Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid.

In the past few years, application of the PCR to the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from patients with encephalitis and meningitis has become standard laboratory practice. However, from an operational perspective, the true diagnostic value of PCR in this setting is yet to be realized because most laboratories subject the amplification products to lengthy probe hybridization procedures by Southern blotting. As alternatives to Southern blotting, we evaluated colorimetric microtiter plate (MTP) systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc. (Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a system developed at the Mayo Clinic with the PCR ELISA system (Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern blotting, the sensitivity and specificity were 63.0 and 100.0%, respectively, for the PrimeCapture system, 98. 2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100. 0%, respectively, for the GEN-ETI-K system, and 100.0 and 96.9%, respectively, for the Mayo system. All four MTP systems had turnaround times 12 to 24 h less than that for Southern blotting. There were no significant differences in costs or technologist time between the Mayo system and Southern blotting. Other features of the Mayo system include type-specific genotypic identification of HSV and the potential for determination of drug resistance by DNA sequencing. Overall, we found that colorimetric MTP systems were likely to improve test turnaround times and patient care at no additional cost.

Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.

Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.

No evidence for Borrelia burgdorferi infection in lesions of morphea and lichen sclerosus et atrophicus in Spain. A prospective study and literature review.

The possible association of Borrelia burgdorferi with morphea and lichen sclerosus et atrophicus has been the focus of research and discussion in dermatology during the last 10 years. To investigate the etiopathogenic role of B. burgdorferi in morphea and lichen sclerosus et atrophicus lesions in Spain, we studied 14 cases: 8 patients with lichen sclerosus et atrophicus and 6 with morphea. For the whole group, a prospective study was performed, including serologic studies by indirect immunofluorescence, histologic evaluation of skin biopsy specimens, culture studies, and polymerase chain reaction with different primers sensitive for detecting virtually all B. burgdorferi strains tested to date. Although one patient with morphea had positive serologic findings at low titer, we were not able to culture or detect borrelial DNA in any of the specimens. These findings do not confirm an association between B. burgdorferi and morphea and lichen sclerosus et atrophicus.

Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in cerebrospinal fluid in Lyme neuroborreliosis.

A polymerase chain reaction (PCR) assay that detects Borrelia burgdorferi DNA in cerebrospinal fluid (CSF) was evaluated as a diagnostic test for acute or chronic Lyme neuroborreliosis. In one laboratory, 102 samples were tested blindly, and 40 samples were retested in a second laboratory. In the first laboratory, B. burgdorferi DNA was detected in CSF samples in 6 (38%) of 16 patients with acute neuroborreliosis, 11 (25%) of 44 with chronic neuroborreliosis, and none of 42 samples from patients with other illnesses. There was a significant correlation between PCR results and the duration of previous intravenous antibiotic therapy. The overall frequency of positive results was similar in the second laboratory, but concordance between the laboratories and among primer-probe sets was limited because many samples were positive with only one primer-probe set. Thus, PCR testing can sometimes detect B. burgdorferi DNA in CSF in patients with acute or chronic neuroborreliosis, but with current methods, the sensitivity of the test is limited.

High level monocyte chemoattractant protein-1 expression in transgenic mice increases their susceptibility to intracellular pathogens.

We have constructed transgenic mice in which the mouse mammary tumor virus long terminal repeat controls the expression of murine monocyte chemoattractant protein-1 (MCP-1). Several independently derived lines of transgenic mice constitutively expressed MCP-1 protein in a variety of organs. Protein extracts from these organs had substantial in vitro monocyte chemoattractant activity that was neutralized by an anti-MCP-1 Ab, indicating that transgenic MCP-1 protein is biologically active. However, no transgenic mouse at any age displayed monocyte infiltrates in MCP-1-expressing organs. Two transgenic lines had circulating MCP-1 levels of 13 to 26 ng/ml, which is a concentration sufficient to induce maximal monocyte chemotaxis in vitro. These transgenic lines showed a 1 to 1.5 log greater sensitivity to infection with Listeria monocytogenes and Mycobacterium tuberculosis. A third transgenic line had lower serum levels of MCP-1 and was resistant to L. monocytogenes. The results suggest that this transgenic model is one of monocyte nonresponsiveness to locally produced MCP-1 due to either receptor desensitization or neutralization of a chemoattractant gradient by high systemic concentrations of MCP-1. Regardless of the mechanism, the data indicate that constitutively high levels of MCP-1 expression do not induce monocytic infiltrates, and that MCP-1 is involved in the host response to intracellular pathogens.

Detection of Borrelia burgdorferi DNA in museum specimens of Peromyscus leucopus.

