Generation of a novel CD30+ B cell subset producing GM-CSF and its possible link to the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-Beffs ) in humans. In this study, we sought to elucidate the generation mechanism of GM-Beffs and also determine a role of this subset in SSc. Among Th-associated cytokines, IL-4 most significantly facilitated the generation of GM-Beffs within memory B cells in healthy controls (HCs). In addition, the profibrotic cytokine transforming growth factor (TGF)-ß further potentiated IL-4- and IL-13-induced GM-Beffs . Of note, tofacitinib, a Janus kinase (JAK) inhibitor, inhibited the expression of GM-CSF mRNA and protein in memory B cells induced by IL-4, but not by TGF-ß. GM-Beffs were enriched within CD20+ CD30+ CD38-/low cells, a distinct population from plasmablasts, suggesting that GM-Beffs exert antibody-independent functions. GM-Beffs were also enriched in a CD30+ fraction of freshly isolated B cells. GM-Beffs generated under Th2 conditions facilitated the differentiation from CD14+ monocytes to DC-SIGN+ CD1a+ CD14- CD86+ cells, which significantly promoted the proliferation of naive T cells. CD30+ GM-Beffs were more pronounced in patients with SSc than in HCs. A subpopulation of SSc patients with the diffuse type and concomitant interstitial lung disease exhibited high numbers of GM-Beffs . Together, these findings suggest that human GM-Beffs are enriched in a CD30+ B cell subset and play a role in the pathogenesis of SSc.

Consensus Paper. Cerebellar Reserve: From Cerebellar Physiology to Cerebellar Disorders.

Cerebellar reserve refers to the capacity of the cerebellum to compensate for tissue damage or loss of function resulting from many different etiologies. When the inciting event produces acute focal damage (e.g., stroke, trauma), impaired cerebellar function may be compensated for by other cerebellar areas or by extracerebellar structures (i.e., structural cerebellar reserve). In contrast, when pathological changes compromise cerebellar neuronal integrity gradually leading to cell death (e.g., metabolic and immune-mediated cerebellar ataxias, neurodegenerative ataxias), it is possible that the affected area itself can compensate for the slowly evolving cerebellar lesion (i.e., functional cerebellar reserve). Here, we examine cerebellar reserve from the perspective of the three cornerstones of clinical ataxiology: control of ocular movements, coordination of voluntary axial and appendicular movements, and cognitive functions. Current evidence indicates that cerebellar reserve is potentiated by environmental enrichment through the mechanisms of autophagy and synaptogenesis, suggesting that cerebellar reserve is not rigid or fixed, but exhibits plasticity potentiated by experience. These conclusions have therapeutic implications. During the period when cerebellar reserve is preserved, treatments should be directed at stopping disease progression and/or limiting the pathological process. Simultaneously, cerebellar reserve may be potentiated using multiple approaches. Potentiation of cerebellar reserve may lead to compensation and restoration of function in the setting of cerebellar diseases, and also in disorders primarily of the cerebral hemispheres by enhancing cerebellar mechanisms of action. It therefore appears that cerebellar reserve, and the underlying plasticity of cerebellar microcircuitry that enables it, may be of critical neurobiological importance to a wide range of neurological/neuropsychiatric conditions.

Tacrolimus is effective for lupus nephritis patients with persistent proteinuria.

OBJECTIVES: To evaluate the safety and potential efficacy of tacrolimus for the treatment of patients with lupus nephritis and persistent proteinuria. METHODS: A total of 23 Japanese patients with lupus nephritis (21 females/2 males) were enrolled in this study. Patients were administered tacrolimus at a dose of 2-3 mg once daily after the evening meal for 6 months. The dose of tacrolimus was unchanged throughout the study period. Concomitant prednisolone therapy was unchanged or gradually tapered, while other immunosuppressants were stopped at the start of tacrolimus treatment. RESULTS: Tacrolimus was well tolerated, and none of the patients developed adverse drug reactions that required discontinuation of the study. Daily urinary protein loss, the U-prot/U-creat ratio, and serum albumin were significantly improved after 4 months, 3 months, and 1 month of treatment with tacrolimus (p<0.05), respectively, and the improvement persisted until 6 months. The serum complement hemolytic activity (CH50), complement C3 level, and CRP level were also significantly improved after treatment with tacrolimus (p<0.05). Improvement of the U-prot/U-creat ratio was most prominent for patients who were in WHO class IV. CONCLUSIONS: Tacrolimus is safe and effective as maintenance therapy for patients with lupus nephritis, at least for 6 months. A larger randomised, controlled trial over a longer period is needed to confirm these results.

