Thymic development in surgically bursectomized embryonic chicken: expression of PCNA, CD3, CD4 and CD8 markers.

Little information is available on the functional relationship between bursa and thymus during chicken embryogenesis. We, therefore, investigated embryonic thymuses taken at 17 days in ovo from chickens bursectomized at 68-72 hours, with histological, histochemical (PAS, Alcian blue), and immunoreaction (anti-cytokeratin B, anti-PCNA/cyclin and anti-CD3, CD4 and CD8 antibodies) methods and compared these data with those from normal and sham-operated chickens of the same age. The bursectomized thymuses distinctly differed from normal and sham-operated thymuses: they were smaller, and the cortical zone was thinner and contained fewer epithelial cells and thymocytes. Only few cortical thymocytes were immunoreactive for PCNA, indicating low proliferative rate. More cortical thymocytes as compared with the normal, expressed CD3 on their cell membrane, whereas the thymocytes at the cortical-medullary border expressing anti- CD4 and anti-CD8 antidodies were less numerous than in normal thymus. The medullary zone contained few epithelial clusters made up of fewer cells than medullary clusters in normal chickens. Some cystic formations were enlarged and contained PAS- or Alcian-blue positive amorphous material. All these data suggest that early bursectomy affects both morphological and functional thymic development.

Functional correlates of nicotine administration: similarity with drugs of abuse.

Changes in the local utilization of cerebral glucose resulting from administered drugs acting on the central nervous system can be evaluated quantitatively by the [14C]2-deoxyglucose method. We report the findings obtained by the [14C]2-deoxyglucose method that contribute to understanding the cerebral functional effects of drugs of abuse and discuss in particular the similarities between nicotine and other addictive drugs. A common consequence of the intravenous administration of psychomotor stimulants and opioids in the rat is the increase in glucose utilization in the shell of nucleus accumbens. This functional change is accompanied by increased local extracellular concentrations of dopamine. Altered functional activity and dopamine neurotransmission in the shell of the nucleus accumbens thus represent distinctive neurobiological markers of the addictive properties of several drugs, independently of the specific neurochemical mechanisms of action. It has recently been shown that the intravenous administration of a pulse of nicotine, at single-unit doses corresponding to those that maintain self-administration in the rat, produces neurochemical and metabolic changes in the shell of the nucleus accumbens that closely resemble those of psychomotor stimulants and opioids. The latter results demonstrate that nicotine shares with highly addictive drugs a distinct neurochemical and functional consequence. They therefore contribute to the neurochemical definition of the addictive nature of nicotine. These neurochemical and functional changes may contribute to the changes in expression of intracellular second messengers and neurotransmitter/receptor systems observed particularly in the shell following the administration of drugs of abuse.

Reduced dopamine in peripheral blood lymphocytes in Parkinson's disease.

The early clinical symptoms of Parkinson's disease (PD) may be difficult to perceive and are frequently overlooked. Thus, interest has focused on the identification of biological or instrumental markers that may contribute to the early diagnosis of PD, with the aim of introducing neuroprotective therapies at the very start of illness. Impairment of nigrostriatal dopamine transmission can be visualized in vivo by functional imaging techniques, but these are rather complex and expensive examinations, available only in selected institutions. Here we show that dopamine content and tyrosine hydroxylase immunoreactivity are reduced in peripheral blood lymphocytes (PBL) in the early stages of PD. These data suggest that PBL may represent a simple and useful tool with which to identify precociously dopamine impairment in PD.

Serum mitogenic activity on in vitro glial cells in Neurofibromatosis type 1.