To determine whether Borrelia burgdorferi was enzootic within the United States at the beginning of the 20th century, ear skin samples taken from museum specimens of the white-footed mouse (Peromyscus leucopus) were examined for evidence of spirochetal DNA. In total, 280 samples from mice collected between 1870 and 1919 were analyzed by a nested polymerase chain reaction protocol. Of these, 2 specimens from the vicinity of Dennis, Massachusetts, during 1894 were reproducibly positive for B. burgdorferi OspA sequences. The remaining 278, representing both currently endemic and nonendemic sites, were negative for spirochetal DNA. These studies suggest that the agent of Lyme disease was present in a suitable reservoir host in the United States before the turn of the century and provide evidence against a hypothesis of recent introduction of this zoonotic agent to North America.

Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis. Identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis.

Monocyte chemoattractant protein-1 (MCP-1) is a monocyte-specific chemoattractant and activator and is a member of the chemokine-beta family of cytokines. To identify regions of MCP-1 which are required for its biological activity, we constructed human MCP-1 mutants that were expressed in eukaryotic cells and tested for their ability to attract monocytes in vitro. Deletion of amino acids 2-8 destroyed activity, suggesting that the amino-terminal region is necessary for activity. Within the deleted region, mutation of aspartate 3 to alanine produced a protein with 9% of wild-type activity, whereas mutation of asparagine 6 to alanine produced a protein with 52.9% of wild-type activity. Mutation of amino acids within the first intercysteine loop yielded variable results. Changing tyrosine 28 to aspartate or arginine 30 to leucine each produced proteins with essentially no monocyte chemoattractant activity. The side chains of these amino acids are predicted to point into a putative receptor binding cleft, and these loss-of-function mutations are consistent with this model. Also consistent is the retention of 60% of wild-type activity after mutation of serine 27 to glutamine, since the side chain of serine 27 is predicted to point away from the binding cleft. However, mutation of arginine 24, which lies outside of this area, to phenylalanine produced a protein with only 5% of wild-type activity, suggesting more complex interactions. Truncations of the carboxyl terminus, as well as mutation of aspartate 68 to leucine, generated proteins with 10-20% of wild-type activity. (Another carboxyl-terminal insertional mutation demonstrated that O-linked carbohydrate in MCP-1 alpha may be added to a threonine in the carboxyl-terminal region.) These findings are consistent with a structural model of dimeric MCP-1 which is similar to interleukin-8, in which amino acids that point into a cleft between the two carboxyl-terminal alpha-helices of the subunits are important for receptor binding. In addition, however, amino acids at the amino terminus and others outside of the interhelical cleft are also essential for activity. The carboxyl-terminal alpha-helix is not required for signaling per se but is required for maximal specific activity. Finally, four mutant proteins partially inhibited the ability of wild-type MCP-1 to attract monocytes in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine-beta (or C-C) family of cytokines. Murine MCP-1, first identified as the JE gene, differs from human MCP-1 in molecular size and extent of glycosylation. We have used Chinese hamster ovary cells to express recombinant murine MCP-1 and find that its predominant form is a microheterogeneous protein of M(r) approximately 25,000. Most of MCP-1's microheterogeneity is due to variable amounts of sialic acid that are terminally attached to a constant number of O-linked oligosaccharide chains per molecule. This carbohydrate, along with a small amount of N-linked carbohydrate, accounts for 50% of the apparent molecular size of murine MCP-1 and is not required for in vitro monocyte chemoattractant activity. Mutational analysis shows that most of the carbohydrate is added to a 49-amino acid C-terminal domain that is not present in human MCP-1 and is not required for in vitro biologic activity, suggesting that murine MCP-1 consists of an N-terminal domain containing monocyte chemoattractant activity and a heavily glycosylated C-terminal domain of as yet unknown function. MCP-1 produced in COS cells contains a small amount of sulfate, but Chinese hamster ovary-produced MCP-1 does not. The absence of sulfate does not alter MCP-1's in vitro chemoattractant properties. In vitro, highly purified murine MCP-1 attracts monocytes, but not neutrophils, with a specific activity similar to human MCP-1 (EC50 approximately 0.5 nM). Equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM. In vivo, injection of up to 1 micrograms murine MCP-1 in a variety of murine strains induces the appearance of a sparse mixed inflammatory infiltrate. The disparity between MCP-1's in vitro and in vivo effects suggests that other factors may be required to elicit a full-blown monocyte chemotactic response to MCP-1 in vivo.

Target imbalance: disparity of Borrelia burgdorferi genetic material in synovial fluid from Lyme arthritis patients.

Lyme arthritis is a late manifestation of Lyme disease that results in episodic synovial inflammation and swelling. Although this process is thought to be driven directly by the spirochetal etiologic agent, Borrelia burgdorferi, the organism itself has been recovered by culture only twice. In contrast, polymerase chain reaction (PCR) studies are usually positive. This apparent discrepancy in 19 culture-negative synovial fluid specimens from 18 patients with Lyme arthritis was investigated. In all 19, DNA sequences characteristic of plasmid-encoded genes OspA and OspB were easily detected. However, despite equivalent or even superior analytic sensitivity for detection of cultured organisms, the reactivity of two genomic DNA targets was often weak or absent altogether in the clinical specimens. This apparent overrepresentation of B. burgdorferi plasmid sequences was found exclusively in clinical specimens and not in cultured organisms. The physiologic imbalance of genomic and plasmid DNA reactivity in B. burgdorferi infection may signal an underlying pathogenetic mechanism.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis.

