GABAergic neurons in cerebellar interposed nucleus modulate cellular and humoral immunity via hypothalamic and sympathetic pathways.

Our previous work has shown that cerebellar interposed nucleus (IN) modulates immune function. Herein, we reveal mechanism underlying the immunomodulation. Treatment of bilateral cerebellar IN of rats with 3-mercaptopropionic acid (3-MP), a glutamic acid decarboxylase antagonist that reduces γ-aminobutyric acid (GABA) synthesis, enhanced cellular and humoral immune responses to bovine serum albumin, whereas injection of vigabatrin, a GABA-transaminase inhibitor that inhibits GABA degradation, in bilateral cerebellar IN attenuated the immune responses. The 3-MP or vigabatrin administrations in the cerebellar IN decreased or increased hypothalamic GABA content and lymphoid tissues' norepinephrine content, respectively, but did not alter adrenocortical or thyroid hormone levels in serum. In addition, a direct GABAergic projection from cerebellar IN to hypothalamus was found. These findings suggest that GABAergic neurons in cerebellar IN regulate immune system via hypothalamic and sympathetic pathways.

Cerebellar fastigial nuclear glutamatergic neurons regulate immune function via hypothalamic and sympathetic pathways.

We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-ß-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.

Modulation of immune function by glutamatergic neurons in the cerebellar interposed nucleus via hypothalamic and sympathetic pathways.

Our recent work has shown that the cerebellar interposed nucleus (IN) contains glutamatergic neurons that send axons directly to the hypothalamus. In the present study, we aimed to demonstrate modulation of cellular and humoral immunity by glutamatergic neurons in the cerebellar IN by means of gene interventions of glutaminase (GLS), an enzyme for glutamate synthesis, and to reveal pathways transmitting the immunomodulation. Injection of GLS-shRNA lentiviral vector into bilateral cerebellar IN downregulated GLS expression in the IN. The silencing of GLS gene in the cerebellar IN decreased interleukin (IL)-2 and interferon (IFN)-γ production, B-cell number, and IgM antibody level in response to antigen bovine serum albumin (BSA). On the contrary, injection of GLS lentiviral vector into bilateral cerebellar IN upregulated GLS expression in the IN. The GLS gene overexpression in the IN caused opposite immune effects to the GLS gene knockdown. Simultaneously, the GLS gene silencing in the cerebellar IN reduced and the GLS overexpression elevated glutamate content in the hypothalamus, but they both did not affect glycine and GABA contents in the hypothalamus. In addition, the immune changes caused by the GLS gene interventions in the IN were accompanied by alteration in norepinephrine content in the spleen and mesenteric lymph nodes but not by changes in adrenocortical and thyroid hormone levels in serum. These findings indicate that glutamatergic neurons in the cerebellar IN regulate cellular and humoral immune responses and suggest that such immunoregulation may be conveyed by cerebellar IN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.

Cerebellar fastigial nuclear GABAergic projections to the hypothalamus modulate immune function.

Our previous work has shown that the cerebellar fastigial nucleus (FN) is involved in modulation of lymphocyte function. Herein, we investigated effect of FN γ-aminobutyric acid (GABA)-ergic projections to the hypothalamus on lymphocytes to understand pathways and mechanisms underlying cerebellar immunomodulation. By injection of Texas red dextran amine (TRDA), an anterograde tracer, into FN, we found that the TRDA-labeled fibers from the FN traveled through the superior cerebellar peduncle (SCP), crossed in decussation of SCP (XSCP), entered the hypothalamus, and primarily terminated in the lateral hypothalamic area (LHA). Further, by injecting Fluoro-Ruby (FR), a retrograde tracer, in LHA, we observed that the FR-stained fibers retrogradely passed through XSCP and reached FN. Among these FR-positive neurons in the FN, there were GABA-immunoreactive cells. We then microinjected vigabatrin, which is an inhibitor of GABA-transaminase (GABA-T) that degrades GABA, bilaterally into FN. The vigabatrin treatment increased both number of GABA-immunoreactive neurons in FN-LHA projections and GABA content in the hypothalamus. Simultaneously, vigabatrin significantly reduced concanavalin A (Con A)-induced lymphocyte proliferation, anti-sheep red blood cell (SRBC) IgM antibody level, and natural killer (NK) cell number and cytotoxicity. In support of these findings, we inhibited GABA synthesis by using 3-mercaptopropionic acid (3-MP), which antagonizes glutamic acid decarboxylase (GAD). We found that the inhibition of GABA synthesis caused changes that were opposite to those when GABA was increased with vigabatrin. These findings show that the cerebellar FN has a direct GABAergic projection to the hypothalamus and that this projection actively participates in modulation of lymphocytes.

