A mouse model of Huntington's disease shows altered ultrastructure of transverse tubules in skeletal muscle fibers.

Huntington's disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation-contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington's disease.

Use of resuscitation promoting factors to screen for tuberculosis infection in household-exposed children in The Gambia.

BACKGROUND: Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children. METHODS: Detailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity. RESULTS: Interferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73-92% and 77-72% respectively, which was similar to and better than 65-75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively). CONCLUSION: RpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB.

Propagation of a laser-driven relativistic electron beam inside a solid dielectric.

Laser probe diagnostics: shadowgraphy, interferometry, and polarimetry were used for a comprehensive characterization of ionization wave dynamics inside a glass target induced by a laser-driven, relativistic electron beam. Experiments were done using the 50-TW Leopard laser at the University of Nevada, Reno. We show that for a laser flux of ∼2 × 10(18) W/cm2 a hemispherical ionization wave propagates at c/3 for 10 ps and has a smooth electron-density distribution. The maximum free-electron density inside the glass target is ∼2 × 10(19) cm-3, which corresponds to an ionization level of ∼0.1%. Magnetic fields and electric fields do not exceed ∼15 kG and ∼1 MV/cm, respectively. The electron temperature has a hot, ringlike structure with a maximum of ∼0.7 eV. The topology of the interference phase shift shows the signature of the "fountain effect", a narrow electron beam that fans out from the propagation axis and heads back to the target surface. Two-dimensional particle-in-cell (PIC) computer simulations demonstrate radial spreading of fast electrons by self-consistent electrostatic fields driven by laser. The very low ionization observed after the laser heating pulse suggests a fast recombination on the sub-ps time scale.

X-ray emission from z pinches at 10 7 A: current scaling, gap closure, and shot-to-shot fluctuations.

We have measured the x-ray power and energy radiated by a tungsten-wire-array z pinch as a function of the peak pinch current and the width of the anode-cathode gap at the base of the pinch. The measurements were performed at 13- and 19-MA currents and 1-, 2-, 3-, and 4-mm gaps. The wire material, number of wires, wire-array diameter, wire-array length, wire-array-electrode design, normalized-pinch-current time history, implosion time, and diagnostic package were held constant for the experiments. To keep the implosion time constant, the mass of the array was increased as I2 (i.e., the diameter of each wire was increased as I), where I is the peak pinch current. At 19 MA, the mass of the 300-wire 20-mm-diam 10-mm-length array was 5.9 mg. For the configuration studied, we find that to eliminate the effects of gap closure on the radiated energy, the width of the gap must be increased approximately as I. For shots unaffected by gap closure, we find that the peak radiated x-ray power P(r) proportional to I1.24+/-0.18, the total radiated x-ray energy E(r) proportional to I1.73+/-0.18, the x-ray-power rise time tau(r) proportional to I0.39+/-0.34, and the x-ray-power pulse width tau(w) proportional to demonstrate that the internal energy and radiative opacity of the pinch are not responsible for the observed subquadratic power scaling. Heuristic wire-ablation arguments suggest that quadratic power scaling will be achieved if the implosion time tau(i) is scaled as I(-1/3). The measured 1sigma shot-to-shot fluctuations in P(r), E(r), tau(r), tau(w), and tau(i) are approximately 12%, 9%, 26%, 9%, and 2%, respectively, assuming that the fluctuations are independent of I. These variations are for one-half of the pinch. If the half observed radiates in a manner that is statistically independent of the other half, the variations are a factor of 2(1/2) less for the entire pinch. We calculate the effect that shot-to-shot fluctuations of a single pinch would have on the shot-success probability of the double-pinch inertial-confinement-fusion driver proposed by Hammer et al. [Phys. Plasmas 6, 2129 (1999)]. We find that on a given shot, the probability that two independent pinches would radiate the same peak power to within a factor of 1+/-alpha (where 0< or =alpha<<1) is equal to erf(alpha/2sigma), where sigma is the 1sigma fractional variation of the peak power radiated by a single pinch. Assuming alpha must be < or =7% to achieve adequate odd-Legendre-mode radiation symmetry for thermonuclear-fusion experiments, sigma must be <3% for the shot-success probability to be > or =90%. The observed (12/2(1/2))%=8.5% fluctuation in P(r) would provide adequate symmetry on 44% of the shots. We propose that three-dimensional radiative-magnetohydrodynamic simulations be performed to quantify the sensitivity of the x-ray emission to various initial conditions, and to determine whether an imploding z pinch is a spatiotemporal chaotic system.

