Endocannabinoids control spasticity in a multiple sclerosis model.

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.

The protective effects of omega-6 fatty acids in experimental autoimmune encephalomyelitis (EAE) in relation to transforming growth factor-beta 1 (TGF-beta1) up-regulation and increased prostaglandin E2 (PGE2) production.

Polyunsaturated fatty acids are known to affect the immune response and administration of the omega-6 fatty acid linoleic acid has been reported to be beneficial in multiple sclerosis (MS) and EAE. In this study we have investigated the effects of oral feeding of plant lipid rich in the omega-6 fatty acid gamma-linolenic acid from Borago officinalis on acute and relapse disease and the immune response in EAE using SJL mice. EAE was induced by an encephalitogenic peptide (92-106) of myelin oligodendrocyte glycoprotein (MOG), and mice were fed the plant lipid daily from 7 days after EAE induction to assess the effects on acute disease and from day 25 to assess the effects on disease relapse. The clinical incidence and histological manifestations of acute EAE, and the clinical relapse phase of chronic relapsing EAE (CREAE) were markedly inhibited by omega-6 fatty acid feeding. A significant increase in the production of TGF-beta1 in response to concanavalin A (Con A) at day 13 and a significant increase in TGF-beta1 and PGE2 to Con A, PPD and MOG peptide (92-106) at day 21 were detected in spleen mononuclear cells from fatty acid-fed mice. There was no difference in interferon-gamma, IL-4 and IL-2 production between the fatty acid-fed and control groups. Significantly higher TGF-beta mRNA expression was found in the spleens of omega-6 fatty acid-fed mice at day 21. There were no differences in spleen cell proliferative response to Con A, PPD and MOG peptide (92-106). Biochemical analysis of spleen cell membrane fatty acids revealed significant increases in the eicosanoid precursor fatty acids dihomo-gamma-linolenic acid and arachidonic acid in response to gamma-linolenic acid feeding, indicating rapid metabolism to longer chain omega-6 fatty acids. These results show that oral feeding of gamma-linolenic acid-rich plant lipid markedly affects the disease course of acute EAE and CREAE and is associated with an increase in cell membrane long chain omega-6 fatty acids, production of PGE2 and gene transcription and, on activation, secretion of TGF-beta1.

Cannabinoids control spasticity and tremor in a multiple sclerosis model.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) is an autoimmune model of multiple sclerosis. Although both these diseases are typified by relapsing-remitting paralytic episodes, after CREAE induction by sensitization to myelin antigens Biozzi ABH mice also develop spasticity and tremor. These symptoms also occur during multiple sclerosis and are difficult to control. This has prompted some patients to find alternative medicines, and to perceive benefit from cannabis use. Although this benefit has been backed up by small clinical studies, mainly with non-quantifiable outcomes, the value of cannabis use in multiple sclerosis remains anecdotal. Here we show that cannabinoid (CB) receptor agonism using R(+)-WIN 55,212, delta9-tetrahydrocannabinol, methanandamide and JWH-133 (ref. 8) quantitatively ameliorated both tremor and spasticity in diseased mice. The exacerbation of these signs after antagonism of the CB1 and CB2 receptors, notably the CB1 receptor, using SR141716A and SR144528 (ref. 8) indicate that the endogenous cannabinoid system may be tonically active in the control of tremor and spasticity. This provides a rationale for patients' indications of the therapeutic potential of cannabis in the control of the symptoms of multiple sclerosis, and provides a means of evaluating more selective cannabinoids in the future.

Response to mucosal antigen challenge in IgA nephropathy.

While IgA nephropathy (IgAN) is characterized by the deposition of glomerular IgA, the source of the deposited IgA is not known, with both the mucosal and systemic IgA systems being implicated. In order to investigate mucosal and systemic antibody production to mucosal antigen challenge in IgAN, 9 patients and 11 controls were immunized intranasally with tetanus toxoid (TT). There was no significant difference in the serum or saliva IgG, IgA, IgA1, or IgA2 antibody production to TT. However, in IgAN there was an increase of in vitro IgA anti-TT production in Epstein-Barr virus transformed cultures of peripheral blood lymphocytes taken after mucosal immunization. This increase in traffic of immunocompetent cells between the systemic and mucosal systems could play a role in the link between the mucosa and glomerulus in IgAN. Systemic immunization with TT following mucosal priming did not result in any difference in the antibody response between patients and controls. There was no evidence from this study that mucosal immunization results in an enhanced antibody response in IgAN or that mucosal priming alters the subsequent systemic antibody response.