The hypothalamus mediates the effect of cerebellar fastigial nuclear glutamatergic neurons on humoral immunity.

OBJECTIVES: We explored effect of glutamatergic neurons in the fastigial nucleus (FN), one of three cerebellar nuclei, on humoral immunity and revealed that this effect was mediated by the hypothalamus via FN-hypothalamic glutamatergic transmission. METHODS: Rats were immunized with bovine serum albumin (BSA). On the third day after the immunization, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase for glutamate synthesis, was microinjected in bilateral FN and D,L-threo-ß-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on plasma membrane, was microinjected in both sides of lateral hypothalamic area (LHA). Glutamate content in the hypothalamus was examined by high-performance liquid chromatography (HPLC). Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to measure B lymphocyte percentage in mononuclear cells of peripheral blood and levels of anti-BSA IgM and IgG antibodies in the serum, respectively. RESULTS: DON injection in bilateral FN reduced B lymphocyte percentage and anti-BSA IgM and IgG levels, and simultaneously decreased glutamate content in the hypothalamus. Combined treatment with DON in the FN and with THA in the LHA elevated B cell number and anti-BSA IgM and IgG levels and increased hypothalamic glutamate content compared with DON treatment alone. However, combined treatment with DON in the FN and with THA in the ventrolateral thalamic nuclei (VL) did not significantly alter DON-dependent changes in B cell number and antibody levels, although the co-treatment altered DON-dependent glutamate content in the thalamus. CONCLUSION: Cerebellar FN glutamatergic neurons participate in modulation of humoral immunity and this effect is mediated by the hypothalamus via FN-hypothalamic glutamatergic transmission.

Effect of Cerebellohypothalamic Glutamatergic Projections on Immune Function.

Our previous work has shown that lesions of the cerebellar interposed nuclei (IN) suppress immune cell functions. Since there is no direct structural connection between the cerebellum and immune system, we explored the pathway mediating the cerebellar immunomodulation at the profile of cerebellohypothalamic projections to understand this modulation. Anterograde tracing of nerve tracts from the cerebellar IN to the hypothalamus was conducted by injection of anterograde tracer dextran-texas red (dextran-TR) in the cerebellar IN. We observed that dextran-TR-labeled nerve fibers, which were sent by cerebellar IN neurons, traveled in the superior cerebellar peduncle (SCP), crossed in SCP decussation, and entered the hypothalamus. In the hypothalamus, the fibers mostly terminated in the lateral hypothalamic area (LHA). Retrograde tracing by injection of retrograde tracer fluoro-ruby (FR) in the LHA found that FR-labeled neurons appeared in contralateral cerebellar IN. Fluorescent immunohistochemistry for glutamate revealed that many of the FR-labeled neurons were glutamatergic. These results demonstrate a direct glutamatergic projection from the cerebellar IN to the LHA. Reduction of the cerebellohypothalamic glutamatergic projections by microinjection of 6-diazo-5-oxo- L-norleucine (DON), an inhibitor of glutaminase for glutamate synthesis, in bilateral cerebellar IN led to suppression of peripheral lymphocyte number, T lymphocyte proliferation, and serum anti-sheep red blood cell IgM level. But the DON injection in the cerebellar cortex that does not send axons to the hypothalamus did not significantly alter all the immune parameters. These findings suggest that cerebellohypothalamic glutamatergic projection modulates immune function, and that via the pathway, the cerebellum implements its immunoregulatory effect.