A dose-escalation study of rituximab for treatment of systemic lupus erythematosus and Evans' syndrome: immunological analysis of B cells, T cells and cytokines.

OBJECTIVE: Accumulating evidence suggests that B-cell depletion therapy by rituximab may be effective for autoimmune disorders. However, an optimal dose of rituximab and a mechanism of its action remain to be established. We performed a dose-escalation study for treatment of Japanese patients with autoimmune diseases including eight with SLE and one with Evans' syndrome. METHODS: Rituximab was infused intravenously, weekly 4 times in a dose-escalating fashion at three different doses of 100, 250 or 375 mg/m(2) to three patients each. Immunological parameters were monitored at certain points until 12 months after the treatment. RESULTS: Rituximab was well tolerated and safe in these patients. Seven out of eight SLE patients and one with Evans' syndrome clinically responded completely or partially to the treatment. Four patients achieved long-term remission (18-30 months) without any additional treatment. In these patients, a significant decrease in circulating B cells continued for 6 months after the treatment. The mean fluorescence intensities of CD19, CD21, CD40 and BR3 on the residual B cells as well as the percentage of CD69+ CD4+ T cells decreased significantly. Serum TNF-alpha levels decreased significantly on day 2. The Th1/Th2 balance of CD4+ T cells gradually shifted towards a Th1 type by 6 months. CONCLUSION: In addition to B-cell depletion, modification of B-cell and T-cell phenotypes as well as cytokine profiles may be involved in the action of rituximab.

Association of MBL gene polymorphisms with major bacterial infection in patients treated with high-dose chemotherapy and autologous PBSCT.

A growing body of evidence indicates that genetic factors are involved in an increased risk of infection. We investigated whether mannose-binding lectin (MBL) gene polymorphisms that cause low levels of MBL are associated with the occurrence of major infections in patients, mainly bearing hematological malignancies, after high-dose chemotherapy (HDT) rescued by autologous peripheral blood stem cell transplantation (auto-PBSCT). A retrospective evaluation of 113 patients treated with HDT and auto-PBSCT revealed that the low-producing genotypes, B/B and B/LXA, were associated with major bacterial infection (P=0.0016, OR 7.9). We next performed a nation-wide large-scale study to assess the allele frequency of the MBL coding mutation in a total of 2623 healthy individuals in Japan. The frequency of allele B was estimated to be approximately 0.2, almost the same in seven different areas of Japan. This common occurrence suggests that MBL deficiency may play an important role in the clinical settings of immunosuppression.

Aberrant HS1 molecule in a patient with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive B lymphocytes, which are supposed to carry aberrant signal transduction after the stimulation of B-cell receptor (BCR). To investigate abnormalities in BCR-mediated signaling pathway in lupus B lymphocytes, we analyzed HS1, a molecule downstream of BCR, in 80 Japanese SLE patients. We identified 37 amino acid deletion of HS1 in a 25-year-old female patient, and the aberrant HS1 lacked a part of a functional motif. Analysis of genomic DNA revealed that the aberrant HS1 was caused by exon skipping. Family study showed that the patient as well as her father and sister are heterozygous for the abnormality. WEHI-231 cell, a mouse B cell line, transfected with the aberrant HS1 displayed a significantly increased cell death upon cross-linking of BCR. Additionally, peripheral B lymphocytes from the patient exerted increased apoptosis after BCR stimulation compared to those from control SLE patients. These data suggest that the aberrant HS1 molecule may transmit an accelerated signal after BCR stimulation and may play a role in the activation of autoreactive B lymphocytes.

Presynaptic inhibition of cerebellar GABAergic transmission by glutamate decarboxylase autoantibodies in progressive cerebellar ataxia.

