Serologic testing for Lyme disease.
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Antibodies, Bacterial↗
Biomedical subjects
B J Johnson
Publications and source records attributed to B J Johnson.
Exploiting secondary growth in Arabidopsis. Construction of xylem and bark cDNA libraries and cloning of three xylem endopeptidases.
The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.
Amino Acid Sequence↗
The activated type 1-polarized CD8(+) T cell population isolated from an effector site contains cells with flexible cytokine profiles.
The capacity of activated T cells to alter their cytokine expression profiles after migration into an effector site has not previously been defined. We addressed this issue by paired daughter analysis of a type 1-polarized CD8(+) effector T cell population freshly isolated from lung parenchyma of influenza virus-infected mice. Single T cells were activated to divide in vitro; individual daughter cells were then micromanipulated into secondary cultures with and without added IL-4 to assess their potential to express type 2 cytokine genes. The resultant subclones were analyzed for type 1 and 2 cytokine mRNAs at day 6-7. When the most activated (CD44(high)CD11a(high)) CD8(+) subpopulation from infected lung was compared with naive or resting (CD44(low)CD11a(low)) CD8(+) cells from infected lung and from normal lymph nodes (LNs), both clonogenicity and plasticity of the cytokine response were highest in the LN population and lowest in the activated lung population, correlating inversely with effector function. Multipotential cells were nevertheless detected among clonogenic CD44(high)CD11a(high) lung cells at 30-50% of the frequency in normal LNs. The data indicate that activated CD8(+) T cells can retain the ability to proliferate and express new cytokine genes in response to local stimuli after recruitment to an effector site.
Animals↗
Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells.
Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.
Animals↗
Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells.
Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.
Animals↗
Comparison of touch imprints with aspirate smears for evaluating bone marrow specimens.
We compared the differential counts of normal and abnormal bone marrow from touch imprints with those from aspirate smears to determine whether the touch imprint was reliable for independent routine use in the examination of bone marrow and the classification of hematologic abnormalities. Normocellular bone marrow specimens were obtained from 87 patients without hematologic abnormality. Abnormal bone marrow specimens were obtained from 173 patients with treated or untreated neoplastic hematologic disease, including acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, non-Hodgkin lymphoma, hairy cell leukemia, myeloma, and acute lymphoblastic leukemia. We found no diagnostic difference in the differential counts from touch imprints and aspirate smears of normocellular bone marrow, and although we found some difference between the differential counts in certain cases of diseased bone marrow, the touch imprint proved to be a reliable diagnostic tool for determining the cellular composition of normal bone marrow and more reliable for the diagnosis of bone marrow involved by a neoplastic hematologic disease. Our findings suggest that evaluating touch imprints should be considered a standard practice in examining bone marrow.
Bone Marrow Cells↗
Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE.
VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.
Animals↗
The Borrelia burgdorferi 37-kilodalton immunoblot band (P37) used in serodiagnosis of early lyme disease is the flaA gene product.
The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The P37 gene was cloned from a B. burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished in Escherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E. coli protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B. burgdorferi anti-P37 reactivity. Lyme disease patient samples serologically negative for the B. burgdorferi P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.
Antibodies, Bacterial↗
Population genetics and phylogenetic analysis of Colorado Borrelia burgdorferi.
Borrelia burgdorferi is transmitted in an enzootic cycle in Colorado between the tick Ixodes spinipalpis and the woodrat Neotoma mexicana. The genetic relationship of Colorado isolates to other B. burgdorferi isolates is unknown nor have relationships among various Colorado isolates been determined. Portions of the flagellin (fla), 66-kD protein, and outer surface protein A (ospA) genes were amplified from 71 Colorado isolates, screened for genetic variability using single strand conformation polymorphism analysis, and unique alleles were sequenced. Colorado isolates were most similar to tick isolates from California and New York isolate 25015. Genetic distances among Colorado ospA sequences were the same or higher than distances among other isolates whereas distances among fla sequences tended to be the same or lower. The index of association (I(A)) was calculated among all loci as a measure of clonality. The I(A) among Colorado isolates was similar to I(A) previously estimated among other United States isolates.
Amino Acid Sequence↗
Association between long periods without high-speed workouts and risk of complete humeral or pelvic fracture in thoroughbred racehorses: 54 cases (1991-1994).
