Edward J. Thompson

Increased levels of circulating ICAM-1 in serum and cerebrospinal fluid of patients with active multiple sclerosis. Correlation with TNF-α and blood-brain barrier damage

The mechanism for the initiation of blood-brain barrier damage and intrathecal inflammation in multiple sclerosis (MS) is poorly understood. We have recently reported that levels of tumor necrosis factor-alpha (TNF-alpha) correlate with blood-brain barrier damage in patients with active MS. Stimulation of endothelial cells by TNF-alpha induces the expression of intercellular adhesion molecule-1 (ICAM-1), which is an important early marker of immune activation and response. We report herein for the first time the detection of high levels of free circulating ICAM-1 in serum and cerebrospinal fluid of patients with active MS. Levels of circulating ICAM-1 in these patients correlated with CSF pleocytosis, TNF-alpha levels and blood-brain barrier damage. These findings have important implications for the understanding and investigation of the intrathecal inflammatory response in active MS.

Poststreptococcal acute disseminated encephalomyelitis with basal ganglia involvement and auto‐reactive antibasal ganglia antibodies

Antibasal ganglia antibodies (ABGA) are associated with Sydenhams chorea and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections. We present 10 patients with acute disseminated encephalomyelitis (ADEM) associated with Group A β hemolytic streptococcal infection. The clinical phenotype was novel, with 50% having a dystonic extrapyramidal movement disorder, and 70% a behavioral syndrome. None of the patients had rheumatic fever or Sydenhams chorea. Enzyme‐linked immunosorbent assay, Western immunoblotting, and immunohistochemistry were used to detect ABGA. Neurological (n = 40) and streptococcal (n = 40) controls were used for comparison. Enzyme‐linked immunosorbent assay results showed significantly elevated ABGA in the patients with poststreptococcal ADEM. Western immunoblotting demonstrated ABGA reactivity to three dominant protein bands of 60, 67, or 80 kDa; a finding not reproduced in controls. Fluorescent immunohistochemistry demonstrated specific binding to large striatal neurones, which was not seen in controls. Streptococcal serology was also significantly elevated in the poststreptococcal ADEM group compared with neurological controls. Magnetic resonance imaging studies showed hyperintense basal ganglia in 80% of patients with poststreptococcal ADEM, compared to 18% of patients with nonstreptococcal ADEM. These findings support a new subgroup of postinfectious autoimmune inflammatory disorders associated with Group A β hemolytic streptococcus, abnormal basal ganglia imaging, and elevated ABGA.

Adaptation of the nitrate reductase and Griess reaction methods for the measurement of serum nitrate plus nitrite levels

Nitrite and nitrate determinations in biological fluids are increasingly being used as markers of nitric oxide production. We have modified a nitrate reductase and Griess reaction method for the measurement of serum nitrate and nitrite in ultrafiltrated samples using a microtitre plate. The recoveries of nitrate and nitrite were 95% (range = 86–113%) and 100% (range = 92–109%), respectively. The intra and inter assay coefficients of variation for nitrate plus nitrite in the concentration range 40–50μM were 9·1% and 7·8%, and in the concentration range of 2·5–10μM 23·4% and 25·5%, respectively. At its lower limit the assay is able to detect 125 pmoles of nitrate plus nitrite in 50μL of sample (2·5μmol/L). A mean serum nitrate plus nitrite level of 32·8μmol/L (SD 12·3) was measured in 24 healthy adult volunteers (12 men and 12 women), no age or sex differences were noted.

A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells.

A quantitative and reproducible technique for establishing primary surface cultures from normal and diseased human muscle is described. Successful cultures were prepared from both fresh muscle and that stored up to 96 hr at 4 degrees C. The CPK activity of the muscle cells ranged between 0.5-3.0 micronmoles creatine per min per mg protein at 30 degrees C, thus indicating a high degree of differentiation. Spontaneous contractions were observed in 4 out of the 22 cultures established. Nerve cells were not required to achieve this level of differentiated function. No gross differences in plating efficiency, rate of myotube formation or CPK specific activity were found for the diseased muscle cells cultured so far. However, a 5--10-fold higher cell yield was obtained from muscles of patients with an inflammatory myopathy. The advantages of this technique for carrying out comparative studies on normal and dystrophic muscle cells are discussed.

Oligoclonal banding of IgG in CSF, blood-brain barrier function, and MRI findings in patients with sarcoidosis, systemic lupus erythematosus, and Behçet's disease involving the nervous system.

A retrospective study of CSF and serum analysis from a total of 43 patients with sarcoidosis, 20 with systemic lupus erythematosus, and 12 with Behçets disease with neurological involvement found local synthesis of oligoclonal IgG using isoelectric focusing and immunoblotting in 51%, 25%, and 8% respectively at some stage in their disease. Blood-brain barrier breakdown, when assessed with an albumin ratio found 47% of patients with sarcoidosis, 30% of those with systemic lupus erythematosus, and 42% of patients with Behçets disease exhibiting abnormal barrier function at some time. Serial CSF analysis showed that clinical relapses were associated with worsening barrier function and in some patients the development of local oligoclonal IgG synthesis; conversely steroid treatment led to a statistically significant improvement in barrier function, and in two patients a loss of oligoclonal IgG bands. A higher proportion of patients had MRI abnormalities than oligoclonal IgG or blood-brain barrier breakdown, MRI being abnormal in 16 of 19 patients with sarcoidosis, three of four patients with systemic lupus erythematosus, and seven of nine patients with Behçets disease, although this may have been due to temporal factors. In the differential diagnosis of chronic neurological disorders, locally synthesised oligoclonal IgG cannot distinguish between diseases, but the loss of bands seen in two patients contrasts with what is seen in multiple sclerosis, and thus may be a useful diagnostic clue.

