Kei-ichi Uemura

Inflammatory cytokine cascade released by leukocytes in cerebrospinal fluid after subarachnoid hemorrhage.

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)-1β, IL-6, transforming growth factor-β1 (TGF-β1) 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-1β and IL-6 were examined by flow cytometric analysis. The molecular weight of TGF-β1 in CSF of 30 patients was examined by Western blot analysis. The TGF-β1 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-1β, whereas a proportion of monocytes and lymphcytes were IL-1β-positive by flow cytometric analysis. TGF-β1 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-β1. The CRP and TGF-β1 levels in CSF are strongly concerned with communicating hydrocephalus after SAH. [Neurol Res 2001; 23: 724-730]

Glycolipids of gastric cancer: The presence of blood group A-active glycolipids in cancer tissues from blood group O patients

Glycolipids were isolated from human gastric cancer tissues and normal mucosae. Sulfogalactosylceramide, ganglioside and neutral glycolipid fractions were separated by DEAE-Sephadex and silica gel column chromatography. Sulfogalactosylceramide contents were higher in the cancer tissues than in the normal mucosae. Ganglioside contents showed considerable variations but in the cancer tissues in mole percentage of ganglioside GM3 was higher than in the normal mucosae. The cancer tissues contained more neutral glycolipids than normal tissues. Glycolipids of lacto-series, including fucolipids, were markedly increased in the cancer tissues. Blood group A-active glycolipids were found in the cancer tissues from two patients with blood group O but not found in the uninvolved tissue associated with the cancer tissue.

SULFATIDE PROLONGS BLOOD-COAGULATION TIME AND BLEEDING TIME BY FORMING A COMPLEX WITH FIBRINOGEN

Sulfatides (galactosylceramide I3-sulfate), which are found in serum lipoproteins of various mammals, effectively increased prothrombin time (anticoagulant effect) and also effectively prolonged bleeding time (anti-platelet effect). When equal volumes of a homogeneous micellar solution of sulfatide and fibrinogen in phosphate-buffered saline were mixed, an insoluble complex precipitated. Analysis of the precipitated complex showed that the molar ratio of sulfatide to fibrinogen was about 400:1. These results indicate that the sulfatide micelle binds tightly to fibrinogen and thereby interferes with both fibrin gel formation (anticoagulant activity) and platelet function.

Heterophile Hanganutziu-Deicher antigen in ganglioside fractions of human melanoma tissues.

Gangliosides were purified from melanoma tissue extracts obtained from 5 patients. GM3 and GD3 were identified as the major components in ganglioside fractions of the melanoma tissues. Following thin-layer chromatography, enzyme immunostaining with Hanganutziu-Deicher (H-D) antigen-specific chicken antisera demonstrated the presence of NeuGc-neolactotetraosylceramide (H-D5) and NeuGc-lacto-N-norhexa-osylceramide (H-D7) in all 5 melanoma extracts.

Mild encephalitogenic activity of basic protein—acidic lipid complex from myelin and detection of immune complexes in experimental allergic encephalomyelitis

Further biochemical and pathological investigation of basic protein--acidic lipid complex from canine cerebral myelin show the presence of sulfatide, phosphatidylserine, ganglioside and basic protein in the molar ratio of approximately 6 : 3 : 1 : 1 and that it is associated with mild encephalitogenic activity in guinea pigs in comparison to intact myelin and basic protein. Circulating immune complexes were detected in the sera of guinea pigs with clinical signs of experimental allergic encephalomyelitis and immunofluorescent staining showed the deposition of immune complexes of immunoglobulin and complement in vessels of white matter and meninges and in the choroid plexus.

Effects of various lysosphingolipids on cell growth, morphology and lipid composition in three neuroblastoma cell lines

The cell numbers of three mouse neuroblastoma cell lines were decreased upon incubation with lysosphingolipids in the following order of effectiveness: lysosulfatide (lysoCS) greater than psychosine (Ps) greater than sphingosylphosphocholine (SPC). The different cell lines showed characteristic sensitivities to various concentrations of lysolipids less than 150 microM. Interestingly, only SPC induced neurite outgrowth and changed the lipid composition, modifying the amounts of cholesterol, sphingomyelin (SM) and ganglioside GM3 in all cell lines. The effect of SPC on these cell lines was comparable to the effect of N-acetyl SPC (NAcSPC) rather than that of SM.

Immunochemical determination of Forssman and blood group A-active glycolipids in human gastric mucosa by inhibition assay of liposome lysis.

A simple liposome immunoassay, liposome immune-lysis inhibition (LILI) assay, is described for quantitative determination of individual glycolipid antigens. Liposomes containing fluorogenic marker, 4-methylumbelliferyl phosphate, were prepared from sphingomyelin, cholesterol, dicetylphosphate and standard glycolipid. Release of trapped markers from these liposomes by antibody and complement (liposome lysis) was inhibited by preincubating the antibody with test glycolipid incorporated into inhibitor liposomes. Based on the competitive inhibition, it was possible to quantitate each glycolipid antigen in less than picomolar amounts. The sensitivity and specificity of the assay were examined with purified glycolipid standards. LILI assay has been applied for the determination of Forssman glycolipid and blood group A-active glycolipid in human gastric mucosa and cancer tissues.

Glycolipid of human pancreatic cancer; the appearance of neolacto-series (type 2 chain) glycolipid and the presence of incompatible blood group antigen in tumor tissues

Glycolipid isolated from normal and cancerous human pancreatic tissues were characterized chemically and immunologically. The major neutral glycolipids in both normal and cancerous tissues were composed of globo-series glycolipids and lacto-series glycolipids. The mole percentage of fucolipids in the total neutral glycolipids of normal tissues was 20-40%, and in general the fucolipids corresponded to blood group glycolipids related to the patients blood group, however, in cancerous tissues the amount of these fucolipids was decreased. Immunostaining revealed that normal tissues contained only lacto-series (type 1 chain) glycolipids. In contrast, cancerous tissues contained the neolacto-series (type 2 chain) glycolipids as well as the lacto-series glycolipids. Incompatible blood group antigens, A active glycolipids in a blood type O patient and B active glycolipids in a blood type A patient, were also detectable in the neutral glycolipid fractions of the pancreatic cancer tissues.

Antibody response of rabbits to nerve ending gangliosides *: Analysis of antibody specificity by liposome lysis and lysis inhibition assays

Rabbits were immunized with nerve ending fraction prepared from guinea pig brain. Serum antibodies to total gangliosides were followed by enzyme-linked immunosorbent assay; their titers were highest at 2 to 3 weeks after immunization and some rabbits showed a response to reinjections. Specific reactivities of the antibodies against each molecular species of gangliosides were analyzed by liposome lysis assay and liposome lysis inhibition assay. Antibody responses were detected against GM1, GD1b, GM3 and GM2, but not against GD1a and GT1b. Natural and immune antibodies to the asialo glycolipids and to galactosylceramide were also observed.

Immunological Properties of Glycolipids Including Some Gangliosides in Mammalian Erythrocyte Membranes

It is well known that various mammalian erythrocyte membranes contain different types of major glycosphingolipid which are considerably species-specific (Table I). These glycosphingolipids are not only structural components of membranes but also important antigenic sites on cell surfaces. Specific antibodies against individual glycosphingolipids are useful for various purposes in immunochemical and membrane research.