X-ray absorption spectroscopy measurements of thin foil heating by Z-pinch radiation.

Absorption spectroscopy measurements of the time-dependent heating of thin foils exposed to intense z-pinch radiation sources are presented. These measurements and their analysis provide valuable benchmarks for, and insights into, the radiative heating of matter by x-ray sources. Z-pinch radiation sources with peak powers of up to 160 TW radiatively heated thin plastic-tamped aluminum foils to temperatures approximately 60 eV. The foils were located in open slots at the boundary of z-pinch hohlraums surrounding the pinch. Time-resolved Kalpha satellite absorption spectroscopy was used to measure the evolution of the Al ionization distribution, using a geometry in which the pinch served as the backlighter. The time-dependent pinch radius and x-ray power were monitored using framing camera, x-ray diode array, and bolometer measurements. A three-dimensional view factor code, within which one-dimensional (1D) radiation-hydrodynamics calculations were performed for each surface element in the view factor grid, was used to compute the incident and reemitted radiation flux distribution throughout the hohlraum and across the foil surface. Simulated absorption spectra were then generated by postprocessing radiation-hydrodynamics results for the foil heating using a 1D collisional-radiative code. Our simulated results were found to be in good general agreement with experimental x-ray spectra, indicating that the spectral measurements are consistent with independent measurements of the pinch power. We also discuss the sensitivity of our results to the spectrum of the radiation field incident on the foil, and the role of nonlocal thermodynamic equilibrium atomic kinetics in affecting the spectra.

New JC virus (JCV) genotypes from papua new guinea and micronesia (type 8 and type 2E) and evolutionary analysis of 32 complete JCV genomes.

The JC virus (JCV) is a ubiquitous human polyomavirus that frequently resides in the kidneys of healthy individuals and is excreted in the urine of a large percentage of the population. Geographic-specific JCV variants, isolated from urine and from brain of progressive multifocal leukoencephalopathy (PML) patients, have been grouped into seven distinct genotypes based on whole genome analysis and by individual polymorphic nucleotides (typing sites) in the VP1 coding region. Mutations in the archetypal regulatory region, sometimes consisting of deletions and/or duplications, are also useful taxonomic characters for further characterizing and subdividing genotypes. Investigation of JCV variation in Papua New Guinea (PNG) revealed three distinct variants called PNG- 1, PNG-2, and PNG-3. These variants exhibited consistent coding region and regulatory region mutations. Evolutionary analysis of 32 complete JCV genomes including six new viral genomes from the western Pacific suggests that the new PNG JCV variants are closely associated with the broad group of Type 2 strains of JCV found throughout Asia, forming a monophyletic group with the Northeast Asian strains (Type 2A). Within the Type 2 clade, however, the PNG JCV variants cluster as two distinct groups and are therefore described here as new JCV genotypes designated Type 2E and Type 8.

Detection of borreliacidal antibodies in dogs after challenge with Borrelia burgdorferi-infected ixodes scapularis ticks.

