Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.

We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.

Retrospective study of the impact of lead-based paint hazard remediation on children's blood lead levels in St. Louis, Missouri.

A retrospective follow-up study was conducted to evaluate the effectiveness of lead-based paint hazard remediation in reducing children's blood lead levels. The authors reviewed existing St. Louis, Missouri, City Health Department records, identified 185 children younger than age 6 years who had blood lead levels > or = 25 micrograms/dl during 1989 or 1990, and compared changes in blood lead levels among children whose dwellings did and those whose dwellings did not undergo remediation. Among 54 children who had not moved or received chelation therapy and whose blood lead levels were measured 10-14 months after diagnosis, the geometric mean blood lead level decreased 23% among children living in remediated dwellings (n = 37) and 12% among children in nonremediated dwellings (n = 17) (p = 0.07, t test). The estimated size of the remediation effect was similar using multiple regression (-13%; 95% confidence interval (CI) -25 to 1; p = 0.06) and an approach based on generalized estimating equations (-16%, 95% CI -25 to -7; p = 0.002), when adjusted for covariates. The effect of remediation was greater among children whose blood lead levels at diagnosis were > or = 35 micrograms/dl (-22%) than among those whose blood lead levels at diagnosis were between 25 and 34 micrograms/dl (-1%). Among lead-poisoned children in St. Louis, children whose dwellings undergo lead-based paint hazard remediation have a greater decline in geometric mean blood lead level than do children whose dwellings do not, but the effect of remediation may be influenced by the blood lead level at diagnosis.

Diagnosis of Kilham rat virus using PCR.

The exclusive use of serology, namely immunofluorescent antibody (IFA), for the diagnosis of rodent parvovirus infection is limited to detecting IgG antibodies which develop some time after the appearance of clinical symptoms, whilst conventional viral isolation, using tissue culture, is both expensive and time consuming. Polymerase chain reaction (PCR) as a diagnostic method has the potential to overcome these problems. However, it requires detailed knowledge of the genetic code of the target organism and careful selection of the primers used. This paper describes preliminary findings in a PCR assay which detected Kilham rat virus, minute virus of mice and Toolan's H1 virus.

Exonuclease cycling assay: an amplified assay for the detection of specific DNA sequences.

An assay is described in which an oligonucleotide probe is specifically digested by lambda exonuclease only when it is annealed to its complementary sequence. In this assay, a cycling effect occurs whereby a small amount of target sequence acts as a specific co-factor in the enzymatic degradation of a larger number of molecules of an oligonucleotide probe. This amplification principle is demonstrated and the effect of the oligonucleotide probe sequence investigated. The necessary steps needed to convert this effect into a useful diagnostic tool are discussed.

Prevalence of radiographic evidence of paint chip ingestion among children with moderate to severe lead poisoning, St Louis, Missouri, 1989 through 1990.

Although experts once believed that ingesting chips of lead-based paint was the major cause of lead poisoning among children, conventional wisdom now holds that lead-contaminated dust and soil are the major routes of exposure. Data from a childhood lead-poisoning treatment clinic were examined to assess the frequency with which children ingest paint chips. For this study, the reports on abdominal radiographs of 90 children with moderate to severe lead poisoning who had received their first chelation treatment during 1989 or 1990 were reviewed. According to a radiologist's evaluation, 13 of 90 abdominal radiographs (14%; 95% confidence interval [CI] 7% to 22%) showed evidence of paint chip ingestion. Of 46 children with blood lead levels greater than or equal to 55 micrograms/dL, 12 (26%) had radiographs that showed paint chips, whereas only 1 (2%) of 44 children with blood lead levels less than 55 micrograms/dL had such radiographs (prevalence ratio = 11.5; 95% CI 1.6 to 84.6). The actual proportion of children with moderate to severe lead poisoning who have consumed leaded-paint chips is likely to be higher than this estimate based on radiographic evidence. While lead-contaminated dust is a major source of lead exposure, ingestion of leaded-paint chips clearly remains an important source of exposure among children with moderate to severe lead poisoning.

Unknown sequence amplification: application to in vitro genome walking in Chlamydia trachomatis L2.

A recently described technique, 'Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. We have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity or purity.

The production and characterization of monoclonal antibodies against the protein III of Neisseria gonorrhoeae.