Role of cerebellohypothalamic GABAergic projection in mediating cerebellar immunomodulation.

Our previous studies have shown that the cerebellar interposed nucleus (IN) modulates lymphocyte functions. As the cerebellum does not have a direct contact with the immune system, it is required to explore the pathway mediating the cerebellar immunomodulation. In this study, both lymphocyte percentage in peripheral leukocytes and lymphocyte proliferation induced by concanavalin A were reduced by the bilateral IN lesions with kainic acid. Anterograde tracing of nerve tracts with biotinylated dextran amine (BDA) from the cerebellum to the hypothalamus revealed that the BDA-labeled fibers from the cerebellar IN neurons traveled through superior cerebellar peduncle (SCP), crossed in SCP decussation, and primarily terminated in lateral hypothalamic area (LHA). Retrograde tracing with wheat germ agglutinin-horseradish peroxidase from the LHA to the cerebellar IN combined with immunohistochemistry for gamma-aminobutyric acid (GABA) or glutamate in the cerebellar sections displayed that the neuronal projections from the cerebellar IN to the LHA mostly were GABAergic. Blockage of GABA(A) receptors in the LHA with hydrastine led to a reduction in the lymphocyte percentage and proliferation, similar to the IN lesions. These results show a direct GABAergic projection from cerebellar IN to LHA and suggest that the projection mediates cerebellar immunomodulation.

Effect of cerebellar fastigial nuclear lesions on differentiation and function of thymocytes.

We have previously shown that the cerebellum regulates functions of T, B and natural killer (NK) cells. Herein, we provide further evidence for cerebellar immunomodulation at the profiles of differentiation and maturation of thymocytes and function of mature T lymphocytes. Neuronal bodies of the fastigial nuclei (FN), one of three cerebellar nuclei, were damaged by microinjection of kainic acid (KA) in the bilateral FN. On days 12 and 32 following the KA injection, percentages of thymocyte subpopulations including CD4(-)CD8(-), CD4(+)CD8(+), CD4(+)CD8(-) and CD4(-)CD8(+) cells, apoptotic DNA fragmentation of thymocytes, and levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the serum were measured by flow cytometry, diphenylamine assay and enzyme-linked immunosorbent assay (ELISA), respectively. In the thymus, the percentage of CD4(+)CD8(-) cells in thymocyte population was elevated by the cerebellar FN lesions on both the 12th and the 32nd days post-lesion. The other thymocyte subsets only significantly changed at the late time point (day 32) post-lesion, with an increase in CD4(-)CD8(-) cells and a decrease in CD4(+)CD8(+) thymocytes relative to control rats with intact FN or saline-infused FN. The cerebellar FN lesions, regardless of the 12th or the 32nd day post-lesion, reduced the percentage of thymocyte DNA fragmentation and elevated the concentrations of IFN-gamma and IL-4 in the serum. However, the cerebellar cortex lesions, as an additional control to show the specificity of the FN-lesion results, did not significantly alter the differentiation and apoptosis of thymocytes. These results reveal that the cerebellar FN lesions accelerate the differentiation of thymocytes into mature helper T lymphocytes in the thymus and enhance function of the helper T cells in the peripheral immune tissue. Collectively, these findings suggest a substantial modulation of immune system by the cerebellum.

CD8 positive T-cell infiltration in the dentate nucleus of paraneoplastic cerebellar degeneration.

Recent reports have discussed the presence of cytotoxic T cells in paraneoplastic cerebellar degeneration (PCD). We report an autopsy case of PCD associated with anti-Hu antibody, in which we revealed infiltration of CD8+ T cells in and around the dentate nucleus but not in the cerebellar cortex, in addition to severe Purkinje cell loss. Some infiltrated mononuclear cells expressed cytotoxic cell marker, Granzyme B. Decrease of neurons and reduced presynapses were demonstrated in the dentate nucleus. This is the first report that suggests the possibility of the dentate nucleus being primarily attacked followed by Purkinje cell loss in PCD.

