Rate and mechanism of enamel demineralization in situ.

In this paper, data are presented on the in situ demineralization of human enamel as a function of the demineralization period. To quantify the mineral loss parameters versus time, it is important to obtain information on the kinetics, and thus on the mechanism of dental caries. The results show that for in situ enamel demineralization, the lesion depth as well as the mineral loss parameter both vary linearly with the demineralization time. This is in contrast to in vitro lesion formation where the third power, or the square power of the lesion depth is linearly related to the demineralization time. In in situ demineralization, the rate-determining step of the demineralization process is the inhibitor-controlled dissolution process at the enamel crystallite surfaces, while the inhibitor content (F-, proteins etc.) in the lesion originating from the plaque, saliva and enamel is high. Furthermore, the study indicates that in in situ demineralization, interprismatic mineral loss is very important.     

Potency of antileukoprotease and alpha 1-antitrypsin to inhibit degradation of fibrinogen by adherent polymorphonuclear leukocytes from normal subjects and patients with chronic granulomatous disease.

We have studied the relative efficacy of antileukoprotease (ALP) and alpha 1-antitrypsin (alpha 1AT) to inhibit the degradation of substrate by polymorphonuclear leukocytes (PMN) attached onto a fibrinogen matrix. PMN elastase activity was assayed by radioimmunoassay of a specific 21-residue cleavage product from the amino terminus of the A alpha chain, A alpha (1-21), of fibrinogen. The adherence of PMN (1.0 x 10(6)) to a fibrinogen matrix was facilitated by incubation with recombinant tumor necrosis factor-alpha (1 nM). Subsequently, the cells were exposed to inhibitors before stimulation with cytochalasin B and formylmethionyl-leucylphenylalanine. Under these conditions, ALP inhibited A alpha (1-21) formation with an IC50 of 85 +/- 30 nM and alpha 1AT gave an IC50 of 220 +/- 98 nM (mean +/- SD). The effect of oxidant production on A alpha (1-21) formation was evaluated by comparing the effect of PMN from normal subjects with PMN from subjects with X-linked NADPH oxidase deficiency. Stimulation of PMN from the latter subjects in a similar fashion as described above resulted in the formation of 40 +/- 4 pmol/ml A alpha (1-21), or approximately twice the amount seen with cells from normal subjects. Preincubation with ALP or alpha 1AT in a concentration range between 10 to 900 nM resulted in an IC50 of 50 +/- 13 nM for ALP compared with 150 +/- 21 nM for alpha 1AT. Both inhibitors are more effective to prevent fibrinogen degradation caused by chronic granulomatous disease (CGD) PMN than by normal PMN despite the fact that CGD PMN generated more A alpha (1-21) than did normal PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

High prevalence of human papillomavirus infections in urine samples from human immunodeficiency virus-infected men

Infection with human immunodeficiency virus (HIV) and the resulting immunosuppression are associated with an increased risk for human papillomavirus (HPV) persistence and related malignancies. In the present study we investigated the prevalence of HPV in urine samples from 104 HIV-infected men with low CD4+ cell counts (<100 per mm(3)) and 115 urine samples from HIV-negative men. A high prevalence of HPV DNA (39.4%) was found in the HIV patients. Most of the HPV types were high risk (81.4%), with HPV 52 as the most prevalent type (12.5%), followed by HPV 18 (6.7%), HPV 35 (5.8%), and HPV 70 (4.8%). Multiple HPV genotypes were observed in 17 (41%) of the 41 HPV- and HIV-positive men. In contrast, only 11 (9.6%) HPV DNA-positive cases were observed among the 115 HIV-uninfected men, and 3 (27.3%) contained multiple genotypes. Quantitative analyses indicated that the HPV viral load, as measured in urine samples, is significantly higher in HIV-positive men compared to HIV-negative men. In the present study we show that urine samples are useful for detecting HPV DNA, there is a high prevalence of HPV in HIV-positive men, and the HPV viral load is substantially higher in HIV-positive than in HIV-negative men. More studies are needed to evaluate the risk and natural development of HPV-related malignancies in HIV-positive men.     

Human herpesvirus 8 load in matched serum and plasma samples of patients with AIDS-associated Kaposi's sarcoma

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma [KS]-associated herpesvirus) is associated with all forms of KS. HHV-8 DNA load in peripheral blood mononuclear cells (PBMCs) of KS patients has been shown to correlate with the clinical stage of the disease. Studies have been done to assess the HHV-8 viral load in different sample types from KS patients and its clinical relevance. This paper describes the design and evaluation of a quantitative real-time (TaqMan) PCR assay for routine diagnosis of HHV-8 infection. The linear dynamic range was 5 to 5 x 10(6) copies of HHV-8 DNA (r(2) > 0.99). The assay is very sensitive, specific, and easily reproducible (less than 2% variability) and can be used for different clinical samples, such as serum, plasma, and PBMCs. The question of which clinical sample, serum or plasma, is preferable for HHV8 DNA testing was addressed, using this newly developed real-time PCR assay. From 85 patients with diagnosed AIDS-KS, matched plasma and serum samples were collected. Of the 85 patients tested, 35 were positive for HHV-8 DNA in both plasma and serum (41%), 8 were positive in serum but not plasma, and 7 had detectable HHV-8 DNA only in plasma. The HHV-8 load was similar in both plasma and serum, and no significant difference was found. However, more inhibition was seen in the plasma samples with the use of a system quality control, seal herpesvirus type 1. Therefore, our results suggest that serum is the preferred material for HHV-8 load testing, since there is less possible hindrance in the amplification than with plasma.     

