Signal transduction pathways of nitric oxide release in primary microglial culture challenged with gram-positive bacterial constituent, lipoteichoic acid.

Between one-third and one-half of all cases of sepsis are known to be caused by gram-positive microorganisms through the cell wall component, e.g. lipoteichoic acid (LTA). Gram-positive bacteria are also known to induce encephalomyelitis and meningeal inflammation, and enhance the production of nitric oxide (NO) via expression of inducible nitric oxide synthase (iNOS) in murine tissue macrophages. It remains to be explored if LTA could activate microglia considered to be resident brain macrophages. We report here that LTA derived from gram-positive bacteria (Staphylococcus aureus) significantly induces NO release and iNOS expression in primary microglia. LTA-induced NO accumulation was detected at 2 h in microglial culture and was significantly attenuated by pretreatment with anti-CD14, complement receptor type 3 (CR3) or scavenger receptor (SR) antibodies. LTA activated mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, p38 MAPK or c-Jun N-terminal kinase in cultured microglia. LTA-elicited microglial NO production was also drastically suppressed by SB203580 (p38 MAPK inhibitor) or pyrrolidine dithiocarbamate (an inhibitor of nuclear factor kappaB), indicating that p38 MAPK and nuclear factor kappaB were involved in microglial NO release after LTA challenge. These results suggest that gram-positive bacterial product such as LTA can activate microglia to release NO via the signal transduction pathway involving multiple LTA receptors (e.g. CD14, CR3 or SR), p38 MAPK and nuclear factor kappaB. The in vivo study further confirmed that administered intracerebrally LTA induced considerable noticeable iNOS, phospho-IkappaB and phospho-p38 MAPK expression in microglia/macrophages.

Effect of levodopa on interleukin-15 and RANTES circulating levels in patients affected by Parkinson's disease.

Parkinson's disease (PD) is an extra-pyramidal neurodegenerative disorder, in which alterations of the immune system are involved. Interleukin (IL)-15 stimulates cellular immune response and induces growth and differentiation of various immune cells. RANTES, promoting leukocyte infiltration to sites of inflammation, mediates the trafficking and homing of immune cells. To clarify the potential effect of levodopa on the immunological network of PD, we analyzed IL-15 and RANTES serum levels in PD patients, treated or not with levodopa, and in healthy donors. Levodopa-treated patients showed significantly higher IL-15 and RANTES circulating levels with respect to healthy controls and higher, although not significantly, levels with respect to untreated patients. So, we hypothesize that the immunological alterations found in PD may be linked, at least in part, to levodopa therapy.

Effect of promoters on cellular immune response induced by recombinant fowlpox virus expressing multi-epitope polypeptides from HIV-1.

HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.

[Evidence for the existence of L-dopa-immunoreactive neurons in the human mesencephalic region].

The existence of L-3,4-dihydroxyphenylalanine (L-DOPA) immunoreactivity is demonstrated for the first time in some neurons in the human mesencephalic region, using an immunohistochemical method with a newly raised, highly specific anti-L-DOPA antiserum. In this study, we have found many L-DOPA-positive/dopamine (DA)-positive neurons. On the other hand, we observed a few L-DOPA-positive/DA-negative cell bodies in the dopaminergic regions in the midbrain. The present results suggest the possibility of the existence of more than one neuronal group in the human mesencephalic ventral tegmental area region. L-DOPA in one group is an intermediate metabolite for decarboxylation to DA and in another group may exist as an end-product. L-DOPA in the latter group could be a neuromodulator and/or neurotransmitter. Thus, we suggest that L-DOPA plays an important role besides being an intermediate of DA in the human mesencephalon.

Immunogenicity studies of a synthetic antigen of alpha methyl dopa.

Since the idiosyncratic liver toxicity of methyl dopa (L-alpha-methyl-3,4- dihydroxy-phenylalanine) may be due to immune mediated mechanisms, immunologic tools are needed to detect methyl dopa induced antibody and antigen. Hapten (methyl dopa)--carrier (albumin) conjugates were synthesized to generate antibodies reactive with this drug. Studies were also conducted to test the immunogenicity of this hapten-carrier conjugate and its cross reactivity with methyl catechol and levodopa. Methyl dopa (MD), levodopa (LD) or methyl catechol (MC) were conjugated to rabbit serum albumin (RSA) under high pH (base) conditions or by a tyrosinase (tyr) catalyzed reaction. Under the base conjugation conditions, MD-RSA, LD-RSA and MC-RSA conjugates were produced at higher hapten: carrier ratios than conjugates produced by the enzyme catalyzed reaction. Rabbits were subsequently immunized with either MD-RSA(base) or MD-RSA(tyr). Antibodies elicited by MD-RSA(base) had marked reactivity to the carrier protein, albumin, whereas antibodies elicited by MD-RSA(tyr) did not. In addition, reactivity of anti-MD antibody was equal to or greater with MC-RSA than reactivity with either MD-RSA or LD-RSA. This work suggests that the conjugation method using the tyr catalyzed reaction produces the optimal immunogen with minimal modification of the carrier protein. In addition, the catechol moiety of MD, MC and LD appears to be the immunogenic epitope on these haptens.