[Effect of cerebellar interposed nuclei on lymphocyte function].

AIM: To increase the cognition of cerebellar functions and the knowledge of neuroimmunology, the effect of cerebellar interposed nuclei (IN), one of three deep nuclei in cerebellum, on lymphocyte function was investigated. METHODS: Kainic acid (KA) was microinjected into bilateral IN for lesions of neuronal bodies in the IN. Control rats was microinjected with saline into their IN. On days 8, 16 and 32 following the IN lesions, the lymphocyte number in the peripheral blood was measured by blood corpuscle counter. Meanwhile, lymphocyte proliferation induced by concanavalin A (Con A), cytotoxicity of natural killer (NK) cells against YAC-1 cells, and anti-SRBC IgM antibody in the serum were examined respectively by methyl-thiazole-tetrazolium (MTT) assay, flow cytometry and ELISA assay. RESULTS: The lymphocyte number in the peripheral blood was significantly reduced on days 8, 16 and 32 following the effective lesions of the bilateral IN in comparison with that of control. The Con A-induced lymphocyte proliferation, the NK cell cytotoxicity to YAC-1 cells, and the titer of anti-SRBC IgM antibody in the serum, were all significantly attenuated on days 8, 16 and 32 following the effective lesions of the bilateral IN in comparison with those of control. There were not remarkable differences between the days 8, 16 and 32 in the decreased lymphocyte number and functions induced by the lesions of the bilateral IN. CONCLUSION: Effective lesions of the cerebellar bilateral IN of rats cause an inhibition in lymphocyte number and functions of T, B and NK cells, strongly showing that the cerebellar IN can modulate lymphocyte functions.

[Involvement of cerebellohypothalamic projections in the modulation of lymphocyte function by cerebellar fastigial nuclei].

AIM: To explore the effect of cerebellar fastigial nuclei (FN)on lymphocyte function and the pathway mediating the effect. METHODS: Kainic acid (KA) was microinjected into bilateral FN of rats to destroy neuronal bodies in the FN. On the eighth day after the surgery, lymphocyte percentage in the peripheral blood and level of sheep red blood cell(SRBC)-specific IgM antibody in the serum were measured by using blood corpuscle counter and enzyme-linked immunosorbent assay (ELISA), respectively.A technology of electrolytic lesion was used to destroy the projections of cerebellar FN neurons to hypothalamus in decussation of superior cerebellar peduncle(xscp). RESULTS: On the eighth day after the microinjection of KA into the bilateral FN of rats, the Nissl-stained neuronal bodies in the FN disappeared and glia could proliferated within the damaged FN. In the nuclei close to FN, the interposed nuclei and the dentate nuclei, Nissl-stained neurons still could be seen. On the control cerebellar sections, in which FN was infused with saline, we could see the normal Nissl-stained neurons in the FN and the other two nuclei.On day 8 following the effective FN lesions, both the lymphocyte percentage in the peripheral blood and the level of anti-SRBC IgM antibody in the serum were significantly increased in comparison with those of control rats infused with saline in the FN. On the eighth day after electrolytic lesion of the fibres in xscp, the FN-hypothalamic projections were damaged and there were no visible BDA-positive endings in hypothalamus. Meanwhile, both the lymphocyte percentage in the peripheral blood and the level of anti-SRBC IgM antibody in the serum were remarkably enhanced relative to those of control rats with sham lesion of xscp. CONCLUSION: The electrolytic lesion of the FN-hypothalamic projections in xscp causes an enhancement of lymphocyte function similar to that of KA lesions of neuronal soma in the FN. These findings suggest that the cerebellohypothalamic projections participate in mediating the modulation of lymphocyte function by the cerebellum.

An update on opsoclonus.

