High-resolution HLA typing for the DQB1 gene by sequence-based typing

BACKGROUND: Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation. RESULTS: TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 micrograms/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells. CONCLUSIONS: Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.     

Synthesis, absolute configuration, and analysis of malathion, malaoxon, and isomalathion enantiomers

Syntheses of the enantiomers of malathion, malaoxon, and isomalathion are reported herein. Malathion enantiomers were prepared from (R)- or (S)-malic acid in three steps. Enantiomers of malathion were converted to the corresponding enantiomers of malaoxon in 52% yield by oxidation with monoperoxyphthalic acid, magnesium salt. The four isomalathion stereoisomers were prepared via two independent pathways using strychnine to resolve the asymmetric phosphorus moiety. The absolute configurations of the four stereoisomers of isomalathion were determined by X-ray crystallographic analysis of an alkaloid salt precursor. A high-performance liquid chromatography technique was developed to resolve the four stereoisomers of isomalathion, and to determine their stereoisomeric ratios.     

Treatment approaches following foot and ankle injury

A complete and effective rehabilitation program begins with a thorough subjective and objective evaluation of the problem and injury presented. Biomechanical changes, whether they are anatomically induced or as a result of a form of compensation, must be identified and properly addressed. During the initial phase of rehabilitation, the issues of pain and swelling often must be resolved before full range of motion and return to normal strength can be achieved successfully during the more advanced stages. It is important to incorporate the entire lower quadrant in exercises that are designed to improve proprioception and function, using both in open and closed kinetic chain exercises. Finally, the demands to which the patient is returning to must be recognized to successfully complete the recovery phase.     

Determinants of genital human papillomavirus infection among cytologically normal women attending the University of New Mexico student health center

OBJECTIVES: To confirm the risk factors for genital human papillomavirus (HPV) infection. GOAL OF THIS STUDY: To investigate risk factors for HPV detection apart from the correlated risk factors for cervical neoplasia. STUDY DESIGN: Cervical human papillomavirus (HPV) DNA was assessed in 357 cytologically normal women attending the University of New Mexico student health center. Cervical swab samples were obtained for HPV DNA detection and typing using a PCR-based DNA amplification system. Possible determinants of cervical HPV were examined including age, ethnicity, history of sexually transmitted disease, oral contraceptive use, smoking, age at first intercourse, lifetime number of sex partners, marital status, and history of pregnancy. RESULTS: A 44.3% overall prevalence of cervical HPV was observed. On univariate analysis, factors associated with increasing HPV prevalence included higher lifetime number of sex partners and single marital status. After adjustment for potential confounding variables, we found that HPV prevalence increased with higher lifetime number of sexual partners. CONCLUSION: These findings, along with those from the companion reports in this issue of the journal, support the sexual route of transmission of the virus.     

Experimental and theoretical comparison of NIR spectroscopy measurements of cerebral hemoglobin changes

Two near-infrared spectroscopy (NIRS) methods are available for measuring changes (Delta) in total cerebral hemoglobin concentration (CHC): 1) a continuous measurement of the changes in total hemoglobin concentration (Delta[Hb]tot) and 2) the difference between two absolute measurements of CHC, each derived from a small, controlled change in inspired O2 fraction. This paper investigates the internal consistency of these two methods by using an experimental and theoretical comparison. NIRS was used to measure [Hb]tot in five newborn piglets before and after a change in arterial PCO2. Delta[Hb]tot demonstrated a low coefficient of variation of 2.8 +/- 2.8 (SD) % which allowed changes in CO2-cerebral blood volume reactivity to be clearly discriminated. However, a high coefficient of variation of 22.8 +/- 3.5% on the DeltaCHC measurements obscured any CO2 reactivity changes. A theoretical analysis demonstrates the effects of optical pathlength, background absorption, scatter, and blood vessel diameter on both methods. For more accurate monitoring of CHC, individual measurements of optical pathlength and more accurate pulse oximetry are required.     

RNA tertiary structure mediation by adenosine platforms

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.     

Concentration-response relationships for adenosine agonists during preconditioning of rabbit cardiomyocytes

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 microM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 microM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 microM CCPA. The APNEA curve appeared moriophasic, the major slope occurring between 1-100 nM; DPCPX (1 microM) shifted the concentration-response curve approximately 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of approximately 5 nM; DPCPX shifted the BNECA concentration-protection relationship approximately 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.     

Ferritin in cultured human cytotrophoblasts: synthesis and subunit distribution

The present study aims at the role of ferritin in the regulation of syncytiotrophoblast free iron levels. The differentiated cytotrophoblast cell in culture is used as a model for this maternal-fetal interface. Cytotrophoblast cells isolated from term placentae are cultured in iron-poor (Medium 199), iron-depleted [desferrioxamine(DFO)] and iron-supplemented [diferric transferrin (hTF-2Fe), ferric ammonium citrate (FAC)] medium. Distribution and de novo synthesis of isoferritins is studied, together with the cellular iron concentration and the ferritin iron saturation. Compared to ferritin isolated from total placenta, ferritin obtained from villous tissue is enriched with acidic isoforms. This observation is in agreement with measured light (L) to heavy (H) subunit ratios < 1 of de novo synthesized ferritin in cultured cytotrophoblast cells. Neither iron-poor culture medium, nor hTf-2Fe supplemented medium affects the cellular iron or ferritin concentration. FAC increased the cellular ferritin iron saturation and (by synthesis) the acidic isoferritin concentrations. The results strongly suggest, that the term syncytiotrophoblast is able to balance transferrin-mediated iron uptake and iron release. In case of FAC supplementation, the syncytiotrophoblast is unable to keep intracellular iron low, and ferritin synthesis is stimulated. The predominance of acidic ferritins and the preferential synthesis of H subunits can be functionally explained by the established fact that iron incorporation in acidic ferritins is faster due to the presence of ferroxidase centres. Damage by free iron catalysed hydroxyl radical formation is therefore minimized.