Glial mitogenic effect was investigated in sera from the following groups of subjects: group (1) 31 patients clinically and genetically affected by Neurofibromatosis type 1 (NF1) belonging to different families; group (2) 42 patients without family history of NF1 affected by sporadic neoplasms of the same histogenetic origin as the proliferative lesions that are present in NF1; group (3) 51 healthy volunteers without family history of NF1 nor of neoplastic disease; group (4) 54 clinically healthy relatives of the NF1 patients included in the first group. All NF1 patients and 3/54 healthy relatives had alterations of exons 31 or 32 of NF1 gene. Glial proliferation, measured by [3H]thymidine incorporation, was significantly increased by sera from all NF1 patients and from 23/54 of clinically healthy relatives, as compared to sera from healthy volunteers. This serum mitogenic activity strongly suggests the existence of soluble glial proliferating molecules in NF1 families. The molecular weight (3-30 kDa), the heat- and freeze-stability and the specificity for glial cells, suggest that the molecules responsible for this mitogenic effect are different from the growth factors previously described in NF1-associated tumor extracts and from lymphokines. Within each NF1 family, the maximal serum dilution stimulating glial proliferation was similar both in affected members and in their clinically healthy relatives. Since none of the clinically healthy relatives showing serum mitogenic activity was positive for the NF1 mutation analysis and, conversely, those having altered exons 31 or 32 of NF1 gene did not show any mitogenic activity; these results suggest that the phenotype expression of NF1 might depend not only on the NF1 mutations per se, but also on other genetic or epigenetic factors, such as serum glial proliferating molecules.

Correlation between P19 presence and MHC class II expression in human fetal astroglial cells cocultured with HTLV-I donor cells.

The possibility of a direct infection of human brain by HTLV-I, has been studied using an in vitro model. Human fetal astroglial cells were cocultivated with irradiated HTLV-I donor cell line MT-2, and assayed for the presence of HTLV-I core protein p19 after 1 week. Fifty-six per cent of GFAP positive astrocytes showed the viral core protein p19 and increased expression of Class II MHC antigens. Electron microscopy of astroglial cells exposed to HTLV-I revealed the presence of vacuoli-like structures containing viral core protein p19. Cell intermediate filament cytoskeleton was also disorganized. Even if this study does not provide direct evidence for virus replication inside astroglial cells, all these findings suggest that HTLV-I can indeed enter the cell and exert a cytopathic effect. Therefore the results of the present study are consistent with the hypothesis that astroglial cells could be involved in demyelination processes occurring in the HTLV-I associated neurological disorders, such as human associated myelopathy and tropical spastic paraparesis.

Effects of haloperidol on the expression of lymphocyte dopamine receptor mRNAs in the rat.

1. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the effects of haloperidol treatment (1 mg/kg/day for 2, 7 or 21 consecutive days) on the expression of D1B and D3 dopamine receptor mRNAs in the rat lymphocytes. 2. The expression of D1B receptor mRNA was significantly decreased after 2 days of treatment and progressively returned toward basal values at the end of treatment. Conversely, haloperidol failed to modify the expression of lymphocyte D3 receptor mRNA. 3. These results indicate short-lasting dynamic changes of expression of lymphocyte D1B dopamine receptor mRNA by haloperidol and suggest that the effects of dopamine and dopaminergic drugs on the immune system might be mediated, at least in part, by direct interaction of these substances with dopamine receptors on lymphocyte membrane.

Anti-beta 2-glycoprotein I antibodies bind to central nervous system.

Anti-beta 2-GPI antibodies (a beta 2-GPI) were found in serum from patients with anti-phospholipid syndrome (APS) and/or systemic lupus erythematosus (SLE). Since a beta 2-GPI are often found in patients with anti-cardiolipin antibodies (aCL), their role in thrombosis as well as other central nervous system (CNS) manifestations in APS is unclear. We, therefore, investigated whether affinity-purified a beta 2-GPI bind the CNS. Astrocyte and neuron cell lines and histological sections were used as CNS substrates. Indirect immunofluorescence and/or streptavidin-biotin-peroxidase techniques revealed that astrocytes, neurons and vascular endothelium were bound by purified a beta 2-GPI (mouse monoclonal, rabbit polyclonal, human serum Ig a beta 2-GPI). This suggests a potential role for a beta 2-GPI in the CNS damage, as a beta 2-GPI might contribute to CNS pathology by either a direct interaction with astrocytes and neurons or an interaction with cerebral vascular endothelial cells. CNS immunoreaction was also demonstrated using six a beta 2-GPI-positive sera from patients (four with neurological manifestations). No binding to CNS was seen using a beta 2-GPI-negative sera, i.e. five from SLE patients (two with CNS involvement) and six healthy donors, or a monoclonal aCL without a beta 2-GPI immunoreactivity. Thus, the CNS reactivity by the a beta 2-GPI-positive sera appears specifically due to a beta 2-GPI and independent from aCL. Because of the presence of aCL in all patient sera, and the CNS involvement in three control patients, it is not possible to attribute a direct role for a beta 2-GPI in neurological diseases in this study.

