Search

In this study, we examined the expression profile of brain-derived neurotrophic factor (BDNF) and its receptor TrkB in adult rat hippocampus following acute administration of diethyldithiocarbamate (DDTC), a neurotoxic compound which was previously shown to induce microglia activation and cell death. Semiquantitative RT-PCR analysis detected significant variations of BDNF mRNA levels in whole hippocampus homogenates, with a peak at 24h after DDTC injection. Increased BDNF protein expression was demonstrated by immunohistochemistry in various hippocampal subfields. The most relevant increase was observed in the hilus of the dentate gyrus where BDNF levels at 120h were found to be almost four times those of basal levels. Full-length TrkB (TrkB.FL) encoding mRNA was also shown to undergo an earlier increase in the hippocampus of DDTC-treated rats. TrkB immunostaining with an antibody binding both full-length and truncated (TrkB.T) isoforms was found to increase at 120h in the hippocampal CA2 and CA3 regions. These results demonstrate that DDTC modulates the expression of BDNF and its receptor in the adult rat hippocampus and suggest a possible involvement of this neurotrophin in the protective response to DDTC-induced neuronal damage.

Dithiocarbamates are compounds commonly used in medicine and in agriculture and their prolonged use is known to result in neurotoxicity. Whether this response may be related to early gene expression has not been investigated. We have addressed this issue by mapping Fos expression in rats acutely injected with diethyldithiocarbamate (DDTC) and correlating these data to neural damage in the hippocampus as determined by pyknotic nuclei count. In comparison to saline injected rats, DDTC treatment induced a marked Fos expression in most brain regions at 1 and 3 h. In the hippocampus, a high Fos expression was followed by a variable number of pyknotic nuclei at 6 h, depending on the subregion. The data suggest that, in this model of neurotoxicity, c-fos induction does not reflect a cell commitment to die or survive, but rather a cell response to the DDTC-induced oxidative disorder.

Dithiocarbamates, a class of compounds widely used in medicine and agriculture, have been reported to impair sleep structure. These effects have been attributed to the decrease in norepinephrine levels induced by these drugs. However, it has also been recently demonstrated that most of the mechanisms by which dithiocarbamates damage cell function involve changes in oxidative environment. To verify the potential relevance of the latter mechanism in the sleep impairment, we examined the sleep response of adult rats to an acute administration of diethyldithiocarbamate (DDTC). At the dose of 0.6 g/kg, DDTC induced fragmentation and a decrease in slow wave sleep (SWS), and a dramatic loss of paradoxical sleep (PS). These changes occurred soon after the treatment (day 0), persisted the following day (day 1), partially recovered on day 3, and regained near basal values on day 6. No sleep anomalies were observed with a lower dose of DDTC (0.06 mg/kg). On the other hand, when the higher dose of DDTC was given in association with either one of two antioxidants, alpha-tocopherol or melatonin, the amounts of SWS and PS significantly improved even on day 1, suggesting that the DDTC effects on sleep involved an impairment of the brain oxidative balance. Likewise, administration of the lower dose of DDTC 5 days before the higher dose induced a much earlier recovery of normal sleep, presumably due to the development of a tolerance to DDTC. On the whole, the data suggest that the brain oxidative environment may play a role in the mechanisms subserving sleep regulation.