Isoelectric focusing of cerebrospinal fluid immunoglobulin G: an annotated update

A revised agarose isoelectric focusing method for detecting oligoclonal IgG in unconcentrated cerebrospinal fluid is presented. The technique is shown to be robust and reproducible and suitable for the detection of intrathecal IgG synthesis.

The potential role of nitric oxide in multiple sclerosis

Nitric oxide (.NO) and its reactive derivative peroxynitrite (ONOO7) have been implicated in the pathogenesis of multiple sclerosis (MS). They are cytotoxic to oligodendrocytes and neurones in culture by inhibiting the mitochondrial respiratory chain (complexes II/III and IV) and inhibiting certain key intracellular enzymes. Recently .NO has been implicated as a possible aetiological factor in reversible conduction block in demyelinated axons. Inducible nitric oxide synthase (iNOS) is upregulated in the central nervous system of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. In some EAE models inhibiting iNOS activity decreases disease severity whilst in other models disease activity is exacerbated. Raised levels of nitrate and nitrite, stable end-products of .NO/ONOO7, are found in the cerebrospinal fluid, serum and urine of patients with MS. CSF levels of nitrate and nitrite correlate with blood-brain-barrier dysfunction, which suggests that .NO may play a role in inflammatory blood-brain-barrier dysfunction. In a longitudinal study on 24 patients with relapsing remitting and secondary progressive MS, raised serum nitrate and nitrite levels correlated with a relapsing course and infrequent relapses. However, no correlation was found between raised serum levels of nitrate and nitrite and MRI activity, disease progression, or the development of cerebral atrophy. In autoimmune mediated CNS demyelinating disease .NO may be a double-edged sword, mediating tissue damage on the one hand and on the other hand modulating complex immunological functions which may be protective.

Elevated serum levels of tumor necrosis factor-α in Guillain-Barré syndrome

Activated T lymphocytes and macrophages play a putative role in the pathogenesis of Guillian-Barre syndrome. Both cell types secrete tumor necrosis factor-α, a cytokine that has well-recognized toxi effects on myelin, Schwann cells, and endothelial cells. We determined serum and cerebrospinal fluid concentrations of tumor necrosis factor α in 26 patients with Guillain-Barre syndrome, 27 patients with other polyneuropathies, 30 patients with neurological diseases of the central nervous system, and 14 helthy control subjects. Markedly increased serum levels were detected in 14 patients(54%) with Guillian-Barre syndrome and to a significantly lesser extent, in patients with other polyneuropathies (26%) and in neurological control subjects(23%). Tumor necrosis factor-α was not detected in the cerebrospinal fluid of patients with Guillain-Barre syndrome or other polyneuropathies. Increased serum concentrations in patients with Guillain-Barre syndrome or other polyneuropathies. Increased serum concentrations in patients with Guillain-Barre syndrome correlated directly with disease severity and these concentrations returned to normal in parallel with clinical recovery. These findings emphasize the complexity of the immune response in patients with Guillain-Barre syndrome and suggest that tumor necrosis factor-αL may be important in the pathogenesis of peripheral demylination in this disorder.

A rapid method for detecting oligoclonal IgG in unconcentrated CSF, by agarose isoelectric focusing, transfer to cellulose nitrate and immunoperoxidase staining

Abstract A method has been developed for determining the electrophoretic distribution of IgG in unconcentrated cerebrospinal fluid by agarose isoelectric focusing, followed by transfer of focused protein from the agarose on to cellulose nitrate membranes. A double antibody immunoperoxidase staining technique is used, which is both rapid and sensitive.

Elevated cerebrospinal fluid and serum nitrate and nitrite levels in patients with central nervous system complications of HIV-1 infection: a correlation with blood-brain-barrier dysfunction

As nitric oxide (.NO) is hypothesised to play a role in the immunopathogenesis of neurological complications associated with inflammation, we compared levels of cerebrospinal fluid (CSF) and serum .NO metabolites in 24 patients with HIV-1 infection, to those in 58 non-HIV infected patients with neurological disorders. Levels of .NO metabolites were correlated with blood-brain-barrier dysfunction. CSF and serum nitrate and nitrite levels were measured by the nitrate reductase and Griess reaction methods. The .NO metabolites, nitrate and nitrite, were raised in the CSF and serum of patients with AIDS and central nervous system complications, when compared to non-HIV infected patients with inflammatory and non-inflammatory neurological disorders (median nitrate and nitrite: CSF=18.3 microM vs. 11.1 microM vs. 7.0 microM, P<0.001, and serum=53.8 microM vs. 50.3 microM vs. 41.4 microM, P=0.04, respectively). CSF nitrate and nitrite levels correlated with the albumin quotient. This study supports the evidence that .NO is a potential mediator of blood-brain-barrier breakdown in inflammatory diseases of the central nervous system.