Detection of human papillomavirus types 6 and 11 E4 gene products in condylomata acuminatum.

Polyclonal antiserum to an Escherichia coli-produced beta-galactosidase/E4 fusion protein of human papillomavirus type 6b (antiserum 256), and affinity purified HPV 11 anti-E4 antibodies were tested for reactivity in Western blots with bacterially expressed trpE/E4 fusion proteins of HPV types 6b, 11, 16, and 18. To further characterize the affinity purified anti-E4 antibodies, a dot-immunobinding assay was performed using overlapping synthetic HPV 11 E1E4 peptides as antigens. Protein extracts of condylomata acuminatum from 18 patients containing HPV type 6 or 11 DNA sequences were tested in Western blots using antiserum 256 or affinity purified HPV 11 anti-E4 antibodies. In the Western blots of the trpE proteins, antiserum 256 identified the HPV types 6b and 11 fusion proteins; the affinity purified HPV 11 anti-E4 antibodies identified only the HPV 11 fusion protein. In the dot-immunobinding assay, three HPV 11 peptides were recognized, each containing a shared 8 amino acid sequence that differs significantly from the corresponding sequences of HPV types 6b, 16, or 18. In the Western blots of protein extracts from 18 condylomata acuminatum samples shown to contain HPV types 6 or 11 DNA, putative E4 gene products were identified in six samples by antiserum 256. The affinity purified HPV 11 anti-E4 antibodies identified putative E4 gene products in one of these same six lesions, which was shown to contain HPV 11 sequences by the Southern blot method. All six samples containing E4 gene products were from women. Three of these women were pregnant, one had serum antibodies to the human immunodeficiency virus, and one was a renal transplant recipient receiving glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pattern of Sequence Variation Across 213 Environmental Response Genes

To promote the clinical and epidemiological studies that improve our understanding of human genetic susceptibility to environmental exposure, the Environmental Genome Project (EGP) has scanned 213 environmental response genes involved in DNA repair, cell cycle regulation, apoptosis, and metabolism for single nucleotide polymorphisms (SNPs). Many of these genes have been implicated by loss-of-function mutations associated with severe diseases attributable to decreased protection of genomic integrity. Therefore, the hypothesis for these studies is that individuals with functionally significant polymorphisms within these genes may be particularly susceptible to genotoxic environmental agents. On average, 20.4 kb of baseline genomic sequence or 86% of each gene, including a substantial amount of introns, all exons, and 1.3 kb upstream and downstream, were scanned for variations in the 90 samples of the Polymorphism Discovery Resource panel. The average nucleotide diversity across the 4.2 MB of these 213 genes is 6.7 × 10^-4, or one SNP every 1500 bp, when two random chromosomes are compared. The average candidate environmental response gene contains 26 PHASE inferred haplotypes, 34 common SNPs, 6.2 coding SNPs (cSNPs), and 2.5 nonsynonymous cSNPs. SIFT and Polyphen analysis of 541 nonsynonymous cSNPs identified 57 potentially deleterious SNPs. An additional eight polymorphisms predict altered protein translation. Because these genes represent 1% of all known human genes, extrapolation from these data predicts the total genomic set of cSNPs, nonsynonymous cSNPs, and potentially deleterious nonsynonymous cSNPs. The implications for the use of these data in direct and indirect association studies of environmentally induced diseases are discussed.     

Eimeria in Fowls: Immunity to Four Species of

Two or three graded infections with oocysts of Eimeria acervulina, E. tenella, E. necatrix and E. maxima produced a resistance to further infection with the immunizing species. The oocyst output after the second infection, in each case, was lower than that after the initial dose indicating the substantial immunizing effect of the initial infection. The species could be placed in a descending order of immunizing activity as follows: E. maxima, E. acervulina, E. tenella and E. necatrix. A solid immunity to the immunizing species in no way prevented the development of an additional infection, here referred to as `cross-infection', with any of the species studied.

Serum precipitins were produced in infections with all four species, the response to infection with E. necatrix being less marked than to the other species. A first challenge of immune fowls with the immunizing species produced some increase in precipitation in agar whereas a second challenge had no such effect; the significance of this lack of response is discussed. Usually, fowls immunized against one species and then infected with an additional one, produced serum precipitins which reacted only with the antigen of the additional species. But E. tenella immunized fowls, when given an additional infection with E. necatrix, produced precipitins that reacted with antigens of both species. The same was also true when E. necatrix immunized fowls were infected with E. tenella.

     

Prevalence of human T-Cell lymphotropie virus infections in Germany

The extent of human T-cell lymphotropic retorvirus HTLV-I and HTLV-II infections in the general population in central Europe has not been investigated fully. Two hundred forty-eight thousand blood donors from southern Germany were examined serologically for antibodies to the human lymphotropic retroviruses HTLV-I and HTLV-II: 0.021% were confirmed postive and 0.056% were “indeterminate”. A limited number of seropositives and “indeterminate” samples were analyzed by polymerase chain reaction (PCR): the seropositives were confirmed as positive and 43% of the “indeterminate” samples were PCR-positive. The range of 0.021% HTLV-positives in 248,000 donors, i.e. about two in 10,000 individuals, mirrors closely the published data for the United States. © 1994 Wiley-Liss, Inc.     