OBJECTIVE: To determine whether a two-month or longer period without official high-speed workouts (lay-up) is associated with humeral or pelvic fracture in Thoroughbred racehorses. DESIGN: Reprospective study. ANIMALS: Thoroughbred racehorses in California that were euthanatized because of a complete humeral or pelvic fracture. PROCEDURE: Age, sex, activity, number of lay-ups, number of days from a race or official timed workout to fracture, number of days from end of last lay-up to fracture, mean duration of lay-ups, and total number of days in race training were compared between horses with humeral fractures and horses with pelvic fractures. A case-crossover study was used to estimate relative risk for fracture of the humerus or pelvis occurring within hazard periods of 10 and 21 days following lay-up, compared with periods following more regular participation in official racing or timed workout events. RESULTS: Horses with pelvic fractures were more often female, older, and had 0 or > or = 2 lay-ups. Horses with humeral fractures were typically 3-year-old males that had 1 lay-up. Horses with pelvic fractures had more total days in race training, fewer days from last exercise event to fracture, and a greater number of days from end of last lay-up to fracture than horses with humeral fractures. Return from lay-up was strongly associated with risk for humeral fracture during hazard periods of 10 and 21 days (relative risk = 71 and 45, respectively). CLINICAL IMPLICATIONS: Risk of humeral fracture may be reduced if horses are cautiously reintroduced into race training after lay-up.
Animals↗
Relationship between race start characteristics and risk of catastrophic injury in thoroughbreds: 78 cases (1992).
OBJECTIVE: To investigate relationships of several racehorse characteristics and race conditions with risk of a catastrophic musculoskeletal injury (CMI) resulting in euthanasia in Thoroughbreds during racing in California in 1992. DESIGN: Retrospective longitudinal study. ANIMALS: Thoroughbreds that incurred CMI during racing and all California race entrants in 1992. PROCEDURE: Necropsy records were reviewed, and race start information was obtained. Incidence risk of CMI/1,000 race entrants was estimated. Relationships between CMI during racing and race-meet, entrant age and sex, race type and length, and racing surface type and condition were evaluated by use of logistic regression. RESULTS: Incidence risk of CMI was 1.7/1,000 entrants. A higher risk of CMI was found at 2 fair race-meets, with incidence risks of 4.9 and 5.5/1,000 entrants. Risk of injury in male horses was 1.7 times greater than that in female horses, and influence of age on risk depended on race type. Risk of injury for horses 2 to 5 years old was two times greater for claiming horses than for maiden horses. Race length or racing surface type (dirt vs turf) or condition (fast, muddy, yielding) were not significantly associated with risk of CMI. CLINICAL IMPLICATIONS: Incidence of CMI was similar among 12 of 14 major and fair race-meets and among various race lengths and racing surface types and conditions, whereas incidence of CMI was influenced by entrant age and sex as well as race type. Investigators should consider controlling for age and sex, race-meet, and race type whenever possible in studies of risk of CMI.
Age Factors↗
Anger management style and the prediction of treatment outcome among male and female chronic pain patients.
Anger is a prominent emotion experienced by chronic pain patients. Anecdotes suggest that anger predicts poor outcome following multidisciplinary pain programs, but no empirical evidence documents this link. We expected that patient anger expression or suppression would predict poor outcome following a pain program and that gender differences would emerge. Pre- to posttreatment measures of lifting capacity, walking endurance, depression, pain severity and activity level were collected from 101 chronic pain patients. An 'anger expression x gender' interaction was found such that anger expression among males was correlated negatively with lifting capacity improvements. 'Anger suppression x gender' interactions emerged such that anger suppression among males was correlated negatively with improvements in depression and general activities. These effects remained significant after controlling for trait anger. Thus, how anger is managed may exert unique influence on outcomes apart from the effects of mere anger proneness, at least among male pain patients.
Adult↗
A case-crossover study of intensive racing and training schedules and risk of catastrophic musculoskeletal injury and lay-up in California thoroughbred racehorses.
The objective of this study was to investigate the relationship between intensive racing and training schedules and risk of either catastrophic musculoskeletal injury (CMI) or lay-up from racing in California Thoroughbreds. Thoroughbred racehorses that sustained a CMI during racing or training and either were subsequently euthanized or died on a California racetrack during 1991 and 1992 were studied using a case-crossover study design. Each study subject (case) provided its own self-matched control information in the form of 'typical' exposure frequency, determined or estimated from historic information. Periods of rapid average daily accumulation of high-speed exercise distance were identified for each horse from official race and training histories with a sliding 60-day window. Those window frames containing an average daily rate of distance accumulation exceeding 75th percentile cutoff values were classified as exposed frames followed by 30 days of increased risk, or hazard periods. All remaining days (excluding periods of layoff from racing) were considered non-hazard time at risk. The relative risk (RR) of CMI within 30 days following a period of rapid accumulation of high-speed exercise distance (during a hazard period) was 4.2 (95% confidence interval [CI] = 3.0, 5.8). Of these 214 cases, 84 (39%) were injured during a hazard period whereas on average only 22% of total career time at risk was hazard time for these horses. A second analysis was performed for horses with at least 1 lay-up period from racing of > or = 60 days. RR for a lay-up beginning during a hazard period was estimated for the last lay-up experienced by each horse. The RR for lay-up beginning during a hazard period was 4.8 (95% CI = 2.9, 8.1). Of these 98 cases, 32 (33%) of the lay-ups began during a hazard period whereas on average, only 22% of at-risk time up to last lay-up was hazard time for these horses.
