No abstract

Subarachnoid hemorrhage (SAH) elicits an inflammatory response in the subarachnoid space, which is mediated by the release of various cytokines. To assess their involvement in post-hemorrhagic complications, we determined the source and time-course of the release of inflammatory cytokines and acute-phase proteins in cerebrospinal fluid (CSF) following SAH. Concentrations of interleukin (IL)- 1beta, IL-6, transforming growth factor-beta1 (TGF-beta1) and C-reactive protein (CRP) in CSF of 36 patients with SAH were measured by enzyme-linked immunoabsorbent assay (ELISA). Floating cells collected from the CSF were centrifuged four to six days after SAH, and examined immunohistochemically. Intracellular IL-1beta and IL-6 were examined by flow cytometric analysis. The molecular weight of TGF-beta1 in CSF of 30 patients was examined by Western blot analysis. The TGF-beta1 levels of patients who had undergone ventriculoperitoneal (VP) shunt (n = 19) was significantly higher than nonshunt group (n = 16). The CRP levels of VP shunt group was significantly higher than nonshunt group. IL-6 concentration was maximal within day 0-1 and it was secreted by neutrophils and monocytes. ELISA showed consistently low levels of IL-1beta, whereas a proportion of monocytes and lymphcytes were IL- 1beta-positive by flow cytometric analysis. TGF-beta1 levels were also maximal on day 0-1 according to ELISA, although it tended to be in the inactive form derived from platelets. A 25 kDa band of TGF-1 was detectable for at least 13 days after SAH, which may have been secreted in part by neutrophils and monocytes. CRP levels in CSF peaked on day 2-3. The present results suggest that leukocytes induced by SAH play an important role in post-hemorrhagic inflammation in the subarachnoid space by releasing IL-6 and TGF-beta1. The CRP and TGF-beta1 levels in CSF are strongly concerned with communicating hydrocephalus after SAH.

Among the pathological effects in man following infection with Mycoplasma pneumoniae is a transient autoimmune disorder characterized by the presence of high-titre erythrocyte autoantibodies (cold agglutinins). These autoantibodies are usually directed against the carbohydrate antigen termed I (ref. 3) which consists of a branched oligosaccharide. The mechanism by which the anti-I antibodies are elicited is unknown. However, sialic acid-containing receptors have been implicated in the adherence of M. pneumoniae to erythrocytes and other cell types, and both I and the related antigen i occur on erythrocytes in sialylated form: i is the predominant antigen on fetal erythrocytes and I is predominant in adults. Anti-I antibodies might arise in M. pneumoniae infection in response to a modification of the 'self' antigen-I as a result of its interaction with this agent. Here we report our study of the specificity of the interaction of M. pneumoniae with human erythrocytes. We found that this interaction is mediated by long chain oligosaccharides of sialic acid joined by alpha 2-3 linkage to the terminal galactose residues of poly-N-acetyllactosamine sequences of Ii antigen type.

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.

Various monosialo- and disialo-gangliosides and their derivatives were examined by delayed ion extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF MS) in the reflector mode with alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid used as the matrix. Native gangliosides were generally found to give good spectra in the negative ion mode. 2,5-Dihydroxybenzoic acid was a better matrix for gangliosides than alpha-cyano-4-hydroxycinnamic acid, because this matrix seemed to minimize loss of sialic acid and carbon dioxide of gangliosides. About 1 pmol of ganglioside was able to be detected with this matrix. When "A-series" gangliosides such as GD1a and GalNAc-GD1a gave undesirable extra peaks probably due to loss of sialic acid besides molecule-related ion peaks, the methyl-esterification of the gangliosides at the carboxyl groups of sialic acids was found to be necessary to obtain good DE MALDI-TOF mass spectra in the positive ion mode. In contrast, "B-series" gangliosides such as GD1b, GD2, and GD3 gave rise to major dehydrated molecule-related ion [M-H2O-H]- peaks in the negative ion mode without the pretreatment of methyl-esterification. The DE MALDI-TOF mass spectrometric analysis enabled us to distinguish between GD1a and GD1b, which have the same molecular weight. It was also found that not only a purified sample, but also a mixed sample of various gangliosides was amenable to the identification of them by DE MALDI-TOF MS.