Dopamine receptor mRNAs in the rat lymphocytes.

It has been suggested that dopamine might play a role in the regulation of the immune system. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of mRNA for the different subtypes of dopamine receptors in the rat lymphocytes. D1, D3 and D5 receptor mRNAs were identified. These results provide further evidence for the interaction of dopamine systems and the immune system, and suggest to further investigate whether the immunosuppressive actions of dopamine and dopaminergic drugs might depend on a direct interaction with dopamine receptors on the lymphocyte membrane. Moreover, they suggest the suitability of this animal species to further investigate the correlation between changes in the expression of central and peripheral dopamine receptors produced by manipulations of the dopamine systems.

Treatment with 6-hydroxydopamine in planaria (Dugesia gonocephala s.l.): morphological and behavioral study.

Morpho-functional and behavioral effects of exposure to 6-hydroxydopamine (OHDA)-HCI (24 microg/ml per day for 24 h and 7 days) were studied in planarias (Dugesia gonocephala s.l.). Exposure to 6-OHDA-HC1 for 24 h produced hypokinesia of the specimens. These behavioral changes were more pronounced, leading to complete immobility, after 7 days of exposure to the neurotoxin. Moreover, specimens exposed to 6-OHDA-HCI for 24 h and 7 days failed to show any behavioral response to nomifensine, thus furnishing evidence of the damage of presynaptic dopamine terminals. Exposure to 6-OHDA-HCl for 24 h significantly reduced cathecolamine content in neuropil region, as demonstrated by histochemistry, and electron-dense presynaptic vesicles, as observed on electron microscopy examination. All these alterations were significantly more pronounced and were accompanied by swelling and strong increase of electron-density in cytoplasm of numerous neurons after exposure to the neurotoxin for 7 days. This appears to be the first demonstration of the neurotoxic effects of 6-OHDA-HCI in flatworms.

Effects of cocaine treatment on the nervous system of planaria (Dugesia gonocephala s. l.). Histochemical and ultrastructural observations.

Acute high dose treatment with cocaine in planaria has been shown to produce hyperkinesia followed by immobilization, thus suggesting progressive neuronal dopamine (DA) depletion. On the contrary, treatment with low doses of cocaine inhibits motor activity in planaria, without producing hyperkinesias. Here we investigated the morpho-functional changes of the DA presynaptic terminals following cocaine treatment in planaria (acute high dose and chronic low dose). Neuronal DA content was determined by means of histochemical methods, and nerve cell ultrastructure was examined by electron microscopy. The effects of cocaine were compared to those of L-dopa, reserpine (used as positive and negative controls, respectively) and normal untreated specimens. Presynaptic vesicles and DA content were significantly reduced by chronic low-dose cocaine treatment. These effects were even more robust when the drug was acutely administered at high dose. Thus, depletion of DA vesicles is produced by cocaine in planaria, as well as in mammals. The behavioral effects of chronic low-dose treatment with cocaine, however, suggest that the drug acts not only as a DA reuptake blocker, but also as a direct agonist on presynaptic DA receptors. Acute high-dose administration of cocaine also produced signs of neuronal suffering, thus providing evidence for a direct neurotoxic effect of the drug.

Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord.

The role of the hyaluronate receptor, CD44, is well known in adult mammal astrocytes where it modulates neuron-glia interactions. However, no data exist regarding its expression in other vertebrates during their development. In order to detect the expression of CD44 in the chicken and its possible involvement in glial precursor migratory patterns during spinal cord development, a monoclonal antibody (MoAb) against the mammalian standard isoform, CD44-H, was used in immunohistochemical and immunoblot assays. With these methods, CD44 hyaluronate receptors were found on mature astrocyte membranes of adult chicken spinal cord. Astrocytes were identified using a MoAb against GFAP. During development, small clusters of CD44 labelled cells were seen lining the central canal starting from embryonic stage E10. These labelled cells were dispersed in the dorsal, lateral and ventral funiculi of the spinal cord in the subsequent stages. After stage E15, the CD44 labelled cells were identified as astrocytes because of their GFAP immunoreactivity. We conclude that CD44 receptors on immature astrocyte precursors should be considered as early astrocyte markers which have a possible role during cell migratory dispersal.