Age Distribution↗
Cognitive and physical capacity process variables predict long-term outcome after treatment of chronic pain.
Cognitive-behavioral and physical therapies are incorporated into multidisciplinary chronic pain programs because changes in pain cognitions and physical capacity may represent therapeutic processes that facilitate favorable outcome. Decreases in depression, however, may explain treatment responses more parsimoniously. Measures of pain helplessness, lifting capacity, walking endurance, depression, pain severity, and activity level were collected from 94 chronic pain patients at pre- and posttreatment and at 3- to 6-month follow-up evaluations. Decreases in pain helplessness were linked to pain severity reduction, whereas walking endurance increases were related to improvements in activity levels and downtime even after controlling for effects of depression decreases. Thus, cognitive and physical capacity changes that occur through pain treatment may make unique contributions to long-term outcome.
Adult↗
Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31.
The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.
Adhesins, Bacterial↗
Preliminary on-road measurement of the effect of oxygenated fuel on CO emissions near Las Vegas, Nevada.
A preliminary measurement of an oxygenated fuel effect for in-use vehicles travelling at freeway speed was conducted near Las Vegas, NV, in January 1991 and May 1992. The experimental design was based on two principal factors: (1) the large volume of traffic that visits Las Vegas from Southern California on three-day holiday weekends and (2) the fact that, at the time of the study, the Las Vegas area used oxygenated fuels in the winter and Southern California did not. Measurements were conducted at carefully selected sites 20 km southwest of Las Vegas near Sloan, NV, and were accomplished with the Fuel Efficiency Automoblie Test (FEAT) remote sensor developed at the University of Denver. The January 1991 measurements were made during the Las Vegas oxyfuel season, while the May 1992 control measurements were made outside the oxyfuel season. Over 24,500 individual CO concentrations were measured; registration data from over 5,500 of these vehicles were obtained from the license plate numbers. After corrections for differences in velocity and mean age, the Las Vegas outbound (oxyfuel) CO emissions on Monday morning of the January holiday weekend showed a difference of -18% +/- 11% compared to the inbound (non-oxyfuel) CO emissions on Friday evening preceding the holiday weekend.
Air Pollutants↗
Differential gene expression in response to adjunctive recombinant human interleukin-2 immunotherapy in multidrug-resistant tuberculosis patients.
Administration of low-dose recombinant human interleukin 2 (rhuIL-2) in combination with multidrug chemotherapy to patients with multidrug-resistant tuberculosis (MDR TB) induces measurable changes in in vitro immune response parameters which are associated with changes in the clinical and bacteriologic status of the patients. To determine the molecular basis of these changes, we have used semiquantitative reverse transcriptase-initiated PCR (RT-PCR) and differential display technology. During rhuIL-2 treatment of MDR TB patients, decreased levels of gamma interferon (IFN-gamma) mRNA in peripheral blood mononuclear cells (PBMC) relative to baseline levels were observed. However, at the site of a delayed-type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD), the expression of cellular IFN-gamma and IL-2 mRNAs was increased during rhuIL-2 therapy. Levels of other cytokine mRNAs were not significantly affected by rhuIL-2 administration. Using differential-display RT-PCR, we identified several genes expressed at the DTH skin test site which were up- or down-regulated during rhuIL-2 treatment. Cytochrome oxidase type I mRNA was increased in response to rhuIL-2 therapy relative to baseline levels, as was heterogeneous nuclear ribonuclear protein G mRNA. CD63, clathrin heavy chain, and beta-adaptin mRNAs, all of which encode proteins associated with the endocytic vacuolar pathway of cells, were also differentially regulated by rhuIL-2 administration. The differential effects of IL-2 were confirmed in vitro by using PBMC obtained from PPD-positive individuals stimulated with Mycobacterium tuberculosis and IL-2. The differential expression of genes may provide a surrogate marker for leukocyte activation at a mycobacterial antigen-specific response site and for the development of an enhanced antimicrobial response which may result in improved outcomes in MDR TB patients.
Adult↗
An in vitro investigation of multi-layer bandages.
This study compares the elastic properties of a number of different bandages both in the non-sterile condition and after sterilisation by autoclaving at 134-138 degrees C. Results suggest that, although some products can withstand sterilisation, the performance of some elasticated and coated bandages is adversely affected by excessive heat.
Bandages↗