Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease.

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.

Molecular cloning, sequence and characterization of cjsT, a putative protease from Rickettsia rickettsii.

The cloning and sequencing of a gene from Rickettsia rickettsii which confers haemolytic activity on Escherichia coli strain TB1 is described. The open reading frame of the haemolysis-promoting gene, cjsT, is 1041 bp and encodes a putative protein with a molecular mass of 33 825 Da. CjsT has high sequence similarity to several bacterial proteases, particularly type IV signal peptidases. Cell lysates from an E. coli clone containing cjsT in pUC19 (pJON1) exhibited greater protease activity in functional assays than found in E. coli containing pUC19 alone. Disruption of the cjsT gene by insertional inactivation with a kanamycin cassette reduced both the protease and haemolytic activities conferred by cjsT. The protease inhibitors antipain and diisopropylfluorophosphate (DFP) both reduced the proteolytic activity of pJON1. The mechanism by which the R. rickettsii cjsT promotes haemolysis in E. coli remains unclear.

The antibacterial effects of tumescent liposuction fluid.

Tumescent liposuction is currently one of the most commonly performed aesthetic procedures. Despite the variable use of preoperative antibiotics, infection is uncommon. Prior works suggest that the low incidence of infection may be due to lidocaine's antibacterial properties. However, these properties have only been demonstrated using concentrations of lidocaine above 0.8%, significantly higher than those used in tumescent liposuction. The purpose of this study was to determine if the commonly used tumescent fluid containing 0.1% lidocaine, 1:1000,000 epinephrine, and 0.012 mEq sodium bicarbonate possesses antibacterial activity. Using the broth microdilution method, the minimum inhibitory concentrations of Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Group A beta-hemolytic Streptococcus were determined after exposure to either lidocaine, epinephrine, bicarbonate, or the combination of all three agents. To determine if there were significant growth differences not detectable by the broth microdilution method, bacterial concentrations were obtained through the use of a spectrophotometer, and significant differences from the controls were calculated by one-way analysis of variance. To determine if prolonged exposure to the tumescent mix would alter bacterial growth, a Killing Time study was also undertaken. The results indicated that the minimum inhibitory concentration of lidocaine was not less than 0.5% for any of the bacteria, whereas the lowest minimum inhibitory concentration of the combined solution was 0.25%. The lowest inhibitory concentration as determined by spectrophotometric analysis for the combined solution was 0.13% (p < 0.01). Analysis of the Killing Time data revealed no inhibition of bacterial growth over time. In conclusion, lidocaine, epinephrine, and bicarbonate do exhibit antibacterial properties at high concentrations. However, the commonly used tumescent mixture containing dilute concentrations of these agents does not significantly inhibit the growth of commonly encountered bacteria.

Efficient adenoviral gene transfer to kidney cortical vasculature utilizing a fiber modified vector.

BACKGROUND: The alpha v integrin is present in vascular endothelium, including that of the kidney. It is also upregulated in the presence of inflammatory cytokines and in some neoplasms. In an effort to transduce vascular endothelial cells, we compare in vitro and in vivo adenoviral gene transfer of a vector with a high affinity peptide ligand to the alpha v integrin incorporated into the fiber coat protein AdZ.F(RGD) to an unmodified vector, AdZ. METHODS: Cell transduction assays were performed on human umbilical vein endothelial cells (HuVEC) and human pulmonary epithelial cells (A549). In vitro competition assays were performed in the presence of either wild type (F5K) or chimeric (F5K(RGD)) soluble recombinant fiber protein. Transduction efficiency was determined by quantitative beta-galactosidase activity. In vivo gene transfer was compared infusing either AdZ or AdZ.F(RGD) into the left renal artery of the rat and assaying beta-galactosidase staining of the kidney. Gene transfer was also evaluated in the presence of a competitive RGD or control RGE peptide. RESULTS: There was a marked increase in transgene expression in HuVEC cells with AdZ.F(RGD) as compared to AdZ. The increased expression with AdZ.F(RGD) was more prominent in the endothelial as opposed to the epithelial cell line. Furthermore pre-incubation of these cells with either F5K or F5K(RGD) soluble fiber protein markedly decreased beta-galactosidase activity of AdZ, whereas only the F5K(RGD) decreased beta-galactosidase activity of AdZ.F(RGD). AdZ.F(RGD) also resulted in significantly enhanced beta-galactosidase expression in the vascular endothelium of the kidney (for comparable amounts of virus injected) as well as significantly higher gene transfer to cortical vasculature. Coinfusion of an RGD peptide with AdZ.F(RGD) blocked gene transfer whereas a control RGD peptide did not. CONCLUSION: We conclude that incorporating a high affinity peptide ligand into the adenoviral fiber protein can preferentially enhance in vitro and in vivo adenoviral transfection efficiency in endothelial cells. Enhancing transfection efficiency will not only broaden the scope of disease processes addressed with adenoviral vectors but also allow the use of lower titer, thereby limiting toxicity. This vector has additional potential to transduce endothelial cells within tumors or ischemic tissue where alpha v integrins are upregulated.