Transient HTLV-I infection of a human glioma cell line following cell-free exposure.

The human T-lymphotropic virus type I (HTLV-I) has been recently associated with cases of tropical spastic paraparesis and human myelopathy. In order to study whether cells of neuroectodermic origin were susceptible to HTLV-I infection, a human glioma cell line T67 was exposed in vitro to HTLV-I by a cell-free method of virus transmission. The presence of HTLV-I proviral DNA was analyzed by polymerase chain reaction 3, 7, and 14 days after infection. The results showed the presence of LTR, pol, and tax sequences within glioma cell line 3 days after the infection. However after 7 and 14 days, detection of HTLV-I sequences remarkably decreased. P19 expression peaked 7 days after infection and decreased in the following week. These data provide evidence that cell-free transmission of HTLV-I results in transient infection of cells of glial origin.

HTLV-I neurotropism. In vitro studies.

The human T cell leukemia lymphoma virus (HTLV-I) has been recently associated to neurological disorders. In order to investigate the interaction between this virus and the central nervous system, cells of neuroectodermic origin have been exposed to HTLV-I through an experimental in vitro model. The results revealed the presence of HTLV-I virus core protein p19 inside glial cells, after a short time exposure to a chronically infected donor cell line. This suggests a possible role of a lymphotropic retrovirus in neurological diseases.

Effect of human T lymphotropic retrovirus-I exposure on cultured human glioma cell lines.

Four different human tumor cell lines of glial origin have been exposed to a human T lymphotropic retrovirus (HTLV-I). All these cell lines were positive for the glial marker glial fibrillary acidic protein (GFAP). The presence of virus RNA was demonstrated by in situ hybridization using an HTLV-I, SStI-SStI viral insert as probe. Virus expression has been monitored through an indirect immunofluorescence assay using a monoclonal antibody against virus core protein p19. All the four glioma cell lines tested became positive for p19 after 2 weeks of co-cultivation and showed a clear alteration of GFAP expression.

Beta2-glycoprotein I (beta2-GPI) mRNA is expressed by several cell types involved in anti-phospholipid syndrome-related tissue damage.

We report here the expression of beta2-GPI mRNA by cell types involved in the pathophysiology of the anti-phospholipid syndrome (APS), i.e. endothelial cells as a target of autoantibodies in the APS, astrocytes and neurones involved in APS of the central nervous system (CNS). Lymphocytes were also included in the study, as it has been demonstrated that patients with systemic lupus erythematosus-associated CNS diseases have serum anti-lymphocyte antibodies cross-reacting with brain antigens, and intrathecally synthesized anti-neurone antibodies. Reverse transcriptase-polymerase chain reaction followed by restriction enzyme digestion of the product obtained demonstrated the presence of beta2-GPI mRNA in all cell types here tested, cultured both in presence and absence of fetal calf serum. In both culture conditions, the same cell types were immunoreactive to an anti-beta2-GPI MoAb, as determined by indirect immunofluorescence technique. Taken together, these results indicate a direct cell synthesis of beta2-GPI, suggesting an antigenic function of beta2-GPI in the APS, including the CNS disease that occurs in this syndrome.

Treatment with cyclosporine A promotes axonal regeneration in rats submitted to transverse section of the spinal cord.

The abortive axonal regeneration in the central nervous system (CNS) of mammals has been attributed to a series of inhibitory factors. Previous reports suggest the development of autoimmune reactions following CNS lesion in mammals. In this study we investigated whether immunosuppressive treatment with cyclosporine A is able to facilitate axonal regeneration in rats submitted to complete transverse section of the spinal cord at the level of T7-T8. Treated animals received daily subcutaneous injections of cyclosporine A (2.5 mg/kg), while control rats were given a similar treatment with saline. Immunosuppression was begun immediately after spinal cord transection. Strong evidence of morphological axonal regeneration was observed 15 days after surgery in all cyclosporine A treated animals. Furthermore, treatment with cyclosporine A markedly reduced the seric immune reaction elicited by the lesion. The results of this study, although preliminary, provide further evidence for the development of autoimmune processes following lesion in the CNS, and suggest that blocking of the immune reaction facilitates axonal regeneration in the rat.

Serum anti-beta2-glycoprotein I antibodies from patients with antiphospholipid antibody syndrome bind central nervous system cells.