PURPOSE OF REVIEW: The aim of this article is to review opsoclonus, with particular emphasis on its immunopathogenesis and pathophysiology. RECENT FINDINGS: Infections (West Nile virus, Lyme disease), neoplasms (non-Hodgkin's lymphoma, renal adenocarcinoma), celiac disease, and allogeneic hematopoietic stem cell transplantation can cause opsoclonus. Newly identified autoantibodies include antineuroleukin, antigliadin, antiendomysial, and anti-CV2. Evidence suggests that the autoantigens of opsoclonus reside in postsynaptic density, or on the cell surface of neurons or neuroblastoma cells (where they exert antiproliferative and proapoptotic effects). Most patients, however, are seronegative for autoantibodies. Cell-mediated immunity may also play a role, with B and T-cell recruitment in the cerebrospinal fluid linked to neurological signs. Rituximab, an anti-CD20 monoclonal antibody, seems efficacious as an adjunctive therapy. Although changes in synaptic weighting of saccadic burst neuron circuits in the brainstem have been implicated, disinhibition of the fastigial nucleus in the cerebellum, or damage to afferent projections to the fastigial nucleus, is a more plausible pathophysiologic mechanism which is supported by functional magnetic resonance imaging findings in patients. SUMMARY: There is increasing recognition that both humoral and cell mediated immune mechanisms are involved in the pathogenesis of opsoclonus. Further studies are needed to further elucidate its immunopathogenesis and pathophysiology in order to develop novel and efficacious therapy.

Cerebellar interposed nucleus lesions suppress lymphocyte function in rats.

We previously reported that the cerebellar fastigial nucleus, output nucleus of the spinocerebellum, modulates lymphocyte function. To further explore the role of the cerebellum in neuroimmunomodulation, we here lesioned bilaterally the cerebellar interposed nuclei (IN) of rats with kainic acid (KA) injections. On days 8, 16 and 32 after IN lesions, lymphocyte percentage in peripheral white blood cells was examined. Furthermore, proliferation of lymphocytes from mesenteric lymph nodes induced by concanavalin A, sheep red blood cell-specific IgM antibody in the serum and cytotoxicity of natural killer cells from spleen against YAC-1 cells were measured by methyl-thiazole-tetrazolium assay, enzyme-linked immunosorbent assay and flow cytometric assay, respectively. On days 8, 16 and 32 after KA injection in the IN, the lymphocyte percentage in the peripheral white blood cells was notably diminished with respect to control rats injected with saline in the IN. Concanavalin A-induced lymphocyte proliferation, serum sheep red blood cell-specific IgM antibody and natural killer cell toxicity of the IN-lesioned rats were significantly attenuated with respect to IN-saline control rats at all the post-lesion time points. The findings reveal that KA-induced neuronal loss in the IN of both sides exerts an inhibitory effect on number and functions of T, B and natural killer lymphocytes, and indicate that the cerebellar IN participates in regulating immune function. Thus, the data suggest that the cerebellum may be an important brain area for neuroimmunomodulation, besides its well-known role in motor control.

[Effect of cerebellar fastigial nucleus lesions on lymphocyte function].

AIM: To investigate effect of cerebellar fastigial nuclei (FN), one of three deep nuclei in cerebellum, on lymphocyte function, and possible central pathway involved in the effect. METHODS: Kainic acid (KA) was microinjected into bilateral FN of rats. On the eighth day after the surgery, lymphocytes from the mesenteric lymph nodes were incubated to measure their proliferative reaction to concanavalin A (Con A) by means of colorimetric assay of methyl thiazole tetrazolium (MTT), and natural killer (NK) cells from the spleen were cultured to evaluate their cytotoxicity to YAC-1 cells with the aid of flow cytometric assay. Simultaneously, glutamate content in the hypothalamus was determined by high performance liquid chromatography (HPLC). As control, 0.9% saline was microinjected into the bilateral FN of rats. When these experiments ended, cerebellar sections and Nissl stain for each rat were made to observe the location and extent of the lesions. If the lesion areas were not in the bilateral FN or not limited in the FN, the results were discarded. RESULTS: On day 8 following the KA injection of FN, the Nissl-stained cerebellar sections showed the neuronal bodies in the FN were effectively damaged by KA. Simultaneously, the lymphocyte proliferation induced by Con A was significantly increased and the NK cell cytotoxicity to YAC-1 target cells was remarkably enhanced when compared with those of the control animals microinjected with saline in their bilateral FN. At the same time as these changes of lymphocyte functions occurred, glutamate content in the hypothalamus was markedly reduced relative to that in the control hypothalamus. CONCLUSION: Effective lesions of cerebellar bilateral FN of rats can cause an enhancement of lymphocyte functions, including increase of proliferation of T cells and cytotoxicity of NK cells. The cerebello hypothalamic glutamatergic projections may be involved in the pathway of cerebellar FN immunomodulation.

