Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and are immunogenic by the oral route

Induction of all forms of protective immunity by oral immunization with subunit vaccines is an ideal goal for the development of novel vaccines, but creates several theoretical problems from the point of view of antigen processing mechanisms. We show here that incorporation of the protein antigen ovalbumin (OVA) in lipophilic immune-stimulating complexes (ISCOMS) induces very strong primary immune responses in mice and requires very small amounts of antigen. OVA ISCOMS were particularly efficient at stimulating T-cell-mediated immunity in vivo, including delayed-type hypersensitivity (DTH) and potent class I major histocompatibility complex (MHC)-restricted cytotoxic T-cell responses. Furthermore, unlike native protein, OVA in ISCOMS was immunogenic when given orally. Thus, ISCOMS seem to allow protein to enter both the endogenous and exogenous pathways of antigen processing and overcome the usual induction of tolerance after feeding antigen. ISCOMS could provide potentially useful adjuvants for the development of oral subunit vaccines.     

The effect of tolbutamide on cerebral blood flow during hypoxia and hypercapnia in the anaesthetized rat

The increase in blood flow in the cerebral cortex of the anaesthetized rat during hypoxia and hypercapnia was investigated. Cerebral blood flow (CBF) was measured using the hydrogen clearance method with acutely implanted platinum electrodes. Hypoxia (PaO₂ 35.3±2.4 Torr) and hypercapnia (PaCO₂ 68.1±5.1 Torr) increased basal CBF from 76.3±9.0 ml/100g/min to 168.1±20.1 ml/100g/min and 162.4±31.9 ml/100g/min respectively. The sulphonylurea tolbutamide (1mM in 1%DMSO) had no significant effect on CBF in hyperoxia or in hypercapnia. However, it attenuated the increase of CBF during hypoxia by 66 ±11% (P<0.01). This suggests that opening of tolbutamide-sensitive potassium channels may be involved in the process of hypoxic vasodilation in the rat cerebral cortex.     

The detection of Alcelaphine herpesvirus-1 DNA by in situ hybridization of tissues from rabbits affected with malignant catarrhal fever.

Tissue sections and cultured lymphocytes from rabbits clinically affected following experimental infection with Alcelaphine herpesvirus-1 (AHV-1) were assessed for the presence of viral DNA by in situ hybridization with the cloned major HindII repeat sequence of this virus. Small numbers of virus-infected cells were consistently detected only in submandibular lymph nodes, while other tissues showed no evidence of viral DNA. Virus titration in culture suggested that there were higher titres of virus in the lymph nodes, spleen and lung of infected animals than in the kidney or peripheral blood lymphocytes and confirmed the low level of virus in these animals. Substantially more viral DNA was detected by in situ hybridization in lymphocytes following at least 24 h of culture, suggesting that viral replication is normally repressed by the host.     

Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification

Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70 degrees C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.     

bcl-2 rearrangement in Hodgkin's disease. Results of polymerase chain reaction, flow cytometry, and sequencing on formalin-fixed, paraffin-embedded tissue.

We examined 81 cases of Hodgkin's disease for evidence of the t(14;18) translocation, using the polymerase chain reaction assay on lysates of formalin-fixed, paraffin-embedded tissue. Seven of 74 amplifiable cases (9%) were positive for the translocation, which involves the bcl-2 oncogene and the immunoglobulin heavy chain gene. Two of these cases were sequenced and the breakpoints had the same pattern found in follicular lymphoma. The nuclei from one of the cases were sorted into large and small subpopulations. The t(14;18) signal was more intense in the large nucleus subpopulation, which contained a greater proportion of Reed-Sternberg-like nuclei. These results are consistent with the hypothesis that Reed-Sternberg cells carry the translocation, but they do not exclude the possibility that the translocation is found in cells representing the reactive component of Hodgkin's disease. The results also demonstrate that routinely processed material is suitable for polymerase chain reaction-based analysis of translocations, although the sensitivity is reduced 10- to 100-fold, compared with fresh tissue.     

Complement fixing antibody responses to virus infection in children with acute lymphoblastic leukaemia.

Thirty children with acute lymphoblastic leukaemia (ALL) were studied and had a virus isolated. Only 50% produced a significant rise in complement fixing (CF) antibody titre compared to 100% of normal children. The failure to produce antibodies was unpredictable. CF antibodies are not a reliable guide to virus infections in children with ALL.     