Biomechanical comparison of semirigid junctional fixation techniques to prevent proximal junctional failure after thoracolumbar adult spinal deformity correction

BACKGROUND CONTEXTAdult spinal deformity patients treated operatively by long-segment instrumented spinal fusion are prone to develop proximal junctional kyphosis (PJK) and failure (PJF). A gradual transition in range of motion (ROM) at the proximal end of spinal instrumentation may reduce the incidence of PJK and PJF, however, previously evaluated techniques have not directly been compared.PURPOSETo determine the biomechanical characteristics of five different posterior spinal instrumentation techniques to achieve semirigid junctional fixation, or “topping-off,” between the rigid pedicle screw fixation (PSF) and the proximal uninstrumented spine.STUDY DESIGNBiomechanical cadaveric study.METHODSSeven fresh-frozen human cadaveric spine segments (T8–L3) were subjected to ex vivo pure moment loading in flexion-extension, lateral bending and axial rotation up to 5 Nm. The native condition, three-level PSF (T11–L2), PSF with supplemental transverse process hooks at T10 (TPH), and two sublaminar taping techniques (knotted and clamped) as one- (T10) or two-level (T9, T10) semirigid junctional fixation techniques were compared. The ROM and neutral zone (NZ) of the segments were normalized to the native condition. The linearity of the transition zones over three or four segments was determined through linear regression analysis.RESULTSAll techniques achieved a significantly reduced ROM at T10-T11 in flexion-extension and axial rotation relative to the PSF condition. Additionally, both two-level sublaminar taping techniques (CT2, KT2) had a significantly reduced ROM at T9-T10. One-level clamped sublaminar tape (CT1) had a significantly lower ROM and NZ compared with one-level knotted sublaminar tape (KT1) at T10-T11. Linear regression analysis showed the highest linear correlation between ROM and vertebral level for TPH and the lowest linear correlation for CT2.CONCLUSIONSAll studied semirigid junctional fixation techniques significantly reduced the ROM at the junctional levels and thus provide a more gradual transition than pedicle screws. TPH achieves the most linear transition over three vertebrae, whereas KT2 achieves that over four vertebrae. In contrast, CT2 effectively is a one-level semirigid junctional fixation technique with a shift in the upper rigid fixation level. Clamped sublaminar tape reduces the NZ greatly, whereas knotted sublaminar tape and TPH maintain a more physiologic NZ. Clinical validation is ultimately required to translate the biomechanics of various semirigid junctional fixation techniques into the clinical goal of reducing the incidence of proximal junctional kyphosis and failure.CLINICAL SIGNIFICANCEThe direct biomechanical comparison of multiple instrumentation techniques that aim to reduce the incidence of PJK after thoracolumbar spinal fusion surgery provides a basis upon which clinical studies could be designed. Furthermore, the data provided in this study can be used to further analyze the biomechanical effects of the studied techniques using finite element models to better predict their post-operative effectiveness.     

A comparison of the in vitro cyto- and neurotoxicity of brominated and halogen-free flame retardants: prioritization in search for safe(r) alternatives

Brominated flame retardants (BFRs) are abundant persistent organic pollutants with well-studied toxicity. The toxicological and ecological concerns associated with BFRs argue for replacement by safe(r) alternatives. Though previous research identified the nervous system as a sensitive target organ for BFRs, the (neuro) toxic potential of alternative halogen-free flame retardants (HFFRs) is largely unknown. We therefore investigated the in vitro (neuro) toxicity of 13 HFFRs and three BFRs in dopaminergic pheochromocytoma (PC12) and neuroblastoma (B35) cells by assessing several cytotoxic and neurotoxic endpoints. Effects on cell viability and production of reactive oxygen species (ROS) were measured using a combined Alamar Blue and Neutral Red assay and a H₂-DCFDA assay, respectively, whereas effects on calcium homeostasis were measured using single-cell fluorescent Ca²⁺-imaging. The majority of the tested flame retardants induced negligible cytotoxicity, except zinc hydroxystannate (ZHS) and zinc stannate (ZS). A considerable fraction of flame retardants affected ROS production (decabromodiphenyl ether (BDE-209), triphenylphosphate (TPP), aluminium trihydroxide (ATH), ammonium polyphosphate (APP), magnesium hydroxide (MHO), ZHS, ZS and melamine polyphosphate (MPP)). Interestingly, ATH, ZHS, ZS and montmorillonite (MMT) increased the basal intracellular calcium concentration ([Ca²⁺]_i), whereas tetrabromobisphenol A (TBBPA), resorcinol bis (diphenylphosphate) (RDP), TPP, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO), ATH, ZHS, ZS and MMT reduced depolarization-evoked increases in [Ca²⁺]_i as a result of inhibition of voltage-gated calcium channels. These combined data on the in vitro (neuro) toxicity of HFFRs in comparison with BFRs are essential for prioritization of safe(r) flame retardants. Though additional data are required for a complete (toxic) risk assessment, our data demonstrate that several HFFRs could be suitable substitutes for BFRs.     

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.