Detection of borreliacidal antibodies is an accurate serodiagnostic test for confirmation of Lyme disease in humans. In this study, 13 pathogen-free beagles, 12 to 26 weeks old, were infected with Borrelia burgdorferi by tick challenge. Dogs were monitored for clinical signs and symptoms of Lyme disease along with borreliacidal antibody production against B. burgdorferi sensu stricto isolates 297 and 50772. Ten (77%) dogs developed lameness in one or more legs within 210 days after attachment of Ixodes scapularis ticks. Eight (80%) of the lame animals had concurrent fever of > or =38 degrees C. Spirochetes were also recovered from the skin and joints of 12 (92%) dogs, but rarely from other organs. Borreliacidal antibodies against B. burgdorferi isolate 297 were detected in only four (31%) dogs, and the levels of killing antibodies remained low for the duration of the infection. In contrast, borreliacidal antibodies against B. burgdorferi isolate 50772 were detected in 13 (100%) dogs within 21 days of infection. Furthermore, the borreliacidal antibody levels correlated with the severity of B. burgdorferi infection. Detection of borreliacidal antibodies, especially against B. burgdorferi isolate 50772, is also a reliable serodiagnostic test for detection of Lyme disease in dogs.

Production of borreliacidal antibody to outer surface protein A in vitro and modulation by interleukin-4.

Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide, but not recombinant OspA. Anti-OspA borreliacidal antibody was detected in supernatants of cultures of lymph node cells obtained on day 7 after vaccination, peaked on day 17, and rapidly declined. The borreliacidal activity was attributable to immunoglobulin G1 (IgG1), IgG2a, and IgG2b antibodies. When lymph node cells were treated with interleukin-4 (IL-4), production of borreliacidal antibody was inhibited but was unaffected by treatment with anti-IL-4 antibodies. These results suggest that other cytokines, but not IL-4, are mainly responsible for production of the secondary borreliacidal antibody response.

Evaluation of whole-cell and OspC enzyme-linked immunosorbent assays for discrimination of early lyme borreliosis from OspA vaccination.

A recombinant Lyme borreliosis vaccine consisting of outer surface protein A (OspA) is commercially available for vaccination of humans against infection with Borrelia burgdorferi. Vaccination with OspA induces an antibody response that makes serologic interpretation of infection with B. burgdorferi difficult, especially by screening tests based on whole-cell preparations of B. burgdorferi. We show that an enzyme-linked immunosorbent assay with B. burgdorferi sensu stricto 50772, which lacks the plasmid encoding OspA and OspB, or a full-length recombinant OspC protein can identify patients infected with B. burgdorferi. We found that 69 and 65% of serum samples from patients with case-defined early Lyme borreliosis had anti-B. burgdorferi sensu stricto 50772 and anti-OspC reactivities, respectively. In addition, little or no reactivity was detected with sera obtained from individuals vaccinated with OspA. Unfortunately, 51 and 33% of sera from healthy patients and sera from patients with other illnesses were also reactive against B. burgdorferi sensu stricto 50772 and OspC, respectively. Although these assays can discriminate B. burgdorferi infection from vaccination with OspA, their lack of specificity highlights the necessity for confirming equivocal or positive reactivities with more specific serodiagnostic tests.

Detection of borreliacidal antibodies in Lyme borreliosis patient sera containing antimicrobial agents.

The borreliacidal-antibody test has been used for the serological detection and confirmation of Lyme borreliosis. However, the presence of antimicrobial agents in serum can confound the accurate detection of borreliacidal antibodies. In this study, we developed a Bacillus subtilis agar diffusion bioassay to detect small concentrations of antimicrobial agents in serum. We also used XAD-16, a nonionic polymeric resin, to adsorb and remove high concentrations of amoxicillin, cefotaxime, ceftriaxone, cefuroxime, doxycycline, and erythromycin without significantly affecting even small concentrations of immunoglobulin M (IgM) or IgG borreliacidal antibodies. High concentrations of penicillin could also be removed by adding 1 U of penicillinase without significantly influencing the levels of borreliacidal antibodies. These simple procedures greatly enhance the clinical utility of the borreliacidal-antibody test.

Borreliacidal antibody production against outer surface protein C of Borrelia burgdorferi.

