New Latex Bead Agglutination Assay for Differential Diagnosis of Cattle Infected with Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis

Extensive studies have shown that the current assays used to identify cattle infected with Mycobacterium bovis or Mycobacterium avium subsp. paratuberculosis are not sufficiently sensitive and specific to detect all infected animals, especially animals recently infected with the pathogens. In the present report we show that these limitations might be overcome with a latex bead agglutination assay (LBAA). With the specific immunodominant epitope (ESAT6-p) of M. bovis, we developed an LBAA and enzyme immunoassay (EIA) for that purpose and compared them with the “gold standard” culture method and skin test for their efficacy in detecting bovine tuberculosis. When sera from control healthy cows (n = 10), M. avium subsp. paratuberculosis-positive cattle (naturally infected, n = 16; experimentally infected, n = 8), and M. bovis-positive cattle (naturally infected, n = 49;experimentally infected, n = 20) were applied to an EIA and an LBAA developed with ESAT6-p, the two tests showed similar sensitivity (97.1% by EIA, 95.7% by LBAA), high specificity (94.2% by EIA, 100% by LBAA), and a positive correlation (kappa value, 0.85; correlation rate, 93.2%; correlation coefficient, 0.64). Receiver operating characteristic analysis of EIA results and comparison with the culture method determined a suitable cutoff value at 0.469, with an area under the curve of 0.991 (95% confidence interval, 0.977 to 1.0). As LBAA didn't show any positive reactions with sera from uninfected control cows or M. avium subsp. paratuberculosis-infected cattle, which were confirmed to be free of M. bovis by culture or PCR, LBAA using the ESAT6-p can be a rapid and useful M. bovis diagnostic assay. The data suggest that rapid, sensitive, and specific assays can be developed with peptides containing immunodominant epitopes present in proteins uniquely expressed in M. bovis or M. avium subsp. paratuberculosis for differential diagnosis of cattle infected with M. bovis or M. avium subsp. paratuberculosis.     

Beziehung zwischen dem UV-Absorptionsmuster einiger Flavone und ihrer gegen L1210-Zellen cytotoxischen Aktivität

The UV-pattern of several flavones, their cytotoxicities against L1210 cell and their Inhibiting effects on ATPase from the cell seem to be correlated. 5,2′-Dihydroxy-6,7,8,6′-tetramethoxyflavone (ED₅₀=2.3 ug/ml) and 5,2′,6′-trihydroxy-6,7,8-trimethoxyflavone (ED₅₀=4.5 ug/ml), the most active flavones studied, have shown a narrow range of the absorbance ratio, Log εII/Log εI=1.073～1.109. They have inhibited the ATPase-activity to the greatest extent. These findings suggest that a certain angle between the flavone rings B and C plays an important role for the inhibition of the enzyme activity and thus the cytotoxicity.     

Neurocysticercosis in Persons with Epilepsy in Medellín,Colombia

Summary: Purpose: A prospective series of 643 persons with epilepsy attending a reference neurologic center in Medellin, Colombia, was examined by computed tomography (CT scan) or serology or both with the enzyme-linked immunoelectrotransfer blot assay (EITB) to assess the prevalence of Taenia solium cysticercosis. Methods: All presenting patients were consecutively enrolled in the study. Five hundred forty-six persons underwent cerebral CT scans; 376 of them also had serum EITB performed. Results: Prevalence of neurocys@ercosis by CT scan was 13.92%. Overall prevalence of T. solium antibodies with EITB was 9.82%, but for those with late-onset epilepsy (onset after age 30 years), prevalence increased to 17.5% and 19% for those who originated from outside urban Medellin. Seroprevalence in individuals with mixed lesions (cysts and calcifications) was 88.2% and 64.10% in those with live cysts. Conversely, only 2.72% of persons with CT findings not related to neurocysticercosis had positive EITB tests. Conclusions: Our study shows that an important proportion of individuals with epilepsy have radiologic or serologic evidence of T. solium infection, suggesting that neurocysticercosis is an important etiology for epilepsy in Colombia.     

Comparative analysis of NK/T-cell lymphoma and peripheral T-cell lymphoma in Korea: Clinicopathological correlations and analysis of EBV strain type and 30-bp deletion variant LMP1

Natural killer/T-cell lymphoma (NKTL) and peripheral T-cell lymphomas (PTCL) are prevalent in the Asian population and exhibit a high association with the Epstein-Barr virus (EBV). Moreover, differentiation of these two groups is often difficult and problematic. We investigated 35 cases of NKTL (22 nasal cases and 13 extranasal cases) and 30 cases of PTCL in terms of their clinical features, immunohistology, EBV positivity, EBV strain-type polymorphism and latent membrane protein 1 (LMP1) deletion variant distribution. Eighteen cases (82%) of nasal NKTL and seven (54%) of extranasal NKTL showed EBV positivity by EBV in situ hybridization. Fifteen cases (50%) of PTCL revealed EBV positivity. EBV strain type A was predominant in NKTL (18:5), and EBV strain types A and B were distributed evenly in PTCL (6:6). EBV-positive patients had significantly shorter survival than EBV-negative patients (P < 0.05), and EBV positivity correlated with advanced clinical stage (P < 0.05). Patients harboring type A EBV showed slightly poorer prognoses than those having type B, though it was not obviously statistically different (P = 0.07). The LMP1 deletion variant was prevalent in both NKTL (three wild-type LMP1, 15 deletion variants) and PTCL (three wild-type LMP1, eight deletion variants, two coexistent forms) patients, but did not have prognostic impact. Our results indicate that EBV acts as a negative prognostic factor in NKTL and PTCL, and that the intrinsic properties of a specific viral strain might influence the clinical behavior of these diseases.     

