Evaluation of the single radial haemolysis (SRH) technique for rubella antibody measurement.

Sera from 1258 individuals have been tested by four laboratories for rubella antibody by both the haemagglutination-inhibition and single radial haemolysis techniques. There was good agreement between the results obtained by the two methods. Although sheep red blood cells were used in the single radial haemolysis plates, no problems were encountered with sera from patients with infectious mononucleosis. The single haemolysis technique was found to be simple, convenient, and reliable, and suited to the rapid screening of large numbers of sera to assess susceptibility to rubella in the context of a vaccination campaign. However, since the technique does not detect anti-rubella IgM, it should not be used as the only test to investigate suspected recent infection.     

Demonstration of carcinoembryonic antigen in bone marrow from patients with carcinoma.

Primary and secondary tumour and bone marrow trephine biopsies from 20 patients with carcinomas were stained for carcinoembryonic antigen by the three stage immunoperoxidase method. Six marrow biopsies contained tumour deposits, five of which were positive for carcinoembryonic antigen. A further five marrow biopsies contained single carcinoembryonic antigen positive cells of uncertain origin. Carcinoembryonic antigen staining may be a useful adjunct to conventional histology in the diagnosis of marrow metastases.     

Evaluation of the Toa E-5000 Automated Hematology Analyzer

A three-phase evaluation of the Toa E-5000 Automated Hematology Analyzer was performed. The first phase consisted of evaluation of linearity, carryover, precisions, accuracy, sample stability, and effectiveness of the mixing and sampling mechanism. The second phase was comparison of erythroid and platelet parameters with results from a Coulter Counter Model S-Plus IV. The third phase consisted of comparison of the trimodal leukocyte differential count of the Toa E-5000 in 300 patients with four other differential methods. The first-phase studies showed excellent performance characteristics. Stability of samples at room temperature was good for a minimum of 12 hours, but storage for longer than 12 hours requires refrigeration to maintain stable values. The erythrocyte and platelet parameters showed good correlation with the Coulter S-Plus IV values except for the erythrocyte distribution width that is calculated differently. The third-phase studies are published elsewhere.     

Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.      

Virulence of Campylobacter jejuni for chicken embryos is associated with decreased bloodstream clearance and resistance to phagocytosis.

The 11-day-old chicken embryo has been shown to be a useful animal model for comparing the virulence of human isolates of Campylobacter jejuni. Virulence in this system is associated with the ability to invade the chorioallantoic membrane and to survive and proliferate in vivo. In this study, the survival and multiplication of C. jejuni in the embryonic host was investigated. It was possible to enhance the virulence of a relatively avirulent C. jejuni strain by passaging it intravenously through the embryos. The resulting isogenic variants demonstrated enhanced abilities to survive in vivo but were still unable to invade when inoculated onto the chorioallantoic membrane. The bloodstream clearance of C. jejuni was studied, and virulent, but not avirulent, strains persisted and multiplied both in the bloodstream and in embryonic liver. Virulent strains also were cleared significantly more slowly from the bloodstream of adult BALB/c mice after intravenous challenge than were avirulent strains. C. jejuni strains which were cleared slowly in vivo were also ingested slowly in vitro by mouse peritoneal macrophages. Clearance studies in mice pretreated with cobra venom factor demonstrated that opsonization by serum complement was not a prerequisite for clearance of campylobacters from the murine bloodstream.     

Development of an injectable,in situ crosslinkable,degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate)

The effect of temporary encapsulation of rat marrow stromal osteoblasts in crosslinked gelatin microparticles on cell viability and proliferation was investigated in this study for microparticles placed on a crosslinking poly(propylene fumarate) (PPF) composite over a 7 day time period. Encapsulated cells were seeded on crosslinking PPF composites at times up to 10 min following initiation of the crosslinking reaction, and also on fully crosslinked PPF composites and tissue culture polystyrene controls, with a cell seeding density of 5.3 x 10(4) cells/cm2. The crosslinked PPF composite exhibited an average gel point of 10.3 min and an average maximum crosslinking temperature of 47.5 degrees C. Cell viability and proliferation were assessed by DNA and 3H-thymidine assays and the results were compared with those for nonencapsulated cells. The results showed that the addition time of cells to a crosslinking PPF composite had a large effect on cell viability and proliferation for both encapsulated and nonencapsulated cells with more surviving cells added at later time points. Most importantly, the temporary encapsulation of cells significantly enhanced cell viability at earlier time points. The data indicate that the presence of gelatin microparticles does not affect the crosslinking of a PPF composite. They further suggest that the temporary encapsulation of cells in crosslinked gelatin microparticles may preserve the viability of cells contained in an actively crosslinking PPF composite used as an injectable polymeric scaffold serving also as a carrier for osteogenic cell populations.     