A murine model for neuropsychiatric disorders associated with group A beta-hemolytic streptococcal infection.

A syndrome of motoric and neuropsychiatric symptoms comprising various elements, including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, can occur in association with group A beta-hemolytic streptococcal (GABHS) infection. We tested the hypothesis that an immune response to GABHS can result in behavioral abnormalities. Female SJL/J mice were immunized and boosted with a GABHS homogenate in Freund's adjuvant, whereas controls received Freund's adjuvant alone. When sera from GABHS-immunized mice were tested for immunoreactivity to mouse brain, a subset was found to be immunoreactive to several brain regions, including deep cerebellar nuclei (DCN), globus pallidus, and thalamus. GABHS-immunized mice having serum immunoreactivity to DCN also had increased IgG deposits in DCN and exhibited increased rearing behavior in open-field and hole-board tests compared with controls and with GABHS-immunized mice lacking serum anti-DCN antibodies. Rearing and ambulatory behavior were correlated with IgG deposits in the DCN and with serum immunoreactivity to GABHS proteins in Western blot. In addition, serum from a GABHS mouse reacted with normal mouse cerebellum in nondenaturing Western blots and immunoprecipitated C4 complement protein and alpha-2-macroglobulin. These results are consistent with the hypothesis that immune response to GABHS can result in motoric and behavioral disturbances and suggest that anti-GABHS antibodies cross-reactive with brain components may play a role in their pathophysiology.

Evidence for a role of dopaminergic mechanisms in the immunostimulating effect of mu-opioid receptor agonist DAGO.

The present study demonstrates that: (1) activation of micro -opioid receptors by systemic administration of a highly selective agonist DAGO (100 microg/kg) results in a significant increase in the number of plaque- and rosette-forming cells in the spleen of CBA mice as well as Wistar rats on the 5th day following sheep red blood cells (5 x 10(8)) immunization, (2) the immunostimulatory effect of DAGO is mediated by central mechanisms including the hypothalamus-hypophysis complex; (3) the postsynaptic dopamine (DA) receptors of D2 type are involved in the DAGO-induced immunostimulation since the combined treatment of animals with haloperidol (2 mg/kg), a blocker of DA D2 receptors, and DAGO abolished this effect; (4) the nuclei caudatus and accumbens of the nigrostriatal and mesolimbic DAergic systems, respectively, are implicated in the immune response stimulation caused by DAGO.

Intrinsic regulation of brain inflammatory responses.

It is now well accepted that inflammatory responses in brain contribute to the genesis and evolution of damage in neurological diseases, trauma, and infection. Inflammatory mediators including cytokines, cell adhesion molecules, and reactive oxygen species including NO are detected in human brain and its animal models, and interventions that reduce levels or expression of these agents provide therapeutic benefit in many cases. Although in some cases, the causes of central inflammatory responses are clear--for example those due to viral infection in AIDS dementia, or those due to the secretion of proinflammatory substances by activated lymphocytes in multiple sclerosis--in other conditions the factors that allow the initiation of brain inflammation are not well understood; nor is it well known why brain inflammatory activation is not as well restricted as it is in the periphery. The concept is emerging that perturbation of endogenous regulatory mechanisms could be an important factor for initiation, maintenance, and lack of resolution of brain inflammation. Conversely, activation of intrinsic regulatory neuronal pathways could provide protection in neuroinflammatory conditions. This concept is the extension of the principle of "central neurogenic neuroprotection" formulated by Donald Reis and colleagues, which contends the existence of neuronal circuits that protect the brain against the damage initiated by excitotoxic injury. In this paper we will review work initiated in the Reis laboratory establishing that activation of endogenous neural circuits can exert anti-inflammatory actions in brain, present data suggesting that these effects could be mediated by noradrenaline, and summarize recent studies suggesting that loss of noradrenergic locus ceruleus neurons contributes to inflammatory activation in Alzheimer's disease.