Identification of L-dopa immunoreactivity in some neurons in the human mesencephalic region: a novel dopa neuron group?

The presence of L-3,4-dihydroxyphenylalanine (L-DOPA) immunoreactivity is reported for the first time in some neurons in the human mesencephalic region, using an immunohistochemical method with a newly raised, highly specific anti-L-DOPA antiserum. We have found many L-DOPA-positive/dopamine (DA)-positive and a few L-DOPA-positive/DA-negative cell bodies in dopaminergic regions in the midbrain. The present results suggest the existence of more than one neuronal group of L-DOPA in the human mesencephalon. L-DOPA in one group is an intermediate metabolite for decarboxylation to DA and in another group may exist as an end-product. L-DOPA in the latter neurons could be a neuromodulator and/or neurotransmitter. Thus, we suggest that L-DOPA plays an important role besides being an intermediate of DA in the human mesencephalon.

Immunolocalization of catecholamine enzymes, serotonin, dopamine and L-dopa in the brain of Dicentrarchus labrax (Teleostei).

Antisera to serotonin (5-HT), dopamine, and L-dopa, and to the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT), were used to localize monoamine containing neurones in the brain of Dicentrarchus labrax (sea bass). In the brain stem, 5-HT-immunoreactive (ir) neurones were recognized in the ventrolateral medulla, vagal motor area, medullary, and mesencephalic raphe nuclei and in the dorsolateral isthmal tegmentum. In the hypothalamus, liquor-contacting 5-HT neurones were seen in various regions of the paraventricular organ. Virtually all regions of the brain contained a dense innervation by 5-HT fibres and terminals. DBH-ir neurones were restricted to three brain stem areas: the locus coeruleus, the area postrema, and the reticular formation of the lower medulla. Neurones in these three groups also displayed TH-ir, and in the latter area, PNMT-ir in addition. In the locus coeruleus and area postrema, TH-ir neurones outnumbered DBH-ir neurones, an observation substantiated by the presence of dopamine-ir neurones. In the forebrain, dopamine- and TH-ir neurones were found in the olfactory bulb, ventral/central telencephalon, periventricular preoptic, and suprachiasmatic areas, dorsolateral and ventromedial thalamus, and posterior tuberal nucleus. In the paraventricular organ, the distribution and morphology of dopamine-ir neurones was similar to that observed with anti-5-HT, but the vast majority of cells were not TH-ir, suggesting accumulation of dopamine by uptake from the ventricle, rather than by synthesis. L-dopa-ir neurones were found only in the central telencephalon, preoptic recess, and dorsolateral thalamus. Fibres and terminals immunoreactive for dopamine, TH, and DBH showed a broadly similar distribution. The results are discussed in relation to the monoaminergic systems previously reported in other teleostean species and the mammalian brain.

Production of specific antibody against L-dopa and its ultrastructural localization of immunoreactivity in the house-shrew (Suncus murinus) lateral habenular nucleus.

An antiserum was raised against L-DOPA bound to bovine serum albumin, purified by affinity chromatography, and its specificities were verified by immunoblotting and enzyme-linked immunosorbent assays. The antiserum did not cross-react with dopamine (DA), tyrosine, tyramine, octopamine, norepinephrine or epinephrine. Immunocytochemical studies using the PAP method revealed that tyrosine hydroxylase- and L-DOPA positive but aromatic L-amino acid decarboxylase- and DA-negative neurons were present in the lateral habenular nucleus of the house-shrew (Suncus murinus) brain. Ultrastructurally L-DOPA immunoreactive products were localized in the cytoplasmic matrix and terminals with vesicles.

[Dopamine antibodies in the pathogenesis of parkinsonism]. / Antitela k dopaminu v patogeneze parkinsonizma.

L-DOPA and dopamine (DA) binding antibodies were found in the blood serum of Parkinsonian patients and middle-aged and elderly normal persons. DA-binding serum gamma-globulins of parkinsonian patients injected into rat caudate nuclei induced the pathogenetic mechanism of Parkinson's syndrome (generator of pathologically enhanced excitation) in these brain part and evoked main parkinsonian symptoms (oligokinesia, rigidity, tremor). The serum gamma-globulins of Parkinsonian patients without Da-antibodies caused less pronounced EEG disturbances. Parkinsonian symptoms developed rarely and were shorter and less pronounced compared with the DA-antibody effect. The DA binding antibodies role in Parkinson's syndrome pathogenesis and is L-DOPA therapeutic tolerance formation was discussed.

