X inactivation as a mechanism of selection against lethal alleles: further investigation of incontinentia pigmenti and X linked lymphoproliferative disease.

Thirty-one females with incontinentia pigmenti (IP), 42 controls, and 11 females from four families segregating for X linked lymphoproliferative disease (XLP) were studied for evidence of skewed X inactivation by analysis of methylation at sites in the HPRT, PGK, and M27 beta (DXS255) regions of the X chromosome. Extensive skewing of X inactivation was present in blood from 4/42 (9.5%) control females and 11/31 (35%) of those with IP. This frequency of skewed inactivation was seen in both familial and sporadic cases of IP. Analysis of inactivation in mother/daughter pairs, both affected and control subjects, showed no familial consistency of pattern, arguing against specific mutations being associated with particular patterns of inactivation. In the only informative family where both mother and daughter were affected by IP and showed skewed inactivation, the IP mutation was on the active X chromosome. This argues against cell selection during early embryogenesis being the explanation for the skewed inactivation observed. These data confirm that skewed inactivation of one X is observed in lymphocytes from a significant minority of normal females, and is seen with raised frequency in IP heterozygotes. It is not, however, a universally observed phenomenon, and the relationship of X inactivity to the IP mutation appears to be complex. In the case of XLP, though skewed X inactivation patterns are seen in most disease carriers, the frequency with which this phenomenon occurs in normal females renders it an unreliable diagnostic marker for XLP carriers.     

Serum PSA and PAP measurements discriminating patients with prostate carcinoma from patients with nodular hyperplasia.

Prostatic specific antigen (PSA) and prostatic acid phosphatase (PAP) are the tumor markers for monitoring disease progression or improvement in patients with prostate adenocarcinoma. The clinical utility of PSA and PAP for early detection of prostate adenocarcinoma, however, requires distinction between prostate adenocarcinoma and prostate nodular hyperplasia. The serum PSA and PAP levels were measured in 20 men with histologically proven prostate adenocarcinoma and 28 men with histologically proven prostate nodular hyperplasia. Patients'' blood samples were collected 1 to 7 days prior to the prostate examination, which included a rectal digital examination, transurethral resection, cytoscopy, and prostate biopsy. Sensitivity, specificity, and predictive values of positive and negative results for the discrimination of prostate adenocarcinoma from prostate nodular hyperplasia were 85%, 89%, 85%, and 29%, respectively, for serum PSA (cutoff level: 10 ng/mL) and 40%, 96%, 89%, and 69%, respectively, for serum PAP (cutoff level: 10 ng/mL). Results indicate that marked elevation of serum PSA suggests prostate adenocarcinoma and that serum PSA can discriminate prostate adenocarcinoma from prostate nodular hyperplasia better than serum PAP.     

Expression of c-myc and c-fms oncogenes in trophoblastic cells in hydatidiform mole and normal human placenta.

AIMS: To compare the expression of c-myc and c-fms proto-oncogenes in the placenta and hydatidiform mole. METHODS: Twelve hydatidiform moles and six induced abortion cases were collected. c-myc and c-fms proto-oncogene expression was analysed by northern blot hybridisation and immunohistochemical staining. RESULTS: The results of northern blot hybridisation analysis showed that c-fms was expressed more strongly in hydatidiform moles compared with normal placenta of similar gestational age. Moreover, c-fms mRNA concentrations increased with more advanced gestational age in moles but not in normal placentas. c-myc expression was very low in hydatidiform moles and normal placentas. Both oncogenes, however, had no direct correlation with the clinical course of the molar pregnancies. CONCLUSION: The difference in c-fms expression between hydatidiform moles and normal placentas suggests that c-fms may have a role in the development of molar pregnancies.     

Do proficiency test results correlate with the work performance of screeners who screen Papanicolaou smears?

We rescreened Papanicolaou smear slides from 40,245 women, which had been examined by 81 cytology screeners, scored the screeners' work performance, and compared these scores with the results of the screeners' performance on glass slide and computer-based proficiency tests. All diagnoses (i.e., from the proficiency tests, the original slides, and the rescreened slides) were classified in the 4 diagnostic categories specified in the Clinical Laboratory Improvement Amendments. The rescreening scores were standardized to account for different distributions of abnormalities in the proficiency tests and rescreened slides. We compared a standardized score with the proficiency test scores. Of the cases, 91% were categorized as normal, benign, or reactive changes when rescreened, and 98% of these agreed with the original diagnosis. Sixteen percent of low-grade and 15% of high-grade intraepithelial lesions were classified as normal. The rank correlation between the rescreening scores and both proficiency tests was 0.24 using a scoring scheme for cytotechnologists. The correlation between the rescreening and proficiency testing scores indicates that performance on a 10-slide test gives some indication of the true performance of screeners. The computer-based test shows promise as an alternative to the glass slide test but needs further development and validation.     