Ehrlichia chaffeensis in Missouri ticks.

A nested polymerase chain reaction specific for Ehrlichia chaffeensis was used to attempt to amplify DNA from extracts of 100 individual ticks collected from 13 counties in central Missouri. Seventeen of 59 Amblyomma americanum and six of 41 Dermacentor variabilis ticks exhibited the characteristic 389-basepair product. This supports the hypothesis that these tick species may be vectors of human monocytic ehrlichiosis.

The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V. cholerae O139.

We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V. cholerae strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection. Susceptibility is restored by mshA complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the mshA pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.

Sp1 is a critical regulator of the Wilms' tumor-1 gene.

We performed deletion analysis of WT1-reporter constructs containing up to 24 kilobases of 5'-flanking and first intron WT1 sequence in stably transfected cultured cells as an unbiased approach to identify cis elements critical for WT1 transcription. Although not a tissue-specific element, a proximate 9-base pair CTC repeat accounted for approximately 80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with their ability to form a DNA-protein complex in gel shifts. Antibody supershift, oligonucleotide competition, and Southwestern studies indicated that the CTC-binding factor is the transcriptional activator Sp1. Sp1 binds the CTC repeat with an affinity, KD = 0.37 nM, at least as high as the consensus GC box. Similar CTC repeats are found in promoters of other growth-related genes. Because Sp1 is important for WT1 expression, we examined Sp1 immunohistochemistry in fetal and adult kidney. In a pattern that precedes that of WT1 message, Sp1 immunostaining was highest in uninduced mesenchyme, early tubules, developing podocytes, and mature glomeruli, but was minimal in mature proximal tubules. This work suggests abundant Sp1 may be a prerequisite for WT1 expression, and that Sp1 may have a wider role in nephrogenesis.

Human monoclonal antibody developed against ovarian cancer cell surface antigen.

BACKGROUND: Murine monoclonal antibodies (MoAb) potentially can be used in the radioimmunodetection and radioimmunotherapy of cancer. However, the administration of these radiopharmaceuticals to humans often leads to induction of human anti-murine antibodies (HAMA). HAMA has many disadvantages, which could decrease efficacy of the murine MoAb. The purpose of this work was to produce human monoclonal antibody against a human ovarian cancer cell surface antigen (OCCSA), which was not present in normal ovarian cells. This 200-kilodalton OCCSA also was used in the present study for characterizing the human monoclonal antibody. METHODS: Human monoclonal antibodies were produced in vitro by fusion of mutant myeloma cells, selected from GM1500, with human lymphoid cells immunized in vitro with purified OCCSA: The human monoclonal antibody was characterized using the following techniques: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, Western blotting followed by protein-A gold staining, immunodiffusion assays, and fluorescent antibody assays. RESULTS: Human monoclonal antibody, TC5 (immunoglobulin G1), was produced and purified. It was found to be specific for ovarian cancer, while also reacting with an early stage breast cancer. TC5 did not react with any normal (i.e., nonneoplastic) cells of the ovary, uterus, cervix, endocervix, or fallopian tube, nor did it react with normal lung, heart, pancreas, liver, or breast tissue. CONCLUSION: Human-human hybridomas produced human monoclonal antibody against OCCSA: The human monoclonal antibody, TC5, was specific for ovarian and breast cancer. TC5 did not react with any normal tissue tested. Future work will focus on the in vivo characterization of the human monoclonal antibody, after labeling with radionuclides.

Detection of ovarian cancer by 198Au-labeled human monoclonal antibody.