Preparation and characterization of a specific antibody for the immunohistochemical detection of L-dopa in paraformaldehyde-fixed rodent brains.

A rat polyclonal antiserum has been obtained after coupling of L-3,4-dihydroxyphenylalanine (L-DOPA) to larger proteins using a low concentration of glutaraldehyde. The antiserum was tested for its affinity and specificity using an enzyme-linked-immunosorbent-assay (ELISA). From competition experiments, the most immunoreactive compound was found to be the non-reduced L-DOPA conjugate. Our specific L-DOPA antiserum enables us to visualize L-DOPA molecule on brain of guinea pigs and rats. We examined the immunohistochemical distribution of the polyclonal L-DOPA antiserum after the fixation of brains with a mixture of paraformaldehyde and picric acid. The presence of L-DOPA-immunoreactive (IR) neurons and fibers was described in the posterior, dorsal and periventricular hypothalamic areas and in the arcuate nucleus. Finally, the distribution of L-DOPA-IR cells was compared to that of tyrosine hydroxylase (TH)-IR cells, by means of a double staining procedure. The presence of two populations of TH-IR cells (TH-positive/L-DOPA-negative and TH-positive/L-DOPA-positive cells) was described in the dorsal part of the hypothalamus.

Specific antisera against the catecholamines: L-3,4-dihydroxyphenylalanine, dopamine, noradrenaline, and octopamine tested by an enzyme-linked immunosorbent assay.

Antisera were raised against L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme-linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catecholamine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the nonconjugated compound was poorly recognized. The nonreduced conjugates of L-DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross-reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L-DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti-NA and anti-OA antibodies, the lateral chain is important.

Antisera against catecholamines: specificity studies and physicochemical data for anti-dopamine and anti-p-tyramine antibodies.

Antibodies against dopamine and p-tyramine were raised in rabbits. The two catecholamines were conjugated to albumin by glutaraldehyde. The specificity of the antibodies was established by equilibrium dialysis competition experiments using an immunoreactive tritiated derivative synthesized by coupling dopamine or p-tyramine to N-alpha-acetyl-L-lysine N-methylamide with glutaraldehyde. Hence, these radiolabelled ligands mimicked the antigenic determinant of conjugated immunogens. A comparison of the data obtained showed the high specificity of each antiserum for its hapten coupled by glutaraldehyde. The anti-dopamine antibodies recognized dopamine-glutaraldehyde but not p-tyramine-glutaraldehyde. The opposite occurred for the anti-p-tyramine antibodies. A slight modification of the molecular structure provided the opportunity for a specific response against that molecule. But this difference was more important when related to the hapten region where the antibody affinity was maximal. The cross-reactivity was observed to be more important dopamine and p-tyramine than between dopamine and noradrenaline on the one hand and between p-tyramine and dopamine than p-tyramine and octopamine on the other hand.

[Effect of fenformin, L-DOPA and para-chlorophenylalanine on the immunological reactivity and chemical carcinogenesis in BALB/c mice]. / Vliianie fenformina, L-DOFA i parakhlorfenilalanina na immunologicheskuiu reaktivnost' i razvitiie khimicheskogo kantserogeneza u myshei BALB/c.

Treatment with 20-methylcholanthrene resulted in the increase in hypothalamic levels of noradrenaline and serotonin in BALB/c mice. Immunization of carcinogen-treated animals with ovine erythrocytes was followed by changes in the hypothalamic levels of central neurotransmitters, opposite to those in intact immunized controls. After correction of the levels of neurotransmitters in the hypothalamus with phenformin, parachlorophenylalanine or L-dopa carried out prior to immunization, the fraction of antibody-producing cells in the spleen of carcinogen-treated mice returned to normal. A combined administration of immunostimulators and drugs for correction of hypothalamic levels of noradrenaline and serotonin potentiated inhibitory effect of the former on chemical carcinogenesis.

Thrombocytopenia associated with long-term levodopa therapy.

A 63-year-old man had severe thrombocytopenia after long-term levodopa therapy. Serologic studies and clinical features indicate that the thrombocytopenia was due to an autoimmune process, presumably similar to that induced by the chemically similar drug methyldopa. Direct allergy to levodopa was ruled out by controlled challenge of the patient receiving levodopa. Combined levodopa-prednisone therapy was then instituted, with good clinical response and no recurrence of thrombocytopenia.