Autoantibodies against glutamic acid decarboxylase (GAD) have been found in stiff-man syndrome, insulin dependent diabetes mellitus, and progressive cerebellar ataxia. A patient with progressive cerebellar ataxia is described who was positive for GAD autoantibodies, and had Sjögren's syndrome. Immunohistochemical studies using CSF and serum samples from the patient showed immunoreactivities in axon terminals of cerebellar GABAergic neurons. A whole cell patch clamp technique recording from rat cerebellar slices showed that the CSF, presumably through GAD autoantibodies, presynaptically inhibited GABAergic transmission. Intravenous administration of immunoglobulin failed to improve clinical symptoms and immunoreactivities examined after therapy. The findings suggest that GAD autoantibodies play a pathogenic part in reducing GABA release in in vitro slices.

Gait disturbances in patients with pontine medial tegmental lesions: clinical characteristics and gait analysis.

OBJECTIVE: To determine the clinical characteristics of gait disorders in patients with pontine medial tegmental lesions. DESIGN: We compared features of gait disorders between patients with infarcts in the medial tegmentum and those with stroke in other areas of the pons (pathological control subjects) by measuring electromyographic results of lower limb muscles and several biomechanical parameters. PATIENTS: Two patients with infarcts in the rostral medial tegmentum and 4 control subjects. Two of the control patients had lesions in the pontine base, while the lesions in the other 2 were in the pontine tegmentum and base (combined lesions). RESULTS: Patients with rostral medial tegmental lesions and controls with pontine base lesions showed unstable walking characterized by irregular angular displacements and foot pressures. However, they differed by the following 3 features. (1) Rostral medial tegmental lesions elicited truncal ataxia without limb ataxia. In comparison, pontine base lesions elicited limb ataxia without truncal ataxia and caused hemiparesis. (2) Instability was more severe and persistent in patients with the former lesions than in those with the latter lesions. Slowness of walking speed and prolongation of the double-support period were clearly observed in the former group. (3) Electromyographic changes characteristic of cerebellar ataxia were clearly evident in patients with rostral medial tegmental lesions. The electromyographic amplitudes of the gastrocnemius and tibialis anterior muscles were almost constant throughout the gait cycle, resulting in the disappearance of the inherent periodic pattern of each muscle. CONCLUSION: Medial tegmental lesions in the rostral pons cause prolonged and severe unstable walking that resembles spinocerebellar ataxic pattern, and impairment of the spinocerebellar loop might be the pathomechanism underlying such a gait disturbance.

Presynaptic impairment of cerebellar inhibitory synapses by an autoantibody to glutamate decarboxylase.

Glutamic acid decarboxylase (GAD), the enzyme responsible for converting glutamate to gamma-aminobutyric acid (GABA), is a target of humoral autoimmunity in stiff-man syndrome and subacute cerebellar ataxia. Recently, we found that an anti-GAD autoantibody in the CSF of an ataxic patient selectively suppressed GABA-mediated transmission on cerebellar Purkinje cells without affecting glutamate-mediated transmission. Here, we examine the mechanism by which the autoantibody impaired the inhibitory transmission, using immunohistochemistry and whole-cell recording in rat cerebellar slices. The present results indicate that CSF immunoglobulins prepared from an ataxic patient acted on the presynaptic terminals of GABAergic interneurons and decreased GABA release onto Purkinje cells.

Characteristics of parkinsonian and ataxic gaits: a study using surface electromyograms, angular displacements and floor reaction forces.

To clarify the characteristics of parkinsonian and ataxic gaits, we analyzed electromyograms (EMGs) of the thigh and leg muscles, angular displacements of the hip and leg joints, and floor reaction forces during free walking for each gait phase in 16 patients with Parkinson's disease (PD) and 14 ataxic patients with cerebellar degenerations. We studied 17 healthy elderly subjects whose walking speed was similar to that of patients with moderate disease. Free walking by PD patients was characterized by low maximum activity of the gastrocnemius/soleus (GC) and tibialis anterior (TA) muscles. Ataxic patients showed high activity of GC and TA during the period when these muscles were not active in normal walking. The ratio of changes of EMG of the distal muscles to changes in angular displacement of the ankle (DeltaEMG/Deltaangle) was reduced in GC of PD patients in ankle dorsiflexion, whereas it was high in GC and TA of ataxic patients in ankle dorsiflexion and plantarflexion, respectively. Changes in DeltaEMG/Deltaangle coincided with those in proprioceptive reflexes reported previously. Our results showed that measurement of EMG for each phase revealed disease-specific factors, and that of DeltaEMG/Deltaangle might be a conventional clue for estimation of reflexes for these gait disorders.