Combined effects of inhaled nitric oxide and a recruitment maneuver in patients with acute respiratory distress syndrome

Nitric oxide (NO) inhalation therapy has been employed in the management of acute respiratory distress syndrome (ARDS), in order to improve oxygenation. Several factors have been implicated as being responsible for the action of inhaled NO. Alveolar recruitment methods, such as prone positioning and a sufficient positive end expiratory pressure (PEEP), have been identified as having a positive impact on the NO response. A Recruitment maneuver (RM) was introduced for the treatment of ARDS, along with a lung protective strategy. Here, we hypothesized that a RM may further augment the oxygenation of patients treated with NO inhalation. Therefore, the effects of the inhalation of NO, either in combination with a RM, or separately, were evaluated on patients with ARDS for their enhancing action. 23 patients with ARDS were enrolled, and divided into three groups. The patients in group 1 (n=11) were treated with 5 ppm NO via inhalation, followed by a RM, applying a sustained inflation pressure of 30 - 35 cmH2O for 30 seconds. Group 2 (n=6) received a RM alone, while group 3 (n=3) was treated with NO inhalation alone. The oxygenation and hemodynamic parameters were obtained prior to, and 2, 12, and 24 h after, the respective treatment procedures. For group 1, the PaO2/FiO2 increased from its initial value of 171.8 +/- 67.8 to 203.2 +/- 90.0 2 h after NO inhalation. Further improvement was noted with the continual application of the RM reaching, 215.5 +/- 74.6 (p=0.05) and 254.2 +/- 109.5 (p < 0.05), after 12 and 24 h, respectively. Initially 7 of the subjects did not respond to NO inhalation, but 3 of these non-responders changed into responders 12 h after the RM. The changes in the PaO2/FiO2 from baseline at each time period were greater in group 1 than in the other groups, but with no statistical significance. The hemodynamics of the patients was not significantly altered during the entire study period. We conclude that the combined application of NO inhalation and a RM could be beneficial and safe for patients with ARDS, showing an enhancing effect in improvement of oxygenation.     

Immunization with Combined HSV-2 Glycoproteins B2:D2 Gene DNAs: Protection against Lethal Intravaginal Challenges in Mice

The immunity of a combined DNA vaccine of HSV-2 glycoproteins B2 (gB2) and D2 (gD2) genes in comparison to individual vaccines was studied with regard to protecting against the HSV infection. Two recombinant DNA vaccines of the pHS2-gB2 or pHS2-gD2 were constructed and formulated. The neutralizing antibody titers appeared higher in the B2:D2 gene cocktail-vaccinated mice than that of the individual B2 or D2 gene-vaccinated group alone, and the positive KOS control induced higher titer of the neutralizing antibody than combined or individual gene vaccines. The mock-immunized mice failed to induce enough. The ranks for the CTL activity and the protection rates against the lethal intravaginal challenge were shown as KOS>B2:D2 cocktail>D2>B2 gene vaccines. The vaginal external diseases in the B2:D2 or D-vaccinated mice were significantly reduced against the challenging dosages. The virus titers in the vaginal secretions of the vaccinated mice significantly reduced with time, and the B2:D2 gene vaccine decreased more than each individual vaccine alone. It can be concluded that the cocktailed vaccines are more effective in the humoral and cellular immune responses in the mice, and in the protection of the mice against the intravaginal challenging dosages when compared with individual gene vaccines. All the DNA vaccines failed to block the latent infection in sensory nerves.     

Somatic mitochondrial DNA mutations in cortex and substantia nigra in aging and Parkinson's disease

Oxidative damage to mitochondrial DNA (mtDNA) increases with age in the brain and can induce G:C to T:A and T:A to G:C point mutations. Though rare at any particular site, multiple somatic mtDNA mutations induced by oxidative damage or by other mechanisms may accumulate with age in the brain and thus could play a role in aging and neurodegenerative diseases. However, no prior study has quantified the total burden of mtDNA point mutation subtypes in the brain. Using a highly sensitive cloning and sequencing strategy, we find that the aggregate levels of G:C to T:A and T:A to G:C transversions and of all point mutations increase with age in the frontal cortex (FCtx). In the substantia nigra (SN), the aggregate levels of point mutations in young controls are similar to the levels in the SN or FCtx of elderly subjects. Extrapolation from our data suggests an average of 2.7 (FCtx) to 3.2 (SN) somatic point mutations per mitochondrial genome in elderly subjects. There were no significant differences between Parkinson's disease (PD) patients and age-matched controls in somatic mutation levels. These results indicate that individually rare mtDNA point mutations reach a high aggregate burden in FCtx and SN of elderly subjects.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.