BACKGROUND: There is no reliable method for the early diagnosis of ovarian cancer. Radiolabeled monoclonal antibodies have potential to assist in early diagnosis, but they are limited by problems that include antibody specificity, stability, and immunoreactivity, as well as patient reactions to the antibodies used. METHODS: Methods were developed to 198Au-label a human monoclonal antibody (TC5 antibody), developed against an ovarian cancer cell surface antigen. Antigen binding sites on the TC5 antibody were protected with sepharose 4B affinity chromatography before 198Au-labeling. The 198Au-labeled TC5 antibody was evaluated with biopsy specimens in a blind study. The immunoreactivity of radiolabeled TC5 antibody also was evaluated in slot-blot experiments with extracts of the biopsy specimens. RESULTS: The 198Au-labeled TC5 antibody had high binding reaction to all biopsy specimens (six of six) pathologically diagnosed as ovarian cancer (serous and endometrioid adenocarcinoma). The radiolabeled TC5 antibody did not bind to any normal (non-neoplastic) specimens (zero in ten), with one exception. One "normal" ovary specimen had high binding of radiolabeled TC5 antibody, and metastatic ovarian cancer was diagnosed 4 months later. The TC5 antibody labeled with 198Au, without protecting antigen-binding sites, did not bind to any biopsy specimens. CONCLUSIONS: The affinity-labeling method was necessary to protect antigen-binding sites and preserve the immunoreactivity of the TC5 antibody. The 198Au-labeling method may be an ideal technique to evaluate monoclonal antibodies in vitro. The TC5 antibody had high sensitivity and specificity for detecting ovarian cancer.

Hyponatremia in the cancer patient.

Hyponatremia is a common metabolic complication in cancer patients. It can be caused by the primary tumor itself, metastasis, by diagnostic or therapeutic interventions, or by a secondary complication. A clear understanding of water and sodium homeostasis is required for evaluation. The initial diagnostic step is to define a hypoosmolar state. The history and physical examination should be tailored to determine the volume status of the patient--volume depleted, euvolemic, or edematous. In general, hyponatremia associated with volume depletion is treated with saline, while fluid restriction is the primary therapy in patients with normal or increased extracellular fluid volume. In symptomatic patients, the duration of the hyponatremia and its causes should be determined. Both overzealous and inadequate treatment put the patient at risk of serious neurologic sequelae.

A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences.

The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever.

Acquired von Willebrand's disease and hypothyroidism: report of a case presenting with menorrhagia.

A 17 year old woman presented with severe anaemia due to menorrhagia. On investigation, she was shown to have abnormalities of her haemostatic mechanism consistent with von Willebrand's disease Type I, although there was no family history of this disorder. In addition, she was shown to have severe primary hypothyroidism. On correction of hypothyroidism with oral thyroxine, her coagulation defects returned to normal and menorrhagia ceased. This is consistent with acquired von Willebrand's disease secondary to hypothyroidism.

A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever.

There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.

Cloning, expression and sequence analysis of the gene encoding the 120 kD surface-exposed protein of Rickettsia rickettsii.

Rickettsia rickettsii (R strain) genomic DNA was partially digested and cloned into the lambda expression vector gt11 generating a genomic clone bank. Transformant plaques were screened with antisera generated against the 120 kiloDalton protein of R. rickettsii to detect those phage expressing the recombinant protein. The gene encoding the 120 kD protein was localized to a 4.3 kilobase SphI-BamHI fragment from the recombinant phage and subcloned into pUC18 and pUC19. Full-length expression of the recombinant protein was achieved with both orientations. The gene and flanking regions were sequenced. The p120 gene consists of 3900 base pairs coding for 1300 amino acids. A distinguishable promoter region was not identified, although there are several 5' sequences that resemble classical prokaryotic promoters. Downstream of the termination codon for this gene lies a 726 base pair open reading frame on the opposite strand with the potential to encode a protein of approximately 27 kD. The identity of this putative gene product is unknown. The two open reading frames are separated by a 106 base pair intergenic region that consists of a stretch of dyad symmetry resembling rho-independent transcriptional terminators.

Stevens-Johnson syndrome with supraglottic laryngeal obstruction.

Patients with the Stevens-Johnson syndrome or erythema multiforme exudativum may present to the otolaryngologist because of involvement of the mucosa of the upper aerodigestive tract. Stevens-Johnson syndrome is a symptom complex of uncertain cause that affects the eyes, skin, and mucous membranes throughout the body. The epithelial lesions progress from macules to bullae that desquamate, form pseudomembranes, and finally heal. Generally there is a prodromal period with fever and symptoms of an upper respiratory tract infection. The literature related to the otolaryngologic manifestations of the disease is reviewed and a case of Stevens-Johnson syndrome with early involvement of the supraglottic larynx is presented. The otolaryngologist should be aware of this syndrome and should be prepared to manage the possible airway compromise.