Early Lyme borreliosis sera with significant titers of anti-outer surface protein C (OspC) borreliacidal antibodies were identified. Human anti-OspC borreliacidal antibodies could be either IgM or IgG. Significant concentrations of borreliacidal activity were detected after vaccination of mice with OspC. Detection of anti-OspC borreliacidal activity was dependent on surface expression of OspC by Borrelia burgdorferi isolate 50772. The ability of OspC to induce borreliacidal antibodies in vivo and after vaccination offers another possible explanation for the ability of vaccination with OspC to protect against infection with B. burgdorferi. Furthermore, detection of anti-OspC borreliacidal antibodies, especially IgM antibodies, in early Lyme borreliosis sera provides additional evidence that borreliacidal antibody detection may be useful for the serodiagnosis of early Lyme borreliosis.

Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine.

Significant borreliacidal antibody was induced in volunteers and hamsters 60 days after primary and secondary vaccination with high concentrations of recombinant outer surface protein A (rOspA). However, the borreliacidal antibody response waned rapidly. Only 1 person had detectable cidal activity 180 days after vaccination. Similarly, the borreliacidal antibody response waned rapidly in hamsters by week 10 of vaccination. By contrast, the total anti-rOspA antibody response remained elevated in volunteers and hamsters. When isolates of Borrelia burgdorferi sensu lato were incubated in sera from vaccinated humans or hamsters, only the vaccine-specific isolate was killed. These results were confirmed by challenging rOspA-vaccinated hamsters with different isolates of B. burgdorferi sensu lato. The results showed that monitoring total rOspA antibody is inappropriate for evaluating the efficacy of an rOspA vaccine. The rOspA vaccine must be improved to yield comprehensive protection and maintain sustained levels of protective borreliacidal antibodies.

Sensitivity and specificity of the borreliacidal-antibody test during early Lyme disease: a "gold standard"?

The serodiagnosis of early Lyme disease has been plagued with problems of sensitivity and specificity. We found that the flow-cytometric borreliacidal-antibody test had a sensitivity of 72% for the detection of patients with early Lyme disease. By contrast, the sensitivity of the enzyme immunofluorescence assay was 28%. The enhanced sensitivity of the borreliacidal-antibody test was due to the use of Borrelia burgdorferi 50772, which lacks OspA and OspB. When B. burgdorferi 297, which expresses both OspA and OspB, was used, the sensitivity of the borreliacidal-antibody test was 15%. Our results also showed that the borreliacidal-antibody test was specific. No borreliacidal activity was detected in normal sera or in sera from patients with mononucleosis, rheumatoid factor, or syphilis. These results demonstrate that the flow-cytometric borreliacidal-antibody test may be the laboratory "gold standard" for the serodiagnosis of Lyme disease.

Detection of borreliacidal antibodies by flow cytometry. An accurate, highly specific serodiagnostic test for Lyme disease.

BACKGROUND: Borreliacidal antibodies can be detected in serum samples from patients with early or late Lyme disease symptoms. When these serum samples are incubated with Borrelia burgdorferi and complement, spirochetes are rapidly killed. Detection of these antibodies can be used as a serodiagnostic test. METHODS: Individual serum samples containing IgM or IgG borreliacidal antibodies were Used to develop a method for detection using flow cytometry. An additional 10 case-defined Lyme disease serum samples and 10 normal serum samples were used to confirm appropriate flow cytometric parameters. To determine specificity, 157 normal serum samples and 104 potential cross-reactive serum samples were tested for borreliacidal activity and antibodies to B burgdorferi using indirect fluorescent antibody or enzyme immunoassay. RESULTS: Flow cytometry can be used to detect borreliacidal activity within 16 to 24 hours after incubation of B burgdorferi organisms. Lyme disease serum, and complement. Significant borreliacidal activity was detected in all Lyme disease serum samples. The percentages of positive normal serum samples were comparable (6% to 10%) using all three assays. In addition, the indirect fluorescent antibody and enzyme immunoassay identified 41 (39%) and 47 (45%) potential cross-reactive serum samples as positive, respectively. In contrast, significant borreliacidal activity was not detected in any potential cross-reactive serum samples. CONCLUSION: Detection of borreliacidal antibody, unlike indirect fluorescent antibody and enzyme immunoassay, is an accurate, highly specific serodiagnostic test for detection of Lyme disease.

Development of destructive arthritis in vaccinated hamsters challenged with Borrelia burgdorferi.

