Astrogliosis during acute and chronic cuprizone demyelination and implications for remyelination

In multiple sclerosis, microglia/macrophage activation and astrocyte reactivity are important components of the lesion environment that can impact remyelination. The current study characterizes these glial populations relative to expression of candidate regulatory molecules in cuprizone demyelinated corpus callosum. Importantly, periods of recovery after acute or chronic cuprizone demyelination are examined to compare conditions of efficient versus limited remyelination, respectively. Microglial activation attenuates after early demyelination. In contrast, astrocyte reactivity persists throughout demyelination and a 6-week recovery period following either acute or chronic demyelination. This astrocyte reaction is characterized by (a) early proliferation, (b) increased expression of GFAP (glial fibrillary acidic protein), Vim (vimentin), Fn1 (fibronectin) and CSPGs (chondroitin sulphate proteoglycans) and (c) elaboration of a dense network of processes. Glial processes elongated in the axonal plane persist throughout lesion areas during both the robust remyelination that follows acute demyelination and the partial remyelination that follows chronic demyelination. However, prolonged astrocyte reactivity with chronic cuprizone treatment does not progress to barrier formation, i.e. dense compaction of astrocyte processes to wall off the lesion area. Multiple candidate growth factors and inflammatory signals in the lesion environment show strong correlations with GFAP across the acute cuprizone demyelination and recovery time course, yet there is more divergence across the progression of chronic cuprizone demyelination and recovery. However, differential glial scar formation does not appear to be responsible for differential remyelination during recovery in the cuprizone model. The astrocyte phenotype and lesion characteristics in this demyelination model inform studies to identify triggers of non-remyelinating sclerosis in chronic multiple sclerosis lesions.     

Estimation of Type-Specific Neutralizing Antibody to Herpes Simplex Virus Type 2 in Uterine Cervical Cancer Patients by a New Absorption Method

A simple method of estimating type-specific neutralizing antibody to type 2 herpes simplex virus (HSV-2) was devised with the use of the microneutralization system. Serially diluted serum was mixed in the well with a constant amount of type 1 virus (HSV-1), and after 3 days' incubation at 37 C, the plate was irradiated with ultraviolet light. The absorbing HSV-1 consisted of culture fluid plus an extract of infected Vero cells not especially concentrated. The well then received indicator HSV-1 or HSV-2, and after being left at 37 C for 1 hr a suspension of dispersed Vero cells was dropped into the wells, following our standard neutralization procedure. Preliminary tests with rabbit antisera showed that even a low level of HSV-2 antibody was detected by this method, unless an exceptionally high titer of HSV-1 antibody originally coexisted with the HSV-2 antibody. Sera from acutely infected persons testified to the specificity of the antibody so detected. It was revealed by means of the new technique that the rate of HSV-2 antibody was significantly higher in uterine cervical cancer patients than in control women. There was no correlation between the clinical stage of cervical cancer and the presence of HSV-2 antibody.     

A new microplate neutralization test for typing of herpes simplex virus.

A microplate serum neutralization test for estimation of complement-requiring neutralizing (CRN) antibody was established as the first step for simplification of typing of herpes simplex virus (HSV). When guinea pigs were immunized with type 2 HSV, the late sera could mostly differentiate the types of HSV better than hyperimmune rabbit sera, the CRN titer against the heterologous type 1 HSV being much lower than the homologous titer. Sera of guinea pigs immunized with type 1 HSV showed about the same level of cross reaction against type 2 HSV as did rabbit antisera. Guinea pig sera having minimal levels of cross reaction were selected, and their high dilution (1:160) and complement were added to serial 10-fold dilutions of virus in the microplate titration of virus infectivity. Selective reduction of virus titer by either antiserum could determine the type of HSV. No equivocal intermediate case was found among a number of stock strains including many fresh isolates. The typing result coincided with that determined by a modification of Yang et al's method based on virus titers obtained with Vero and primary chick embryo cells. The typing based on plaquing in chick embryo cells sometimes failed to identify type 1 HSV.     

Studies on the neutralization of herpes simplex virus. VIII. Significance of viral sensitization for inactivation by complement.

Early and late IgG of rabbits immunized with herpes virus showed, respectively, 8-fold and 2-fold enhancement of neutralization endpoint in the presence of complement (C). Kinetic curve experiments employing an appropriate amount of virus revealed that both neutralization and sensitization followed first-order reaction, and each IgG possessed a certain range of concentration where neutralization was negligible while sensitization was marked. Dose responses of neutralization and sensitization velocities demonstrated that the C enhancement of late IgG was about 7-fold and that of early IgG more than 20-fold. These facts suggested that the IgGs contained two different entities of complement-requiring (CRN) and non-requiring neutralizing (N) antibodies at different proportions, only the former being responsible for sensitization. The different CRN: N ratios obtained by the endpoint and kinetic methods may mean either that the two antibodies differ in avidity for the virus or that the number of critical sites per virion for CRN antibody is greater than that for N antibody. In this interpretation, sensitization by CRN antibody as well as neutralization by N antibody is thought to result from attachment of a single antibody molecule to the viral critical site. Alternative explanations, ascribing the mechanism of neutralization to steric hindrance of critical sites or to multiple hit of those sites by antibody, were denied by analyses of the present data.     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.     

Stereoselective Synthesis of (S,E)-6-Isopropyl-3,9-dimethyl-5,8-decadienyl Acetate,the (S)-Enantiomer of the Yellow Scale Pheromone

The (S)-enantiomer of the sex pheromone of the yellow scale (Aonidiella citrina), (S,E)-6-isopropyl-3,9-dimethyl-5,8-decadienyl acetate, was stereoselectively synthesized from (R)-(+)-citronellic acid.     

An analysis of factors influencing the isolation rate of herpes simplex virus.

Attempts were made to improve the rate of isolation of herpes simplex virus (HSV) from clinical specimens by minimizing loss of virus infectivity during transportation and employing the most sensitive cells for isolation. Basical analyses using standard strains of type 1 and type 2 HSV indicated that virus titer decrease was marked even at low temperatures in environments free of proteinous stabilizer such as normal serum or tissue extract, negating the generally held concept that HSV is stable in distilled water. YLE (Earle-lactalbumin HYDROLYSATE-YEAST EXTRACT) medium containing 20% inactivated calf serum was determined to be a transport medium of choice, because degradation of suspended virus during storage and freeze-thawing was negligible and loss of virus during Millipore filtration was minimal. Special coating of the membrane could also be obviated by the use of this solution. In a cell susceptibility test using clinical specimens, secondary rabbit kidney (SRK) cells were the most sensitive, showing a quick development of cytopathic effect. Vero and RK-13 cells were the second best, whereas monkey kidney, HeLa and L cells were far less sensitive. A total of 136 specimens from suspected cases, sent by dermatologists, were tested using SRK cells, and 99 strains of type 1 and 15 strains of type 2 HSV were isolated. Excluding one case from which vaccinia virus was isolated, the isolation rate of HSV was 84.4%.