An increasing body of evidence suggests a role for activated microglia in the pathogenesis of neurodegenerative disorders. Hence, it would be useful to have a better understanding of the significance of microglial activation for neuronal damage. Unfortunately, most models of microglial activation use invasive or long-lasting insults, which make it difficult to evaluate the role played by microglia. We have instead developed a model for microglial activation by using brief exposure to the widely available neurotoxin diethyl-dithiocarbamate (DDTC). Despite evidence for the neurotoxic nature of this substance, microglia involvement has not been hitherto investigated. After acute i.p. administration of DDTC at two different doses, microglia were already activated in selected areas of the rat brain (hippocampal dentate gyrus, entorhinal-pyriform cortex and hypothalamus) after 1 hour, reaching a peak at 3-6 hours and subsided within 6-48 hours, depending on the brain region. Microglia activation was associated with interleukin-1 beta immunopositivity between 3 and 6 hours and with up-regulation of major histocompatibility complex class II expression between 24 and 48 hours. No significant changes in astrocyte immunostaining were detected between 6 hours and 6 days. The TUNEL procedure revealed the death of a limited number of cells in the above-mentioned structures that peaked at 6h and then declined rapidly. Cell death was detected in sites with major, minor, or no microglial activation, indicating that these two events can occur concomitantly or independently. The study shows that the administration of DDTC provides a useful model for studying the implications of region-specific reactivity of microglia and its differential interaction with neuronal damage., (Copyright 2002 Wiley-Liss, Inc.)

Effects of microenvironmental changes were examined in the microglial cell line BV-2. In serum supplemented medium cells were ameboid shaped and exhibited thin cytoplasmatic processes at lower concentration or in absence of serum. High levels of acetylated low-density lipoprotein (LDL) receptor and of phagocytic and proliferative activity were detected. Lipopolysaccharide (LPS) and the neuropeptide substance P (SP) induced secretion of interleukin-6. Low interleukin-3 secretion was detected only occasionally and was not influenced by LPS and SP. In defined medium, "process-bearing" cells were evident. Compared to cultures in serum supplemented medium, the cells expressed lower acetylated LDL-binding and phagocytic activity while actively proliferated, the response to LPS was reduced and to SP absent. Granulocyte/macrophage colony-stimulating factor increased the number of process-bearing cells, of acetylated LDL-binding and of IL-6 secretion induced by LPS. Cell morphology was not influenced by neurotrophins like nerve growth factor and brain-derived neurotrophic factor. The described phenotypical and functional plasticity makes the BV-2 cell line a useful model to investigate mechanisms of microglial activation.

The expression of neurotrophin and neurotrophin receptor mRNAs in human granulocytes and bone marrow cells was examined using ribonuclease protection assay and reverse transcription-polymerase chain reaction. The granulocytes expressed mRNA coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3). Moreover, the inflammatory mediator leukotriene B4 (LTB4) up-regulated the expression of NT-4 mRNA in granulocytes, but did not affect the expression of other neurotrophin mRNAs. Granulocytes generally lacked expression of mRNA coding for neurotrophin receptors. In contrast, human bone marrow cells consistently expressed mRNA for trkB (the BDNF and NT-4 receptor) and displayed variable expression of mRNA coding for trkA (the tyrosine kinase NGF receptor) and LNGFR (the low-affinity NGF receptor), whereas mRNA for trkC (the NT-3 receptor) was not expressed. Contrary to granulocytes, normal bone marrow cells generally expressed only low levels of mRNA encoding BDNF and NT-4. Expression of mRNA encoding NGF and NT-3 was not detected. However, significantly increased expression of BDNF mRNA was observed when bone marrow cells from patients with chronic myeloproliferative disorders (MPD) were analyzed. The results suggest that neurotrophins may act as granulocyte-derived effector molecules and that human bone marrow cells may be targets for these compounds, in particular BDNF and NT-4.

The expression of neurotrophin and neurotrophin receptor mRNAs was examined using RNase protection assays and Northern-blot analysis in rat thymus, spleen tissue and immunocompetent mononuclear cells purified from these two organs. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4 mRNAs were all expressed in thymus and spleen tissue although at different levels, while immunocompetent cells expressed neurotrophin-3 and neurotrophin-4 mRNAs. Thymus and spleen tissue expressed mRNAs encoding the low-affinity nerve-growth-factor receptor, the non-neuronal TrkA I receptor, the truncated (kinase deficient) and full-length TrkB, and the TrkC receptor. Low-affinity nerve-growth-factor receptor and non-neuronal TrkA I mRNAs were detected in both thymus and spleen immunocompetent cells. In addition, thymus cells expressed neuronal TrkA II mRNA and spleen cells expressed truncated TrkB mRNA. The expression of TrkA I and TrkA II mRNAs was enhanced in both thymus and spleen cells after cell culture. Enhanced levels of neurotrophin-4 mRNA were observed in spleen immunocompetent cells after adrenalectomy. Moreover, the expression of neurotrophin-4 mRNA was up-regulated after stimulation of immune cells with the mitogens concanavalin A or lipopolysaccharide or with the inflammatory mediator leukotriene B4. This suggests that neurotrophin-4 could be secreted by immunocompetent cells and may be involved in inflammatory processes.

