The fluorolysis assay,a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard 51Cr-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few (> or =6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers (> or =25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies.     

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.     

Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.     

The incorporation of iodine in thyroid hormone may stem from its role as a prehistoric signal of ecologic opportunity: an evolutionary perspective and implications for modern diseases

To optimize fitness under conditions of varying Darwinian opportunity, organisms demonstrate tremendous plasticity in their life-history strategies based on their perception of available resources. Higher-energy environments generally promote more aggressive life-history strategies, such as faster growth, larger adult size, greater genetic variation, shorter lifespan, larger brood sizes, and offspring ratio skewed towards the larger-sized gender. While numerous mechanisms regulate life-history plasticity including genetic imprinting, methylation, and growth factors, evidence suggests that thyroid hormone plays a central role. Given the pivotal adaptive role of thyroid hormone, the teleology of its dependence on dietary iodine for production remains unexplained. We hypothesize that iodine may have emerged as a substrate for production of thyroid hormone in prehistoric ecosystems because the former represented a reliable proxy for ecologic potential that enabled the latter to modulate growth, reproduction, metabolic rate, and lifespan. Such a scenario may have existed in early marine ecosystems where ocean-surface vegetation, which concentrates iodine for its antimicrobial and antioxidant properties, formed the basis of the food chain. Teleologic parallels can be drawn to the food-chain accumulation of antimicrobials that also exhibit antioxidant properties and promote adult size, brood size, and offspring quality by modulating central hormonal axes. As each higher species in the food chain tunes its life-history strategy based on iodine intake, the coupling of this functional role of iodine with its value as a resource signal to the next member of the food-chain may promote runaway evolution. Whereas predators in prehistoric ecosystems successfully tuned their life-history strategy using iodine as a major input, the strategy may prove maladaptive in modern humans for whom the pattern of iodine intake is decoupled from resource availability. Iodine acquired through sodium iodide supplementation may independently contribute to some biologic dysfunctions currently attributed to sodium.     

Rapid identification and susceptibility testing using the VITEK(R) 2 system using culture fluids from positive BacT/ALERT(R) blood cultures

BACKGROUND AND PURPOSE: In order to reduce the turnaround time for laboratory diagnosis of bacteremia, the efficacy of identification and antimicrobial susceptibility testing using samples taken directly from positive BacT/ALERT(R) standard aerobic and standard anaerobic blood culture bottles was evaluated. METHODS: 160 positive blood culture bottles were examined and incubated at 35 degrees C in 5% carbon dioxide for 4-24 h, and an aliquot of the culture fluid was Gram stained. Samples containing Gram-negative bacilli were inoculated on VITEK(R) 2 ID-GNB (identification-Gram-negative bacilli) and AST (antimicrobial susceptibility testing)-GN04 cards, and those containing Gram-positive cocci were inoculated on ID-GPC (identification-Gram-positive cocci) and AST-P526 cards. The same samples were also examined by the standard method, involving subculture from positive BacT/ALERT standard blood culture bottles. RESULTS: Eighty seven of 97 Gram-negative bacilli (89.7%) and 21 of 63 Gram-positive cocci (33.3%) were correctly identified to the species level. For antimicrobial susceptibility testing, the direct method had an overall error rate of 5.4% for Gram-negative bacilli, with 0.9% very major, 0.9% major, and 3.6% minor discrepancies compared to the standard method. The overall error rate in antimicrobial susceptibility testing for the 13 Staphylococcus spp. was 10.3%, with 6.0% very major, 2.6% major, and 1.7% minor discrepancies. CONCLUSION: These data suggest that VITEK 2 cards inoculated with samples taken directly from positive Bact/ALERT blood culture bottles would provide acceptable identification and antimicrobial susceptibility testing results for Gram-negative bacilli, but not for Gram-positive cocci. Compared to the standard method, the direct method would reduce turnaround time by at least 24 h.     

Invasive pulmonary aspergillosis: high incidence of disseminated intravascular coagulation in fatal cases.

BACKGROUND AND PURPOSE: Disseminated intravascular coagulation (DIC) is a rarely described finding in invasive pulmonary aspergillosis (IPA) with unclear impact on mortality. METHODS: This study included patients with positive cultures of Aspergillus spp. from respiratory specimens, serological evidence of aspergillosis, or lung biopsy findings supporting aspergillosis treated at National Taiwan University Hospital from January 1999 to June 2005. IPA was defined based on the consensus of the European Organization for Research and Treatment of Cancer, and the Mycosis Study Group of the National Institute of Allergy and Infectious Diseases. Univariate logistic regression analysis was used to evaluate the factors associated with mortality. RESULTS: Proven or probable IPA was diagnosed in 26 patients. Hematological malignancy was found in 11 patients (42%) and immunosuppressive agents had been administered to 17 patients (65%). Among 20 culture-proven infections (77%), the most frequently encountered fungi were Aspergillus fumigatus (46%) and Aspergillus flavus (23%). The overall mortality rate was 62%. Univariate and multivariate analyses revealed that DIC was the only factor that was significantly associated with death attributable to IPA (p<0.01). CONCLUSIONS: IPA is associated with a high mortality rate, particularly for patients with DIC.     

Free-feeding and free-drinking patterns of male rats following treatment with opiate kappa agonists.

Three experiments investigated the effects of PD117302 and U50,488H on the patterns of food and water intake by male rats. Experiment 1 demonstrated early dose-related suppression of food and water intake after PD117302 (0, 1.25, 2.5, 5 mg/kg). The initial suppression of drinking was followed by a sustained increase 4-12 h after drug administration. Experiment 2 demonstrated that 2.5 mg/kg PD117302 failed to increase food intake whether given at the beginning of the night (high baseline food intake) or the beginning of the day (low baseline food intake). Experiment 3 showed that 0.5 mg/kg U50,488H significantly enhanced meal size but, at doses of 0.5, 1.0, and 2.0 mg/kg, had no effect on overall food intake. U50,488H also produced delayed, dose-related increases in water intake. The results suggest kappa receptors may have limited importance in modulating ad lib food intake and demonstrate the behavioural characteristics of increased drinking after excessive urine output.     

Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB.

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.     

Loss of mucosal CD4 lymphocytes is an early feature of HIV infection.

T cell subsets in the gut mucosa are distinct populations and their imbalance in HIV has specific implications in infection. Alterations in T cell subsets in duodenal biopsies were investigated in 17 asymptomatic HIV patients, 24 AIDS patients and 10 controls with non-ulcer dyspepsia. Immunohistochemistry and immunofluorescence using MoAbs to CD3, CD4, CD8, CD68, CD45RA, CD45RO and gp120 were performed on frozen sections. In the lamina propria, there was a significant depletion of CD4+ cells at all stages of HIV, but the density of CD8 lamina propria cells was increased. Intraepithelial lymphocytes were decreased in AIDS patients. There was a significant correlation between cellular density and mucosal CD3+ lymphocytes, and between mucosal CD3+ and CD8+ lymphocytes. Although mucosal CD4,CD45RO+ 'memory' cells were decreased, CD8,CD45RO+ 'memory' cells were increased. Mucosal CD4+ lymphocyte depletion occurred early in HIV, and thus their role in mucosal protection against opportunistic infection should be revised. Mucosal CD8+ lymphocytes initially increased, but decreased when CD4 blood counts were depleted, perhaps contributing to loss of host protection against infection. Intraepithelial lymphocyte depletion may also contribute to opportunistic infection.