Sera from 20 patients with antiphospholipid syndrome (APS), primary or secondary to systemic lupus erythematosus (SLE), or with SLE, were assayed by immunoblot analysis for anti-beta2-glycoprotein I antibodies (abeta2-GPI), and by indirect immunofluorescence (IIF) technique for reactivity with astrocyte and neuron cell lines and with histological sections of human brain biopsies and monkey cerebellum. Six sera from healthy donors were studied as a control. Eleven out of the 20 patient sera contained abeta2-GPI and were immunoreactive with astrocytes and neurons, both in culture and in the histological sections, and with the endotheliocytes of the microvessels present in the histological sections. Cell localization and the pattern of immune reaction were similar to those obtained with a monoclonal antibody abeta2-GPI. Eight of the remaining patient sera, found abeta2-GPI-, did not react with the nervous substrates (and the control sera), while one exhibited immunoreactivity analogous to the abeta2-GPI+ sera. The interference of anticardiolipin antibodies (aCL) in the immunoreactivity with the nervous substrates was excluded since aCL were present in all patient sera and no immune reaction was observed in the histological sections incubated with a monoclonal aCL. Therefore, the binding of abeta2-GPI from patients to cells of the central nervous system (CNS) occurs independently from aCL. This issue may be relevant to further evaluate the potential pathogenetic role of abeta2-GPI in the CNS damage of APS-like conditions.

Phosphorylated and non-phosphorylated neurofilament epitopes are co-distributed in the fish Mauthner axon.

The neurofilament (NF) polypeptides of the fish giant Mauthner cell axons (MAs) and their degree of phosphorylation were investigated in the adult by means of immunoblot and immunohistochemical staining. Fasciculus longitudinalis medialis (FLM) axons of much smaller caliber, among which MAs run in the ventral spinal cord, were also analyzed in two teleost fish belonging to continuously growing species. To detect NF polypeptide subunits, commercially available monoclonal antibodies against mammalian NF-L (68 kDa), NF-M (160 kDa), phosphorylated (P) and non-phosphorylated (nP) NF-H (200 kDa) epitopes were used. These antibodies labelled bands of the identical molecular weight in fish cervical spinal cord total protein immunoblots. A peculiar NF composition of the MAs was observed, following immunohistochemical staining i.e. a low NF-M expression and a codistribution of P and (nP)-NF-H epitopes. Moreover, on the basis of immunoperoxidase staining in ultrathin longitudinal MA sections, we suggest that P NF-H are arranged in bundles, whilst (nP)-NF-H are likely to be free in the axoplasm. By contrast, FLM axons were found reactive with antibodies only against P NF-H. These results confirm that in carp and trout, NF have epitopes cross-reacting with monoclonal antibodies directed against the mammalian NF subunits. Furthermore, as regards the co-distribution of phosphorylated and non-phosphorylated NF-H epitopes in the M-cell axons, these might be considered as not yet completely mature axons taking into account that carp and trout belong to continuously growing species.

Dopamine transporter immunoreactivity in peripheral blood lymphocytes in Parkinson's disease.

There is increasing interest in the identification of biological markers for the early diagnosis of Parkinson's disease (PD). Previous studies indicate changes of dopamine content, tyrosine hydroxylase immunoreactivity and dopamine receptors in peripheral blood lymphocytes (PBL) in PD. Here we demonstrate a reduction of dopamine transporter immunoreactivity in PBL in the early clinical stages of the disease. These findings contribute to our understanding of the peripheral dopamine system in PD.

Effects of 17 beta-estradiol, progesterone and tamoxifen on in vitro proliferation of human pituitary adenomas: correlation with specific cellular receptors.

Six human pituitary adenoma cultures, characterized for estrogen and progesterone (Pg) receptors, were treated with 17 beta-estradiol (17 beta-E2), Pg and tamoxifen (TAM) at different concentrations, alone and in combination, for 2, 4 and 8 days. Cell proliferation data showed in most cases a stimulating effect of 17 beta-E2 and an inhibitory effect of Pg on cell growth, which appeared to be correlated with specific receptor expression, but independent of pituitary cell hormone content. A marked inhibitory effect of TAM on cell proliferation was present in all cases, but, on the contrary, was independent of estrogen receptor expression.