Increased immunoglobulin A and immunoglobulin A1 cells in bone marrow trephine biopsy specimens in immunoglobulin A nephropathy.

The origin of mesangial immunoglobulin A (IgA) in IgA nephropathy remains unknown. To investigate potential abnormalities within the bone marrow in this condition, bone marrow trephine biopsy specimens from seven patients and matched controls were studied using two-color immunofluorescence. In addition, serum levels of IgA and IgA1 were determined by radial immunodiffusion. Serum levels of IgA and IgA1 were higher in patients than in controls (4.53 +/- 1.38 g/L v 2.56 +/- 1 g/L, P < 0.01 and 3.68 +/- 1.11 g/L v 1.92 +/- 0.7 g/L, P < 0.005, respectively). In addition, patient trephine biopsy specimens contained an increased percentage of IgA plasma cells (61.6% +/- 4.4%) compared with controls (47.3% +/- 2.5%) (P < 0.02). The proportion of IgA plasma cells bearing subclass IgA1 was also greater in the patient biopsy specimens (91.6% +/- 1.9%) compared with controls (81.4% +/- 2.7%) (P < 0.01). In patients a positive correlation between the percentage of marrow IgA plasma cells and serum IgA levels was found (r = 0.94, P < 0.002). However, our studies failed to demonstrate a similar correlation between serum IgA1 levels and IgA1 marrow cells. These findings support the hypothesis that mesangial IgA may derive from the bone marrow.

In vitro immunoglobulin isotype suppression in immunoglobulin A nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial IgA deposition, and up-regulation of the IgA system is frequently described. This study investigated in vitro immunoglobulin isotype production by peripheral blood mononuclear cells from patients with IgAN and controls, without mitogenic stimulation and under the influence of cell cycle inhibitors and cyclosporin A (CyA). In controls, only IgM production was suppressed by cell cycle inhibitors, and no isotype was affected by CyA. This suppressible IgM production may represent antigen-independent 'natural antibody'. In IgAN, IgM, IgA and IgG were all suppressed by cell cycle inhibitors, and CyA suppressed IgA production. This state of altered immune activation in IgAN demonstrates T-cell-independent IgA production, suggesting that it is IgA 'natural antibody'. In conclusion, we suggest that mesangial IgA deposition may not be antigen dependent but due to some non-immune characteristic of the IgA molecule.

Expression of J chain mRNA in duodenal IgA plasma cells in IgA nephropathy.

Glomerular IgA in IgA nephropathy (IgAN) is at least in part polymeric, and is thought to derive from the mucosal IgA system in view of the association between mucosal infection and haematuria in this condition. To investigate this hypothesis, an in situ hybridization (ISH) technique was developed for the detection of J chain mRNA, the expression of which has been correlated with the secretion of high level polymeric immunoglobulin (pIg). Endoscopic duodenal biopsies from ten patients and matched controls were examined by: (i) two color immunofluorescence (IF); (ii) ISH; and (iii) combined ISH and IF, to permit simultaneous identification of plasma cell type. IF revealed a reduction in the percentage of IgA plasma cells (P < 0.02) and increased absolute numbers of IgG cells (P < 0.02) in patient biopsies. ISH demonstrated fewer J chain mRNA expressing plasma cells (P < 0.005) with lower signal intensity (P < 0.002) in patients' biopsies compared with controls. Combined ISH and IF confirmed a reduction in J chain mRNA-positive IgA plasma cells in the patient biopsies (P < 0.02). The reduction in J chain mRNA expression in duodenal IgA plasma cells in IgAN argues against the gastrointestinal lamina propria as the source of glomerular pIgA.

Increased IgA and decreased IgG production by Epstein-Barr virus transformed B cells in culture in IgA nephropathy.