Synaptic localization of the 67,000 mol. wt isoform of glutamate decarboxylase and transmitter function of GABA in the mouse cerebellum lacking the 65,000 mol. wt isoform.

Subcellular localization of the 67,000 mol. wt isoform of glutamate decarboxylase and neurotransmitter function of GABA were investigated in the cerebellum of the mice lacking the 65,000 mol. wt isoform of glutamate decarboxylase. The GABA content decreased by 25% in the cerebellum. Putative GABA-releasing terminals from basket/stellate and Golgi cells were immunostained with glutamate decarboxylase-67 antibody. Basket cell-derived inhibitory postsynaptic currents in Purkinje cells and the high potassium-induced release of GABA were not significantly affected. Although previous investigations have suggested that glutamate decarboxylase-65 is mainly involved in transmitter synthesis and that glutamate decarboxylase-67 is transported to the nerve terminals only after association with glutamate decarboxylase-65, the present results indicate that glutamate decarboxylase-67 is independently concentrated in the nerve terminals and provides GABA for synaptic transmission in the absence of glutamate decarboxylase-65.

Selective suppression of cerebellar GABAergic transmission by an autoantibody to glutamic acid decarboxylase.

Humoral immune response to glutamic acid decarboxylase (GAD) has been implicated in the pathogenesis of stiff-man syndrome and cerebellar ataxia, but the underlying pathomechanism is unclear. Using a whole-cell patch-clamp technique with rat cerebellar slices, we found that immunoglobulins present in the cerebrospinal fluid of an ataxic patient acted presynaptically to cause a selective suppression of GABAergic transmission. This synaptic depression was most likely elicited by an autoantibody to GAD.

Monoaminergic long-term facilitation of GABA-mediated inhibitory transmission at cerebellar synapses.

Long-term facilitation of neurotransmission by monoaminergic systems is implicated in the cellular mechanism of memory and learning-related processes at invertebrate synapses. Using whole-cell recording and rat cerebellar slices, we have examined whether mammalian monoamine-containing neurons play analogous roles in synaptic plasticity, and our results suggest that serotonin and noradrenaline are critically involved in short- and long-term modulation of GABAergic transmission in the cerebellar cortex. Exogenously applied serotonin and noradrenaline selectively induced a short-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells, their effect subsiding in 30 min. Successive amine applications converted this effect to long-term facilitation lasting more than 2 h. During the monoamine-induced short- and long-term facilitation, spontaneously occurring miniature inhibitory synaptic responses increased in frequency, without significant changes in their mean amplitude and amplitude distribution, as well as the GABA receptor sensitivity of Purkinje cells. The actions of the two amines on the inhibitory transmission were mimicked by forskolin and blocked by kinase inhibitors, H-7, H-89 and Rp-adenosine 3',5'-cyclic monophosphothioate. Thus, serotonin and noradrenaline are likely to activate cyclic-AMP- and protein kinase-dependent pathways in GABAergic interneurons, thereby reinforcing the inhibitory transmission on to Purkinje cells. Repetitive electrical stimulation within the molecular layer mimicked the facilitatory effect induced by exogenous monoamines: namely, neural stimulation selectively elicited long-lasting enhancement of GABAergic transmission in a manner sensitive to the monoamine receptor antagonists, methiothepin and propranolol, and an uptake inhibitor, imipramine. Synaptically released monoamines thus appear to induce cyclic-AMP- and protein kinase-dependent long-term facilitation of cerebellar GABAergic transmission, thereby providing a likely mechanism of synaptic plasticity associated with motor coordination within the mammalian cerebellar system.

Differential response of macrophage subpopulations to myelin degradation in the injured rat sciatic nerve.