We present the first direct evidence that adverse effects, particularly severe destructive arthritis, can develop in vaccinated hamsters after challenge with Borrelia burgdorferi sensu lato isolates. Hamsters were vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant. A severe destructive arthritis was readily evoked in vaccinated hamsters challenged with the homologous B. burgdorferi sensu stricto isolate C-1-11 before high levels of protective borreliacidal antibody developed. Once high levels of C-1-11 borreliacidal antibody developed, hamsters were protected from homologous challenge and development of arthritis. Vaccinated hamsters, however, still developed severe destructive arthritis when challenged with other isolates of the three genomic groups of B. burgdorferi sensu lato (B. burgdorferi sensu stricto isolate 297, Borrelia garinii isolate LV4, and Borrelia afzelii isolate BV1) despite high levels of C-1-11 specific borreliacidal antibody. Vaccines that contained whole spirochetes in adjuvant induced destructive arthritis, but this effect was not dependent on the isolate of B. burgdorferi sensu lato or the type of adjuvant. These studies demonstrate that caution is necessary when employing whole spirochetes in adjuvant for vaccination to prevent Lyme borreliosis. Additional studies are needed to identify the antigen(s) responsible for the induction and activation of arthritis and to define the immune mechanisms involved.

Ability of canine Lyme disease vaccine to protect hamsters against infection with several isolates of Borrelia burgdorferi.

We used flow cytometry to determine levels of borreliacidal antibodies in hamsters after vaccination with a commercially available canine Lyme disease vaccine. In addition, we evaluated the ability of vaccinated hamsters to resist infection with several isolates of Borrelia burgdorferi. Borreliacidal antibodies could be detected 1 week after a primary vaccination, peaked at weeks 3 to 5, and then rapidly declined. One week after a booster vaccination, borreliacidal activity was detected at a dilution of 1:10,240, and it decreased fourfold by week 10 after the booster vaccination. Vaccinated hamsters were protected against infection with < or = 10(6) B. burgdorferi 297 organisms during the peak borreliacidal response (5 weeks after primary vaccination or 2 weeks after booster vaccination). However, hamsters were not fully protected from development of Lyme arthritis when the titer of borreliacidal antibodies was < 1:5,120. In addition, no significant borreliacidal activity was induced against B. burgdorferi C-1-11, LV4, or BV1, which belong to three other seroprotective groups. These studies demonstrate that vaccination with the canine Lyme disease vaccine induces protective antibodies against B. burgdorferi 297. However, significant levels of borreliacidal antibodies are not produced until 5 weeks after vaccination, and protection is short-lived. In addition, no borreliacidal activity was induced against other isolates of B. burgdorferi. Because of this, the incorporation of multiple isolates or protein subunits may be necessary to increase the effectiveness of future vaccines.

Lyme disease: laboratory diagnosis and serologic testing.

Although identified less than 20 years ago, Lyme disease has proved to be the most common tick-borne disease in the United States: some 10,000 cases were reported in 1992. In some cases the disease may be transitory and of little consequence but in others it may become chronic and severely disabling. Accurate diagnosis is, therefore, of great importance but, as this article shows, laboratory testing techniques still need improvement.

Recovery of Borrelia burgdorferi by filtration.

Bacterial contamination frequently interferes with successful recovery of the Lyme spirochete from cultures of tissue from Borrelia burgdorferi-infected humans, rodents, or ticks. We used 0.20- and 0.45-microns-pore-size syringe-tip filters to recover spirochetes from cultures contaminated with other bacteria. Low concentrations (1 to 10/ml) of B. burgdorferi organisms could be recovered from cultures seeded with 1 x 10(8) to 4 x 10(8) Staphylococcus aureus, Streptococcus faecium, Escherichia coli, or Bacillus subtilis organisms per ml. We also used this technique to recover B. burgdorferi from contaminated environmental and clinical cultures of B. burgdorferi. We conclude the filtration is an efficient method for recovering Lyme spirochetes from contaminated samples and increasing the number of successful isolations of B. burgdorferi.