Interleukin-6 (IL-6) activity was measured in the cerebrospinal fluid (CSF) of patients at different stages of human immunodeficiency (HIV) virus infection and of patients with multiple sclerosis (MS) or other inflammatory (OID) and noninflammatory neurological diseases (OND). In the advanced stages of HIV infection and in OID, IL-6 was detected more frequently (80 and 75% of the cases) and at higher concentrations than in the early stages of HIV infection. MS and OND (44, 48, and 44% of cases). Analysis of CSF and paired sera indicated that IL-6 production can be compartmentalized to either of the fluids. Evidence that altered blood-brain barrier functions can, at least in part, influence the CSF IL-6 levels was found in OID patients. No association was evident between intrathecal immunoglobulin synthesis and CSF IL-6 levels. Interleukin-1 (IL-1) levels were detectable in a minority of the samples from neurological patients; one OID patient had high levels of both CSF IL-1 and IL-6.

Substance P (SP) has recently been reported to induce interleukin 1 (IL-1) production by human monocytes. This was confirmed in our experiments with human monocytes cultivated in the presence of SP or SP together with lipopolysaccharide (LPS). In addition, a wide variability of cell response to the neuropeptide was noticed. Three out of twelve cell cultures were directly stimulated by SP to release IL-1, while four additional cultures needed prestimulation with suboptimal doses of LPS, and no effect was seen in the five remaining experiments. The data may suggest that preferentially activated monocytes respond to SP. The production of IL-1 by SP-stimulated monocytes is of great interest considering the broad spectrum of activity of IL-1 and the increasing evidence of sensory neuron involvement in acute and chronic inflammatory responses.

The mobility of 17 different proteins in CSF and serum on isoelectric focusing was investigated by subsequent immunofixation using monospecific antisera. Individual proteins yielded identical, often complex band patterns in normal CSF and serum, except transferrin which gave one to two additional bands between pH 5.8-6.4, and the low molecular wieght beta-trace protein and gamma-trace protein, which gave three bands at pH 7.4, 8.0, and 8.4, and a single band at pH 9.5, respectively, on investigation of CSF but not serum. Polyclonal IgC migrated as multiple bands between pH 4.7-8.6. Oligoclonal IgG in CSF in multiple sclerosis and neurosyphilis migrated between pH 8.6-9.5 and was easily discriminated from other proteins.

Agarose gel electrophoresis and immunofixation of CSF and serum from 39 patients with multiple sclerosis (MS) revealed oligoclonal IgG in the CSF in all cases and oligoclonal IgA and IgM in 1 patient each. IgG kappa bands only were found in 10 patients, while no patient had IgG lambda bands alone. IgG kappa bands predominated in 20 patients and IgG lambda bands in 5, while 4 patients had the same number of IgG kappa and IgG lambda bands. Twenty-seven patients also displayed IgG bands with kappa and lambda present simultaneously. Bands of free lambda chains were found in 7 patients, while free kappa chain bands were not seen. One or 2 faint IgG bands in 4 patients constituted the only serum abnormality. In 4 additional MS patients selected on the basis of normal findings on agarose gel electrophoresis of the CSF, immunofixation did not reveal oligoclonal Ig, while isoelectric focusing showed bands in 1. Immunofixation is recommended for proving the presence of oligoclonal Ig in CSF and for characterizing oligoclonal Ig into classes and types of light chains.

