of 38 HIV-1-infected subjects was tested while asymptomatic for comparative cytotoxic lymphocyte responses to the Gag and envelope antigens of HIV-1. Twenty of the 38 patients had no detectable primary cytotoxic T lymphocyte (CTL) response to Gag, and this was associated with a relative risk of 1.89 for progression to ARC or AIDS during the subsequent 3 to 40 months of observation when compared with patients who had Gag-specific CTL activity at the beginning of the observation period. In contrast, no significant association was observed between envelope-specific cytotoxic activity and disease progression. Other patient characteristics, including CD4+ T lymphocyte counts and antibody levels to the p24gag protein, measured at the start of observation, did not correlate with disease progression during the observation period. This suggests that the anti-Gag CTL response may be protective during HIV-1 infection.

Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins.

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.

[Erythrocyte deformability in chronic respiratory insufficiency]. / La deformabilità dei globuli rossi nell'insufficienza respiratoria cronica.

The red blood cell deformability was evaluated in 10 patients suffering from chronic respiratory failure and in 10 normal volunteers. Patients with chronic respiratory failure showed a pH of 7.36 +/- 0.04, a pCO2 of 48.7 +/- 7.3 mmHg and a pO2 of 61.2 +/- 10.2 mmHg and had a decreased red blood cell deformability if compared with normal volunteers (p less than 0.001). The red blood cell deformability of patients suffering from chronic respiratory failure showed a weak correlation with pO2 (r = 0.50, p less than 0.05).