The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species

Ataxia-telangiectasia (A-T) is an autosomal recessive disorderinvolving cerebellar degeneration, immunodeficiency, radiationsensitivity, and cancer predisposition. A-T heterozygotes aremoderately cancer prone. The A-T gene, designated ATM, was recentlyidentified in our laboratory by positional cloning, and a partialcDNA clone was found to encode a polypeptide with a PI-3 kinasedomain. We report here the molecular cloning of a cDNA contigspanning the complete open reading frame of the ATM gene. Thepredicted protein of 3056 amino acids shows significant sequencesimilarities to several large proteins in yeast, Drosophilaand mammals, all of which share the PI-3 kinase domain. Manyof these proteins are involved in the detection of DNA damageand the control of cell cycle progression. Mutations in theirgenes confer a variety of phenotypes with features similar tothose observed in human A-T cells. The complete sequence ofthe ATM gene product provides useful clues to the function ofthis protein, and furthers understanding of the pleiotropicnature of the A-T mutations.     

Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA

Nucleic acid sequence-based amplification (NASBA) is a technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique has been developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The NASBA technique described below is rapid and specific for the identification of influenza A subtype H5 viruses of the Eurasian lineage. More importantly, it can be used to distinguish highly pathogenic and low pathogenic strains of the H5 subtype.     

Solid-phase Clq-binding fluorescence immunoassay for detection of circulating immune complexes.

A fluorescence immunoassay for detection of immune complexes bound to solid-phase C1q was developed. The method was standardized by using human aggregated immunoglobulin G (IgG) to simulate immune complexes. A linear relationship existed between the concentrations of the aggregated IgG standards and the resulting fluorescent intensity. The method was found to be reproducible and capable of detecting as little as 10 micrograms of aggregated IgG per ml of heat-inactivated human serum. Antigen-antibody complexes prepared in vitro were detectable from equivalence to moderate antigen excess. Endogenous serum C1q inhibited the binding of aggregated IgG to solid-phase C1q. Pretreatment of test sera with EDTA was ineffective in eliminating this competitive effect. Heating the sera at 56 degrees C alleviated, but did not abolish, interference of endogenous C1q. Elevated levels of immune complexes were detectable in sera fro seven of nine patients wit systemic lupus erythematosus, provided the samples were heat inactivated before testing. Heparin and DNA were also found to interfere with the detection of aggregated IgG added to human serum. Assay values were falsely decreased due to competitive inhibition by these anions. Lipopolysaccharides from a variety of bacterial preparations produced no detectable interference. A comparative study was conducted on samples that had previously been tested by fluid-phase C1q-binding radioimmunoassay. The two methods were concordant in assigning normal or elevated levels of immune complexes in 70% of the samples tested. This solid-phase fluorescence immunoassay is proposed as a possible alternative to radioimmunoassay for the detection of circulating immune complexes.     

Differential activation of human and guinea pig complement by pentameric and hexameric IgM

Human and mouse IgM can be polymerized as a hexamer in addition to a pentamer. Our previous work with mouse IgM measured activation of guinea pig complement by highly enriched preparations of hexamer and pentamer and showed that hexamer is >100-fold more active than pentamer. In this report pentamer and hexamer were compared for their capacity to activate complement in a homogeneic system, i.e. chimeric mouse V/human Cmu IgM pentamer and hexamer were assayed separately for their capacity to activate human (and guinea pig) complement. In both the homogeneic and the xenogeneic systems hexamer was more active than pentamer, but the magnitude of the difference between hexamer and pentamer depended on the complement source. Whereas chimeric hexamer activated guinea pig complement >100-fold more efficiently than did chimeric pentamer, this hexamer was only 4-13-fold more active than pentamer when assayed with human complement. Similarly, mouse hexamer, which was >100-fold more active than mouse pentamer with guinea pig complement, was only approximately 2-fold more active than mouse pentamer with human complement. Mouse hexameric and pentameric IgM were each approximately 20-fold more active with human complement than were the corresponding chimeric isoforms of IgM.     

The history of contraction of the wrist flexors can change cortical excitability

Many freshwater turtles in temperate climates may experience winter periods trapped under ice unable to breathe, in anoxic mud, or in water depleted of O₂. To survive, these animals must not only retain function while anoxic, but they must do so for extended periods of time. Two general physiological adaptive responses appear to underlie this capacity for long-term survival. The first is a coordinated depression of metabolic processes within the cells, both the glycolytic pathway that produces ATP and the cellular processes, such as ion pumping, that consume ATP. As a result, both the rate of substrate depletion and the rate of lactic acid production are slowed greatly. The second is an exploitation of the extensive buffering capacity of the turtle's shell and skeleton to neutralize the large amount of lactic acid that eventually accumulates. Two separate shell mechanisms are involved: release of carbonate buffers from the shell and uptake of lactic acid into the shell where it is buffered and sequestered. Together, the metabolic and buffering mechanisms permit animals to survive for 3–4 months at 3 °C with no O₂ and with circulating lactate levels of 150 mmol l⁻¹ or more.     

Effects of Escherichia coli and Porphyromonas gingivalis lipopolysaccharide on pregnancy outcome in the golden hamster.

This report describes the effects of two gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) preparations on hamster pregnancy outcome variables. Single intravenous challenges with Escherichia coli and Porphyromonas gingivalis LPS on day 8 of pregnancy produced dose-dependent effects on fetal weight malformation and fetal resorption with E. coli LPS having potent embryolethal effects. Premating maternal exposure to P. gingivalis produced embryolethal effects similar to those of E. coli. These data suggest that maternal exposure to P. gingivalis LPS prior to and during pregnancy can induce deleterious effects on the developing fetus.