Development of a larval bioassay for susceptibility of cat fleas (Siphonaptera: Pulicidae) to imidacloprid

Strategies for controlling cat fleas, Ctenocephalidesfelisfelis (Bouché), have undergone dramatic changes in the past 5 yr. With the advent of on-animal treatments with residual activity the potential for the development of insecticide resistance increases. A larval bioassay was developed to determine the baseline susceptibility of field-collected strains of cat fleas to imidacloprid. All four laboratory strains tested showed a similar level of susceptibility to imidacloprid. Advantages of this bioassay are that smaller numbers of fleas are required because flea eggs are collected for the test. Insect growth regulators and other novel insecticides can also be evaluated. Using a discriminating dose, the detection of reduced susceptibility in field strains can be determined with as few as 40 eggs.     

Single radial immunodiffusion test for detecting antibodies against surface antigens of intracellular and extracellular vaccinia virus.

Antibodies to surface antigens of intracellular naked vaccinia virus (INV) and in limited studies extracellular enveloped virus (EEV) were determined by single radial immunodiffusion tests (SRDT) with immobilized virions in agarose gels. Antibodies to INV were demonstrable in rabbit hyperimmune sera (one to four visible zones), smallpox convalescent sera and sera from re-vaccinated individuals. A difference in specificity of antibodies reacting with INV and EEV was detectable by SRDT. The SRDT provides a simple, reproducible and specific test for detection of antibodies against vaccinia or variola virus, but the technique requires large quantities of virions.     

Molecular analysis of de novo germline mutations in the von Hippel-Lindau disease gene

VHL disease is a dominantly inherited familial cancer syndromewith variable expression and age-dependent penetrance. The diagnosisof isolated cases is often delayed compared with familial cases,and estimates of the new mutation rate have varied more than20-fold. To investigate the frequency and origin of de novoVHL gene mutations we have analysed: (i) families with identicalmutations to determine if there is a common haplotype, and (ii)apparent new mutation cases to determine whether the clinicaldiagnosis of such cases is reliable and to define the parentalorigin of de novo VHL gene mutations. Haplotyping of 12 VHLmutations occurring in two or more families (total 42 kindreds)revealed that for most mutations there was no evidence of afounder effect. A marked bias for a paternal origin of new mutationshas been reported in other familial cancer syndromes such asneurofibromatosis type 1 (NF1), multiple endocrine neoplasia(MEN) 2B and bilateral retinoblastoma, but it is unclear whetherthis bias results from a greater susceptibility for mutagenesisduring male gametogenesis because of the larger number of celldivisions compared with that in oogenesis, or from genomic imprintingeffects. Analysis of 13 de novo VHL mutations in which the parentof origin could be established, showed no evidence for a biasfor a paternal origin (seven paternal, six maternal), and differedsignificantly from that reported in NF1, MEN2B and bilateralretinoblastoma. This result demonstrates that an increased susceptibilityto paternal allele mutation is not a universal finding in autosomalgenetic diseases and that the origin of new mutations may beinfluenced by both genomic imprinting effects and the increasednumber of cell divisions in spermatogenesis compared with oogenesis.     

Evaluation of the Coulter Counter model S-Plus IV

Evaluation of linearity, carry-over, precision, and accuracy of a Coulter Counter model S-Plus IV prototype showed that they meet the manufacturer's specifications. The instrument also was compared with an earlier model. Correlations of the lymphocyte and granulocyte values in the three-part differential count with manual eye-count and a Technicon Hemalog D90 results were very close to the correlation of eye-count to Hemalog D90 results. The percentage of mononuclear cells showed acceptable correlation to the manual eye-count when monocytes were combined with blasts, eosinophils, and basophils. The medical effectiveness of the three-part differential was determined by comparison with manual eye-counts in 1,084 samples. The false positive and false negative values were 7.38% and 7.84%, respectively. The instrument has acceptable limits of operation. When combined with analysis of histograms by trained personnel, the three-part differential count is capable of screening for abnormalities that require further analysis.