BACKGROUND: Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists despite antibiotic therapy. METHODS: Using the polymerase chain reaction (PCR), we attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period. The samples were tested in two separate laboratories with four sets of primers and probes, three of which target plasmid DNA that encodes outer-surface protein A (OspA). RESULTS: B. burgdorferi DNA was detected in 75 of 88 patients with Lyme arthritis (85 percent) and in none of 64 control patients. Each of the three OspA primer-probe sets was sensitive, and the results were moderately concordant in the two laboratories (kappa = 0.54 to 0.73). Of 73 patients with Lyme arthritis that was untreated or treated with only short courses of oral antibiotics, 70 (96 percent) had positive PCR results. In contrast, of 19 patients who received either parenteral antibiotics or long courses of oral antibiotics (> or = 1 month), only 7 (37 percent) had positive tests (P < 0.001). None of these seven patients had received more than two months of oral antibiotic treatment or more than three weeks of intravenous antibiotic treatment. Of 10 patients with chronic arthritis (continuous joint inflammation for one year or more) despite multiple courses of antibiotics, 7 had consistently negative tests in samples obtained three months to two years after treatment. CONCLUSIONS: PCR testing can detect B. burgdorferi DNA in synovial fluid. This test may be able to show whether Lyme arthritis that persists after antibiotic treatment is due to persistence of the spirochete.

The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis.

We describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-alpha. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.

DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa.

In Neurospora, five structural and two regulatory genes mediate the initial events in quinate/shikimate metabolism as a carbon source. These genes are clustered in an 18 x 10(3) base-pair region as a contiguous array. The qa genes are induced by quinic acid and are coordinately controlled at the transcriptional level by the positive and negative regulators, qa-1F and qa-1S, respectively. The DNA sequence of the entire qa gene cluster has been determined and transcripts for each gene have been mapped. The qa genes are transcribed in divergent pairs and two types of transcripts are associated with each gene: basal level transcripts that initiate mainly from upstream regions and are independent of qa regulatory gene control, and inducible transcripts that initiate downstream from basal transcripts and are dependent on qa-1F binding to a 16 base-pair sequence. We discuss how both types of transcription relate to the organization of the qa genes as a cluster and how this may impose constraints on gene dispersal.

Alteration of hsp82 gene expression by the gypsy transposon and suppressor genes in Drosophila melanogaster.

Several mutations in Drosophila result from insertion of the gypsy retrotransposon. Gypsy insertion mutagenesis and its modulation by allele-specific modifier genes were investigated by inserting gypsy or fragments of it into the intron of the Drosophila hsp82 heat shock gene. With gypsy in the parallel orientation, nearly all transcripts in transfected cells and transformed pupae were truncated in the 5' long terminal repeat (LTR). Truncation also occurred in or near the 3' LTR. The 5' LTR polyadenylation signal was strongly potentiated by a downstream 326-bp internal gypsy segment in either orientation. Anti-parallel gypsy reduced the amount of normal transcript to a much smaller extent, and a low level of truncation occurred within gypsy. No evidence was found for effects of the gypsy insertions on the hsp82 promoter. Mutations in the allelespecific modifier genes su(f) and su(w alpha) had effects on the amounts of readthrough transcripts consistent with their genetic behavior, whereas the effects of mutations in su(Hw) were only partly in accord with genetic expectations.

Genetic interactions of modifier genes and modifiable alleles in Drosophila melanogaster.

We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.

Molecular characterization of the qa-4 gene of Neurospora crassa.

The qa-4 gene of Neurospora crassa encodes 3-dehydroshikimate dehydratase, which catalyzes the third step of the quinic acid (qa) catabolic pathway. The enzyme has previously been purified and characterized as a monomer of approx. 37 kDal. The nucleotide sequence of the qa-4 gene is presented here and the amino acid composition and tentative NH2-terminal sequence confirm the identification of the coding region within the qa-4 DNA sequence. There are no introns in the qa-4 coding region. By S1 nuclease mapping and primer extension analysis three distinct regions of transcription initiation were identified. Heterogeneity was also observed in the 3' ends of qa-4 mRNA. 5' and 3' untranslated regions of the qa-4 gene were compared with the corresponding regions in other Neurospora genes. Genomic blot analysis of twenty previously isolated qa-4 mutants revealed that two mutants, MC150 and MC191, have restriction patterns altered from wild type. In each strain the alteration occurs in the 3' half of the qa-4 coding region.