A modified technique of isoelectric focusing on thin-layer polyacrylamide gel followed by immunofixation with monospecific antisera was used to identify individual cerebrospinal fluid (CSF) and serum proteins and to define the oligoclonal reaction observed in multiple sclerosis (MS). "Normal" IgG gave about 20 to 30 bands at pH 3.5 to 9.5, IgA about 10 bands at pH 3.5 to 6.4, beta-trace protein a smear at pH 3.5 to 8.5, and gamma-trace protein 1 or 2 bands at pH 8.0, 9.5 or both. Up to 11 oligoclonal IgG bands migrating between pH 6.5 and 9.5 were found in CSF from 26 of 27 consecutive patients with MS and also in 20 of the corresponding sera, although at lower numbers and concentrations. In 26 patients, 1 or more of the bands corresponding to normal polyclonal IgG were stronger in CSF than in serum. These data support the hypothesis that two colonies of lymphocytes are activated intrathecally, one of them synthesizing oligoclonal and the other polyclonal IgG. Up to 11 mostly faint bands of free light chains, predominantly of lambda type and migrating between pH 3.5 and 9.5, were found in 8 of 9 CSF specimens from patients with MS.

The mobility of the immunoglobulins G, A and M, beta-trace protein and gramma-trace protein on isoleectric focusing of serum and CSF was determined by immunofixation using specific antisera. Polyclonal IgG migrated as multiple bands between pH 4.7--8.6, polyclonal IgA as multiple bands between pH 4.9--6.1 in CSF and serum. IgM could not be identified in normal CSF or serum. beta-trace protein gave three bands at pH 8.0, 8.4 and 7.4--7.5, respectively, while gamma-trace protein gave one single band at pH 9.5--greater than 9.5. Oligoclonal IgG in CSF in multiple sclerosis and neurosyphilis migrated between pH 8.6--greater than 9.5 and was easily discriminated from other proteins.

Serum and CSF from 32 patients with idiopathic ALS, 30 age-matched controls and 30 MS patients were investigated regarding immunoglobulin concentration and virus-specific antibodies, the lymphocytes in the peripheral blood and lymphocyte subsets were also investigated. ALS patients' results were compared with findings in MS and controls. The ALS patients had significantly higher IgG concentration in serum than the controls, marked lymphopenia, reduction of CD2, CD8 and Leu 7 positive cells and increase of the CD4/CD8 ratio and of SIg-positive lymphocytes. Compared with the MS patients, the ALS patients showed similarity in T-subset distribution with a lower standard deviation. No HTLV-I and HIV antibodies were found in any group and no significant differences in antibody distribution to Toxoplasma G, herpes simplex, cytomegalovirus, measles and mumps viruses were evident. All ALS patients were investigated at an early disease stage, therefore, our findings seem to support the conclusion that the immune alterations are related to the mechanisms of the disease and not to complications of its evolution.

Isoelectric focusing and agarose gel electrophoresis of CSF and serum revealed similar frequencies of oligoclonal bands in multiple sclerosis (100% with both methods), infectious CNS disorders (38 and 23%) and other neurological diseases (8 and 10%). In selected cases with unsure CSF oligoclonal bands on agarose gel electrophoresis, isoelectric focusing displayed definite oligoclonal bands. In contrast to agarose gel electrophoresis, isoelectric focusing revealed evidence for oligoclonal bands in serum as well as in CSF in 41% of the multiple sclerosis patients, indicating diffusion from CSF to serum. In 4 cases with gammaglobulin bands appearing in both CSF and serum on agarose gel electrophoresis, isoelectric focusing revealed normal CSF and serum protein patterns at pH above 6.4 where most IgG is migrating.

Serum and cerebrospinal fluid (CSF) from eight patients with subacute sclerosing panencephalitis (SSPE), 21 with multiple sclerosis (MS) and 16 controls were analyzed for IgG subclass pattern of anti-viral and anti-bacterial antibodies. In CSF of SSPE and MS patients IgG1 and IgG4 antibodies to measles and IgG1 to mumps were increased compared to the controls. In addition, the SSPE patients had elevated levels of IgG1 to PPD, teichoic acid, and to dextran in CSF. The group of MS patients had decreased levels of IgG1 antibodies to Staphylococcus aureus alpha-toxin.