Despite many studies describing IgA upregulation both in vivo and in vitro in IgA nephropathy (IgAN), the underlying mechanism of increased IgA production is not known. In this study, Epstein-Barr virus was used to transform B cells in vitro in a T-cell-independent manner in order to investigate immunoglobulin production by B cells in IgAN. B cells from patients with IgAN produced more IgA and less IgG in culture than controls. While both IgA subclasses contributed to the increase in IgA production, only IgA1 synthesis was significantly higher than controls. These results of increased IgA production in patients with IgAN, with a concomitant decrease in IgG production, demonstrate hyperactivity of B cells restricted to the IgA isotype in IgAN.

Low antibody affinity restricted to the IgA isotype in IgA nephropathy.

Antibody affinity affects the handling and behaviour of immune complexes, and experimental studies have shown that animals which produce predominantly low-affinity antibody are prone to immune complex deposition resulting in glomerulonephritis. In order to investigate the potential role of antibody affinity in the pathogenesis of IgA nephropathy, affinity of both IgA and IgG antibody isotypes during secondary response to systemic immunization with tetanus toxoid was studied in 20 patients with IgA nephropathy. Patients with IgA nephropathy produced IgA antibodies of significantly lower affinity than controls (P < 0.001), whereas IgG antibody affinities were similar. Contrasting with controls, patients' IgA antibody affinity was inversely related to antibody concentration, with higher responders producing large amounts of low-affinity antibody. IgG antibody affinity increased with time, and maturation of IgG antibody affinity was similar in both controls and patients. IgA affinity in controls decreased with time, and this lack of IgA affinity maturation may explain the relative unimportance of IgA in normal systemic immunity. This temporal decrease in IgA affinity was not observed in patients with IgA nephropathy. The production of low-affinity IgA in IgA nephropathy may provide an explanation for the predominant deposition of IgA in this disease.

Antineutrophil cytoplasm antibodies (ANCA) of IgA isotype in adult Henoch-Schönlein purpura.

ANCA are associated with certain forms of systemic vasculitis, and have been reported previously to be of the IgG and IgM isotype. We examined the possible association between IgA ANCA and the IgA-related diseases Henoch-Schönlein purpura (HSP) and IgA nephropathy (IgAN). IgA and IgG ANCA were detected by isotype-specific solid-phase assays with a crude neutrophil extract, and their presence was confirmed by antigen-specific fluid-phase competitive inhibition tests and by indirect immunofluorescence. The possible interference by IgA rheumatoid factor was excluded. IgA ANCA were detected in sera from 11/14 HSP patients (79%), from 1/30 IgAN patients (3%), from 1/40 patients with vasculitides classically associated with IgG ANCA (2.5%), and in none of 60 sera from healthy blood donors. IgG ANCA were present with IgA ANCA in three patients with HSP. Only one HSP serum had anti-myeloperoxidase (MPO) activity by both IgA and IgG isotype-specific ELISA, and none was positive for proteinase 3 (PR3). Western blot analysis performed with neutrophil extract showed that the four strongest IgA ANCA-positive HSP sera reacted with a 51-kD protein; Western blot performed on cellular fractions showed that this protein is primarily membrane-associated, and different from fibronectin. Our study suggests that adult HSP is closely associated with circulating IgA ANCA, which may be directed against a different autoantigen than that recognized by IgG ANCA.

Systemic and mucosal IgA responses to systemic antigen challenge in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by the deposition of glomerular IgA. The source of the deposited IgA is not known, but both the mucosal and systemic IgA systems have been implicated. In order to investigate mucosal and systemic antibody production to systemic antigen challenge in IgAN, 20 patients and 20 controls where immunized with tetanus toxoid (TT). While patients with IgAN responded with a similar serum IgG, IgA, IgA1, and IgA2 antibody response to controls, they did, however, produce more IgA1 antibodies relative to IgA2 (P < 0.05). No salivary IgA antibody response was observed to systemic immunization in controls; however, there was a significant IgA response to TT in the saliva of patients with IgAN. IgA antibodies were produced in vitro by Epstein Barr virus (EBV)-transformed peripheral blood lymphocytes (PBLs) obtained from control blood only when taken shortly (1 or 2 weeks) after immunization. Patients with IgAN produced significantly more IgA anti-TT positive cultures than controls and for a longer period (P < 0.01) after immunization. In contrast, IgG anti-TT was produced in EBV-transformed cultures at all time points, but with no difference between IgAN and controls in the proportion of IgG producing cultures. These results demonstrate increased IgA antibody production in both the systemic and mucosal IgA systems following systemic immunization in IgAN and suggest an abnormal overlap between the two systems in IgAN.