Evaluation of hyperforin analogues for inhibition of 5-lipoxygenase

The acylphloroglucinol hyperforin, a constituent of the herb Hypericum perforatum (St. John's wort), was recently identified as potent and direct inhibitor of 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes. In this study, naturally occurring analogues of hyperforin, isolated from H. perforatum, as well as a series of synthetic derivatives obtained by chemical modification of hyperforin by acylation, alkylation or oxidation, were analysed for the inhibition of 5-LO. The efficacies of these compounds were evaluated in intact human polymorphonuclear leukocytes, but also the inhibitory effects on isolated recombinant human 5-LO were investigated. Our data show that some of the oxidised hyperforin derivatives possess even improved efficacy, whereas alkylation and acylation have detrimental effects.     

USH2A mutation analysis in 70 Dutch families with Usher syndrome type II

Usher syndrome type II (USH2) is characterised by moderate to severe high-frequency hearing impairment, progressive visual loss due to retinitis pigmentosa and intact vestibular responses. Three loci are known for USH2, however, only the gene for USH2a (USH2A) has been identified. Mutation analysis of USH2A was performed in 70 Dutch USH2 families. Ten mutations in USH2A were detected, of which three are novel, c.949C>A, c.2242C>T (p.Gln748X) and c.4405C>T (p.Gln1468X). Including 9 previously published Dutch USH2a families, estimates of the prevalence of USH2a in the Dutch USH2 population were made. Mutations were identified in 62% of the families. In 28% both mutated alleles were identified, whereas in 34% the mutation in only one allele was found. It is estimated that about 28% of the Dutch USH2 families have a different causative gene. Analysis of deduced haplotypes suggests that c.1256G>T (p.Cys419Phe) is a Dutch ancestral mutation, occurring in 16% of the alleles.     

An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.     

Amnesia-producing drugs affect hippocampal frequency potentiation

The effects of intravenous injections of lorazepam, scopolamine and propranolol upon hippocampal potentiation produced by commissural stimulation have been investigated in rats anaesthetized with urethane. Administration of 250 micrograms/kg or 500 micrograms/kg lorazepam significantly delayed the onset of secondary potentiation (frequency potentiation) of the population spikes recorded in subfields CA1 and CA3 of the dorsal hippocampus. Scopolamine also delayed the onset of frequency potentiation in CA1, but only at high dose (10 mg/kg). No other measured parameters of frequency potentiation, paired-pulse potentiation or post-tetanic potentiation were affected by any of the drugs. Lorazepam (greater than or equal to 250 micrograms/kg) and propranolol (3 mg/kg) reduced the severity of hippocampal after-discharge. Rhythmic entrainment of after-discharges was occasionally observed. The results are discussed in relation to the possible link between hippocampal potentiation and memory processes.     

Comparison of two ELISAs for the determination of Hsp70 in serum

We have compared a previously developed in-house Sandwich-ELISA with a commercial kit for the determination of heat shock protein (Hsp) 70 in serum. Samples from 64 participants were tested and there was a significant correlation between results obtained using the two assays (r = 0.807, p < 0.0001). Additionally, when ranking samples on a categorical scale, the agreement was good (72%). In the commercial test system Hsp70 was detectable in 42 (66%) of the sera, compared with 61 (95%) in the in-house ELISA method. The three samples with undetectable levels of Hsp70 in the in-house ELISA were among the 22 samples with undetectable levels of Hsp70 in the commercial ELISA kit. The apparent serum concentrations detected were different in the two systems. This dissimilarity can be ascribed to differences in the matrix used. We conclude that the in-house ELISA is more economical and performs well when measuring physiologically high, as well as low, concentrations of Hsp70.     

Marginal zone and germinal center development in the spleens of neonatally thymectomized and nonthymectomized young rats

Spleens from neonatally thymectomized and nonthymectomized young rats were studied histologically and histochemically to elucidate the development of the splenic immune system with and without thymus. In intact animals primitive germinal center activity could be elicited with antigen as early as 13 days of age. More definitive germinal centers lacking tingible body macrophages were observed at 18 days of age. Germinal centers containing tingible body macrophages did not develop until 35 days of age in response to antigenic stimulation. This coincided with maximal development of the marginal zone of medium-sized lymphocytes and the mature development of nodular macrophages possessing strong acid phosphatase activity. Neonatally thymectomized rats developed marginal zones and germinal centers similar to control littermates when the young animals were maintained on tetracycline. Thymectomized animals not given tetracycline showed disturbances in splenic development. These are discussed. The results suggest that the thymus may be critical to the immune system in rats from birth to about 30 days of age but is not essential to its function beyond this period. Marginal zone lymphocytes and germinal center cells proliferate normally and mature to the plasma cell stage in the absence of a thymus if the animals are maintained on tetracycline beyond this critical age.