Monoclonal antibodies were produced against gonococcal protein III. Antibodies of two different specificities were obtained. One reacted with all Neisseria species tested (N. gonorrhoeae, N. meningitidis and five non-pathogenic species), whereas the other was specific for Neisseria gonorrhoeae and may provide the basis for improved diagnostic reagents.

Neisseria gonorrhoeae: subdivision of auxogroups by genetic transformation.

Genetic transformation was used in an attempt to subdivide the most prevalent auxotypes of Neisseria gonorrhoeae in local isolates. The large proline requiring (Pro-) group could be divided into two genetic types, as could the less common arginine requiring (Arg-) group. The large arginine, hypoxanthine, and uracil requiring (Arg- Hyp- Ura-) group could not be subdivided by this method. The genetic relation between these and other auxotypes was investigated.

Western blot analysis of gonococcal serogrouping reagents.

The W class antisera used in the coagglutination method of serogrouping Neisseria gonorrhoeae were analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot transfers. All were found to contain antibodies to the homologous protein II as well as antibodies to protein I group antigens. Examination of local isolates showed that some strains owed their reaction with coagglutination reagents to epitopes on their protein II not their protein I. How this may lead to difficulties when using coagglutination patterns to subdivide the W groups is discussed.

Epidemiological studies on Neisseria gonorrhoeae isolated in the United Kingdom.

Seven hundred and seventy nine isolates of Neisseria gonorrhoeae from the County of Avon, United Kingdom, were examined by auxotyping and penicillin susceptibility testing. Prototrophic strains which were heterogeneous as judge by penicillin susceptibility were the most commonly isolated and increased in prevalence over the three year period. The group most generally susceptible to penicillin required ArgHypUra for growth and decreased in prevalence over the three year period. Ten strains failed to grow on the auxotyping medium. Serogrouping was undertaken on 408 isolates. All strains reacting with the WI reagents were ArgHypUra requiring. However auxotypically similar strains (ArgHypUra) reacted with WII/WIII reagents suggesting a distinct and separately evolved group. Twenty four strains did not type with the serogrouping reagents. Using contact pairs, W-class serology was found to be a stable marker during natural transmission. However auxotypes were found to be unstable in 22 of 104 contact pairs and of these 18 involved a single requirement for proline. This very high incidence of instability with respect to this marker means it is not possible to reliably differentiate between the prototrophic group and the group with a single requirement for proline.

Neisseria gonorrhoeae: stability of typing markers after natural transmission.

The gonococcal isolates from 15 contact pairs and three large contact groups were examined using various methods to assess the stability of different typing markers. With the exception of one contact group which showed variable proline requirements, the auxotypes were stable during natural transmission. Serogrouping using the coagglutination method to detect W and M antigens was undertaken. The lipopolysaccharide M antigens were readily lost and gained during transmission whereas the protein W antigens represented stable markers and are thus useful for epidemiological studies.

Auxotyping of Neisseria gonorrhoeae isolated in the United Kingdom.

One hundred and twenty consecutive isolates of Neisseria gonorrhoeae from the County of Avon, UK, were examined by auxotyping and penicillin susceptibility testing. Requirements for proline (Pro), arginine (Arg), hypoxanthine (Hyp), uracil (Ura) and methionine (Met) were determined with a modified Heckels' medium. Prototrophic strains accounted for 29.9% of isolates and were a heterogeneous group as judged by penicillin susceptibility testing. The group most generally susceptible to penicillin required Arg-Hyp-Ura, and represented 27.5% of isolates. The Pro-Argo-Ura auxogroup (10% of isolates) had the narrowest range of penicillin susceptibility. Prototrophic strains had a reduced growth rate on arginine-free medium. Investigations of this phenomenon and suitable methods for auxotyping are reported.

Immunological responses in late syphilis.

Thirty-one serum samples from 18 patients with clinically established late syphilis and 1319 from patients at other stages of the disease were fractionated by density gradient ultracentrifugation and examined for antilipoidal and antitreponemal antibodies of the IgM and IgG classes. Sera from the patients with late syphilis always showed persistent concentrations of antilipoidal IgM and IgG and of antitreponemal IgG but never yielded detectable concentrations of antitreponemal IgM. When treated, these patients' antibody titres did not decline. Patients with secondary or latent syphilis also showed this serological picture after treatment but only transiently; their antibody titres continued to decline in a way which clearly distinguished them from the cases of late syphilis. It is suggested that patients whose sera persist in showing the stable pattern described may develop late symptomatic syphilis.