Deficiency of IgG subclass antibody response to tetanus toxoid associated with high serum IgA levels in IgA nephropathy.

In order to investigate IgG subclass response in IgA nephropathy (IgAN), 20 patients and 20 age and sex matched controls were systemically immunized with tetanus toxoid (TT). Nineteen/20 controls and 19/20 IgAN made a serum IgG anti-TT response of similar magnitude. However, significantly more patients with IgAN had undetectable amounts of at least one IgG subclass antibody to this antigen than controls (7/19 IgAN, 1/19 controls, p < 0.05). All individuals with an IgG subclass anti-TT deficiency lacked IgG1 and/or IgG4. Two patients made no IgG3 anti-TT (as well as no IgG1 and IgG4 anti-TT) but all individuals who responded to TT made IgG2 anti-TT. Total IgG subclass levels in IgAN did not differ from controls and no patient with IgAN had a total IgG subclass deficiency. Total serum IgA was significantly raised in IgAN (p < 0.002) and 6/7 IgAN with an IgG anti-TT subclass deficiency had a serum IgA level of over 3.2 milligrams compared to only 2/12 IgAN with no IgG subclass anti-TT deficiency (p < 0.01). The association of high serum IgA levels with IgG subclass deficiency to TT may be due to an abnormality in switching from IgG to IgA production in IgAN, or a manifestation of a defect of immunoregulation analogous to that proposed in IgA deficiency.

Elevation of IgA in IgA nephropathy is localized in the serum and not saliva and is restricted to the IgA1 subclass.

Glomerular deposits in IgA nephropathy (IgAN) are predominantly IgA1 but their origin is not known. Previous studies have analysed serum or saliva IgA, but not in the same patients. To investigate whether IgA and IgA subclass anomalies occur in IgAN at both mucosal and systemic sites, blood and saliva from 20 patients and 20 age- and sex-matched controls were studied. Patients with IgAN had significantly increased serum IgA (P < 0.002), and this elevation was restricted to the IgA1 subclass. Serum IgA1 was also increased significantly (P < 0.001) but IgA2 was not. By contrast salivary IgA, IgA1, and IgA2 did not differ significantly from the controls. These results demonstrate that the elevated serum IgA is predominantly IgA1 and is likely to be of systemic origin. Further studies should consider the bone marrow as a potential source of the elevated IgA1.

Increased and prolonged production of specific polymeric IgA after systemic immunization with tetanus toxoid in IgA nephropathy.

IgA nephropathy (IgAN) is a chronic form of glomerulonephritis which is characterized by the deposition in the glomerular mesangium of polymeric IgA (pIgA), the source of which is unknown. In order to investigate the production of pIgA in IgAN, patients were immunized systemically with tetanus toxoid (TT). Two weeks after immunization patients and controls responded to TT with an IgA response of similar magnitude. HPLC separation of sera showed that patients with IgAN produce significantly more pIgA anti-TT than controls (7.7 versus 2.88 arbitrary units; P less than 0.04). At this time, 33% of serum IgA anti-TT produced by patients with IgAN was polymeric, compared with 21% produced by controls (P less than 0.02). Monomeric IgA (mIgA) anti-TT levels were similar in both groups. Four weeks after immunization the proportion of pIgA anti-TT in controls and patients was significantly reduced from the 2 week level (from 21% to 0%, P less than 0.02 for controls; and from 33% to 8%, P less than 0.001, for patients). Only four out of 12 controls had any detectable pIgA anti-TT at this time compared with nine out of 10 patients with IgAN (P less than 0.05), and IgAN patients produced proportionally more pIgA anti-TT than did controls (median 8%, interquartile ranges (IQR) 4-10% versus 0% IQR 0-3%; P less than 0.01). HPLC analysis under acid conditions did not alter the pattern of pIgA and mIgA anti-TT, suggesting that the high molecular weight IgA fraction was not due to complexes. These data indicate that circulating pIgA results (at least in part) from a systemic response to antigen, which may be exaggerated in IgAN.