Association of oestrogen receptor alpha gene polymorphisms with postmenopausal bone loss, bone mass, and quantitative ultrasound properties of bone

Background: The gene encoding oestrogen receptor α (ESR1) appears to regulate bone mineral density (BMD) and other determinants of osteoporotic fracture risk.

Objective: To investigate the relation between common polymorphisms and haplotypes of the ESR1 gene and osteoporosis related phenotypes in a population based cohort of 3054 Scottish women.

Results: There was a significant association between a common haplotype "px", defined by the PvuII andXbaI restriction fragment length polymorphisms within intron 1 of the ESR1 gene, and femoral neck bone loss in postmenopausal women who had not received hormone replacement therapy (n = 945; p = 0.009). Annual rates of femoral neck bone loss were ~14% higher in subjects who carried one copy of px and 22% higher in those who carried two copies, compared with those who did not carry the px haplotype. The px haplotype was associated with lower femoral neck BMD in the postmenopausal women (p = 0.02), and with reduced calcaneal broadband ultrasound attenuation (BUA) values in the whole study population (p = 0.005). There was no association between a TA repeat polymorphism in the ESR1 promoter and any phenotype studied, though on long range haplotype analysis subjects with a smaller number of TA repeats who also carried the px haplotype had reduced BUA values.

Conclusions: The ESR1px haplotype is associated with reduced hip BMD values and increased rates of femoral neck bone loss in postmenopausal women. An association with BUA may explain the fact that ESR1 intron 1 alleles predict osteoporotic fractures by a mechanism partly independent of differences in BMD.

     

Image analysis derived ploidy and proliferation indices in soft tissue sarcomas: comparison with clinical outcome.

AIMS--To compare prognostic information obtained by image analysis cytometry of paraffin wax embedded soft tissue sarcomas with conventional assessment. METHODS--A CAS 200 image analyser was used to determine DNA content of Feulgen stained cytology preparations and tissue sections and to quantify immunostaining by Ki67 and PC10 antibodies. A mitotic count in 50 high power fields was undertaken and histological grade assigned by the Trojani system. Clinical details including follow up and outcome were obtained by case note review. The Kruskal-Wallis one way analysis test, Spearman rho significance test, Kaplan-Meier method, and log-rank test were applied in statistical analysis. RESULTS--Ploidy status, DNA index, 2.5c exceeding rate, 5c exceeding rate, mitotic count and Trojani grade all correlated significantly with clinical outcome. The relation between Ki67 index and outcome did not reach significance. The PC10 index and outcome were not related. Only 2.5c exceeding rate, 5c exceeding rate, and mitotic count correlated significantly with Trojani grade. CONCLUSIONS--DNA content determination of soft tissue sarcomas by image analysis provides quantifiable information of benefit in prediction of outcome. Larger series are required to determine the independent value of ploidy. In this study quantification of anti-Ki67 and anti-PC10 immunostaining was not of prognostic benefit) by contrast with mitotic count and Trojani grade.     

Immunological identification of Neisseria gonorrhoeae with monoclonal and polyclonal antibody coagglutination reagents.

The reliability of immunological identification of Neisseria gonorrhoeae using polyclonal and monoclonal antibody coagglutination reagents has been evaluated. When clinical isolates of neisseriae were tested in an "in use" trial the sensitivity and specificity of each reagent were similar and the overall agreement with carbohydrate utilisation was 97.9% (141/144) for the polyclonal antibody reagent and 97.2% (140/144) for the monoclonal reagent. When results of testing 13 stock cultures of N lactamica and five stock cultures of beta-lactamase producing Branhamella catarrhalis were combined with the results for clinical isolates of non-gonococcal neisseriae the agreement with carbohydrate utilisation was 86.5% (64/74) for the polyclonal reagent and 97.3% (72/74) for the monoclonal reagent: this difference is statistically significant at the 5% level. Calculation of positive and negative predictive values showed differences in the reliability of the coagglutination reagents when testing Gram negative diplococci isolated from various anatomical sites. The value and limitations of the polyclonal and monoclonal reagents were similar with respect to anogenital isolates: N gonorrhoeae was confirmed by a positive result but not excluded by a negative result. The monoclonal reagent was superior for testing throat isolates; although a negative result with either reagent confirmed Gram negative diplococci as non-gonococcal neisseriae, a positive result with the monoclonal reagent was more reliable (predictive value 93%) than a positive result with the polyclonal reagent (predictive value 86%).