Molecular mechanisms of myelin removal by macrophages were explored by examining the immunophenotypes of macrophages following injury of rat sciatic nerve, using a combined method of immunohistochemistry and confocal laser microscopy. In the crush injury model, the involvement in myelin clearance of a cytoplasmic antigen specific for monocytes/macrophages, ED1, was evident. The obvious recruitment of ED1-immunoreactive (-ir) cells was detected first at the crush injury site and then in the distal stump within which Wallerian degeneration had occurred. Double labelling revealed that the ED1-ir cells, except for monocyte-like round cells, always phagocytosed myelin basic protein-ir myelin debris. On the other hand, the expression of ED2, a surface antigen specific for resident macrophages, was significantly different; ED2-ir cells also increased while myelin removal was progressing from day 3 to day 7, but only some of the cells were engaged in myelin phagocytosis. The poor capacity of myelin phagocytosis by ED2-ir cells was supported by the transection model, in which the proximal stump was ligated to suppress regeneration. ED2 may be involved in events other than myelin removal, providing a local environment conducive to axonal regeneration. Our findings thus seem to suggest that ED1 is one of the most reliable markers for cells carrying out myelin phagocytosis, whereas ED2 may participate in entirely different functions. The expression of complement receptor type 3, OX42, was similar to that of ED1 in terms of the swift recruitment of immunopositive cells, their distribution with close association to myelin debris and their high phagocytotic capacity. This supports previously reported in vitro evidence that myelin phagocytosis by macrophages may be complement-mediated.

[Kinematic and EMG pattern of parkinsonian gait].

To clarify characteristic pattern of parkinsonian gait, gait analysis was performed by measuring floor reaction forces, angular displacements, and surface electromyograms on patients with Parkinson's disease (PD) and normal controls. PD patients walked with slow speed, shortened stride, prolonged double support period, and restricted movement of the hip, knee, and ankle joints. Muscle activity of the gastrocnemius and tibialis anterior muscles reduced, and correspondingly pressures for step-in and kick-off decreased. These changes were correlated with severity of clinical scale. Despite these alterations, reciprocal activity of the distal antagonistic muscles was preserved. We conclude that these characteristic alterations reflect distorted mechanisms for gait control in PD and could be one of useful parameters for evaluation of pathophysiology of parkinsonian gait.

[A physiological study on instability in Parkinson's disease].

To clarify roles of impairment of proprioceptive reflexes in instability of parkinsonian gait, delta EMG/delta angle of the gastrocnemius and tibialis anterior muscles was measured during the stance phase on patients with Parkinson's disease and normal controls. This ratio decreased in the gastrocnemius muscle, while it was unchanged in the tibialis anterior muscle. delta EMG/delta angle is likely to be correlated with muscle stiffness(delta muscle force/delta muscle length) that reflects from stretch reflexes. Thus our results suggest an impairment of stretch reflexes of the extensor muscle during parkinsonian gait.

Long-lasting facilitation of inhibitory transmission by monoaminergic and cAMP-dependent mechanism in rat cerebellar GABAergic synapses.

Roles of monoaminergic neurons in synaptic plasticity are not well clarified at mammalian central synapses. We therefore examined the actions of serotonin and noradrenaline on cerebellar excitatory and inhibitory synapses using the whole-cell recording with rat cerebellar slices. Applications of the two amines selectively elicited long-lasting facilitation of gamma-aminobutyric acid (GABA)-mediated inhibitory transmission, possibly through a presynaptic mechanism. The facilitatory action of the amines on GABAergic transmission was mimicked by forskolin and blocked by protein kinase inhibitors, suggesting the involvement of cyclic AMP (cAMP)-dependent mechanism in their action. A monoamine uptake inhibitor, imipramine, enhanced the GABAergic transmission, whereas it was suppressed by monoamine receptor antagonists, propranolol and methiothepin, suggesting that endogenous monoamines released by stimulation enhance the inhibitory transmission at cerebellar GABAergic synapses in sustained and activity-dependent manner. Such monoaminergic facilitation may provide a likely mechanism for synaptic plasticity in motor coordination within the mammalian cerebellar function.