T-cell receptor analysis in anti-Hu associated paraneoplastic encephalomyelitis.

OBJECTIVE: To analyze the T-cell receptor (TCR) repertoire in the inflammatory infiltrates of the nervous system and tumor of patients with anti-Hu associated paraneoplastic encephalomyelitis and sensory neuronopathy (PEM/SN). BACKGROUND: In PEM/SN, the pathogenic role of the infiltrating T cells is speculative. TCR analysis may establish whether these lymphocytes are attracted nonspecifically by a proinflammatory environment or are driven by a specific antigen or superantigen. METHODS: We examined frozen tissues of seven patients with PEM/SN using immunohistochemical and PCR analysis. Of 62 tissue blocks from seven patients, 19 blocks from five patients had >100 CD3+ cells per section infiltrating the nervous system or tumor. These infiltrates allowed screening of the TCR Vbeta family repertoire using a panel of 18 antibodies that recognize family-specific regions of most TCR Vbeta families against which antibodies have been generated. To distinguish between antigen-driven clonal and superantigen-driven family expansion, we extracted RNA from frozen tissue and performed reverse transcriptase (RT)-PCR analysis followed by subcloning and sequencing of the antigen-specific CDR3 region of the TCR Vbeta chain. RESULTS: All five patients showed a limited Vbeta repertoire. An overrepresentation (>10% of total CD3+) of certain Vbeta families was identified in three patients (as high as 45% of total CD3+), which consisted mainly of CD8 + cells. CDR3 sequences obtained from one patient revealed an in situ expansion of two clones in the amygdala (one at a frequency of 57%) and four clones in the tumor. CONCLUSION: These findings suggest that an antigen-driven oligoclonal cytotoxic T-cell response plays a role in the pathogenesis of anti-Hu associated PEM/SN.

Protein L-isoaspartyl methyltransferase: developmentally regulated gene expression and protein localization in the central nervous system of aged rat.

We have cloned a cDNA encoding protein L-isoaspartyl methyltransferase (PIMT) and characterized gene expression in the development, maturation, and the aging process of the central nervous system by RNA blot analysis, western blot analysis, and immunohistochemistry. PIMT transcript was detected in rat embryonic brain and showed a linear up-regulation during the maturation of the brain and maintained its level in aged rat brain. Immunoblot analysis also supported a linear increase in the amount of PIMT in the maturation process of rat brains. An immunohistochemical study showed that PIMT is strongly expressed in neurons and weakly but definitively in glial cells and oligodendrocytes. These immunoreactivities significantly increased in some neurons of the hippocampus, cerebral cortex, and the brain stem of aged rat brain. The present results suggest that the expression of PIMT is associated with the amount of racemized/isomerized proteins accumulated during the developmental and aging process of the central nervous system.

The new monodendritic neuronal type within the adult human cerebellar granule cell layer shows calretinin-immunoreactivity.

The distribution of calretinin immunoreactive structures within the granule cell layer of the adult human cerebellar cortex was studied using the avidin-biotin peroxidase method. Immunoreactivity is found in numerous fibers and glomerular formations, in Golgi- and Lugaro cells as well as in a recently described novel neuronal type, the monodendritic cell. The soma of the monodendritic neuron contains a faintly stained nucleus and issues a single short dendrite terminating in a tuft. Most probably, the tuft contributes to the formation of a glomerulum. Soma and tuft are of about the same size (diameter 10-18 microns). The number of monodendritic neurons is higher in the vermal than in the hemispheric part of the lobulus (lobulus VII) and is higher in lobulus X than in lobulus VII of the vermis.