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF), a very high capacity method for separating immunoglobulins (Ig), was performed on cerebrospinal fluid (CSF) and serum. It was followed by antigen immunofixation with measles, mumps, herpes simplex (HSV), and rubella virus antigens and anti-human Ig autoradiography in order to demonstrate viral antibodies in separated Ig zones. Two of 11 control patients and 21 of 25 patients with multiple sclerosis (MS) displayed one or more zones of viral antibodies in the CSF without any counterpart, or with distinctly fainter zones, in the serum. Such reaction patterns were taken to indicate the possibility of intrathecal antibody synthesis. Antibody synthesis to measles was found in one to five zones in 76% of the patients with MS; antibody zones were found to HSV in 36% of the patients, to mumps in 12%, and to rubella in 12%. In 36% of the patients, two or three different antibody specificities (of which one was always measles) were found simultaneously in individual autoradiogram zones. For all viral antibodies detected in the CSF autoradiograms, their counterparts in oligoclonal or polyclonal IgG zones (or both) were demonstrable by PAG IEF of the corresponding CSF. The majority of patients with MS also had one or more oligoclonal CSF IgG zones without known antibody specificity. Antigen immunofixation and autoradiography are mainly qualitative. It is not known whether the viral antibodies present in oligoclonal or polyclonal IgG zones in MS CSF reflect a polyclonal B cell activation, a disease-specific immune reaction, or both.

Substance P (SP) at a concentration of 10(-7) M significantly increased the number of IgG-producing cells induced by the polyclonal activator Staphylococcus aureus protein A (SpA) in 11 out of 22 cultures of enriched human blood B lymphocytes, in nine cultures SP did not significantly affect the SpA response and in three cultures IgG secretion was decreased in the presence of SP. Stimulation by SP was observed in cultures at days 6 and/or 8. In 3 out of 4 cell cultures depleted of monocytes SP did not affect the cell response to SpA stimulation. SP antagonists inhibited the enhancing effect of SP on B-cell antibody secretion induced by SpA. SP alone did not stimulate B lymphocytes. Neurokinin A (NKA) had similar effects as SP and enhanced the IgG secretion induced by SpA in 5 out of 9 experiments, in two experiments was inactive, and in one decreased the IgG secretion. The effect of SP and NKA on B lymphocytes suggest that the neuropeptides interact with the regulation of the immune response.

Several techniques are now available for the quantitative and qualitative examination of CSF proteins modified during the course of neurological disease. The CSF and serum of 42 patients suffering from various neurological diseases and of 7 controls were examined using quantitative methods--single radial immunodiffusion, nephelometric analysis--and qualitative methods--isoelectric focusing on polyacrylamide gel and on agarose gel, isoelectric focusing of unconcentrated CSF followed by transfer to cellulose nitrate membranes and immunoperoxidase staining and double antibody peroxidase labeling and avidin-biotin amplification. In the present study the results and the advantages of different methods are described and compared.

The occurrence of viral antibodies in relation to IgG separated by thin-layer polyacrylamide gel isoelectric focusing was studied in CSF and serum from 24 patients with mumps meningitis by immunofixation with viral antigens and autoradiography. Eleven of the patients displayed on the autoradiograms evidence of locally in the central nervous system synthesized mumps virus antibodies which were related to oligoclonal IgG bands in all 5 patients who displayed this CSF abnormality, otherwise to polyclonal IgG bands. Local synthesis of mumps virus antibodies was detectable in 43% of specimens obtained 1-13 days after onset, and in 75% obtained 27-47 days after onset. Only one patient displayed local synthesis of antibodies to other viruses (measles and herpes simplex) which could then be traced to polyclonal IgG bands.