脂阿拉伯甘露糖抗体检测对AIDS合并肺结核患者的诊断价值

目的 探讨脂阿拉伯甘露糖(lipoarabinomanna,LAM)抗体检测在获得性免疫缺陷综合征(acquired immune deficiency syndrome,AIDS)合并肺结核患者中的诊断价值.方法 收集我院2013年3月至2016年3月就诊的AIDS感染肺结核患者38例(研究组)及无肺结核的AIDS患者38例(对照组),对两组患者均进行LAM抗体检测及PPD试验,比较两组患者LAM抗体阳性率及PPD阳性率.同时对所有患者行CD4+T淋巴细胞计数.根据CD4+T淋巴细胞计数,将研究组患者进一步分为CD4+T淋巴细胞≥200/μl组和CD4+T淋巴细胞＜200/μl组,比较两组患者LAM抗体阳性率及PPD阳性率.比较LAM抗体检测及PPD试验对AIDS合并肺结核的诊断价值.结果 研究组患者LAM抗体检测阳性率为84.2％,对照组LAM抗体检测阳性率为l0.5％,两组患者比较差异有统计学意义(P＜0.05).研究组患者PPD阳性率为42.1％较对照组(23.7％)高,但差异无统计学意义(P＞0.05).CD4+T淋巴细胞≥200/μl组患者LAM抗体阳性率92.9％,较CD4+T淋巴细胞＜200/μl组(79.2％)高,但差异无统计学意义(P＞0.05).CD4+T淋巴细胞≥200/μl组患者PPD阳性率为92.9％,较CD4+T淋巴细胞＜200/μl组(12.5％)高,且差异有统计学意义(P＜0.05).LAM抗体检测诊断AIDS合并肺结核的敏感度为84.2％,特异度为89.5％,阳性预测值为94.1％、阴性预测值为85.7％、阳性似然比为15.9、阴性似然比为0.17.LAM抗体诊断AIDS合并肺结核阳性似然比＞10,即表示LAM抗体诊断AIDS合并肺结核的可能性显著增加.结论 LAM抗体检测在AIDS患者合并肺结核的诊断中特异度及敏感度高,有利于AIDS合并肺结核患者的诊断.值得临床进一步地推广应用.     

结核和肺吸虫抗体快速联检的鉴别诊断价值

目的探索结核抗体、肺吸虫抗体快速联合检测技术的诊断和鉴别诊断价值。方法结核分枝杆菌脂阿拉伯甘露糖(LAM)抗原、肺吸虫成虫可溶性抗原分别点样在硝酸纤维素膜上,以胶体金-蛋白A结合物为检测标志物,建立结核抗体、肺吸虫抗体联合检测的金标免疫渗滤法(TB/Pw-DIGFA);用TB/Pw-DIGFA检测肺结核病人、肺吸虫病人及健康人血清样本,以敏感性、特异性、灵敏度等指标考核其诊断价值。结果TB/Pw-DIGFA检测涂阳肺结核病人、涂阴肺结核病人和肺吸虫病人抗体的敏感性分别为85.9%(134/156)、83.0%(44/53)和95.6%(22/23);对肺结核病和肺吸虫病的特异性分别为92.6%(100/108)和99.1%(107/108);与TB-Dot平行检测结核病人和健康人群血清共94人份,符合率达96.8%(91/94),经Kappa检验,两者有高度一致性(K=0.935,P<0.001);与ELISA肺吸虫抗体检测试剂盒平行检测肺吸虫病人和健康人群血清共90人份,检测结果完全一致;未发现结核病和肺吸虫病人血清之间有交叉反应。结论TB/Pw-DIGFA诊断肺结核病和肺吸虫病具有较高的敏感性和特异性;该方法一次操作同时完成结核和肺吸虫二项特异性抗体测定,不仅检测效率高而且便于临床医生鉴别诊断。     

脂阿拉伯甘露聚糖的提纯及其对肺结核的诊断价值

目的 初步建立提取纯化结核分枝杆菌（M .TB）脂阿拉伯甘露聚糖（lipoarabinomanan,LAM）的方法 ,并将其用于血清标本检测,评价其对肺结核的诊断价值。 方法 M .TB菌体经冰浴超声破碎,乙醇回流,DNase I 酶RNase酶除去DNA和RNA, 热酚水法去蛋白,氯仿甲醇去酚和脂质,得到粗制的脂多糖。经Sephacryl-100凝胶过滤层析分离纯化,得到LAM。将其作为抗原,通过免疫金渗滤法检测涂阳肺结核和对照血清标本中的LAM抗体。 结果 所得LAM经SDS-PAGE银染,与对照标准品大小一致,为37 kDa。血清LAM抗体检测对肺结核诊断的敏感性、特异性、阳性预测值、阴性预测值、和涂片符合率分别为91.7%、87.8%、92.7%、86.2%、90.2%。 结论 成功提取到LAM抗原,用其检测血清结核抗体显示出理想的敏感性和特异性,有望成为结核病血清学快速诊断方法之一。     

Influence of acyl chain fluidity on the lipopolysaccharide-induced activation of complement

Lipopolysaccharides (LPSs, endotoxins) are the major amphiphilic constituents of the outer leaflet of the outer membrane of Gram-negative bacteria. They are known to activate the complement cascade to form lytic membrane pores. Here, we study the influence of the fluidity of the acyl chains of LPSs and lipid As on the formation of lytic pores. To this end, we have performed electrical measurements on asymmetric planar endotoxin/phospholipid bilayers as a reconstitution model of the outer membrane using two deep rough mutant LPSs (from Escherichia coli strains WBB01 and WBB25) and two lipid As (from E. coli WBB25 and Rhodobacter sphaeroides). The two LPSs and the two lipid As each differ in their acylation pattern which is correlated with the fluidity. The addition of human serum to the endotoxin side of the bilayers led to the formation of membrane pores, and pore formation correlated in each case with acyl chain fluidity, i.e. time required for the first lytic pore to be formed was shorter for the more fluid endotoxin. Furthermore, in the case of LPSs, the activation rate was higher for the more fluid membrane and the respective bacteria had a higher susceptibility to the growth inhibitory action of serum.     

Vesicles surfing on a lipid bilayer: self-induced haptotactic motion

Haptotaxis is a mechanism proposed at the end of the 1960s to explain cell motility. It describes cell movement induced by an adhesion gradient. In this work, we present evidence for self-induced haptotaxis using negatively charged giant vesicles interacting with positively charged supported lipid bilayers, which has not been previously described. Depending on the charge of the vesicle, we observed different behaviors. At low charge, no adhesion occurs. At high charge, the vesicle adheres but does not move. In a restricted range of intermediate charge densities, we found that the vesicle moves spontaneously with velocities of the order of a few micrometers per second over distances of >100 microm. We show that a local lipid transfer between the giant vesicle and the supported lipid bilayer takes place during the adhesion, breaking the symmetry and inducing a lateral charge gradient. This charge gradient polarizes the giant vesicle and induces its motion. To explain our observations, we propose a scaling model that relates the adhesion energy to the velocity of vesicle motion and to the characteristic lipid transfer time. Our measurements indicate that the effective adhesion energy is strongly reduced by counterions, which are dynamically trapped between the vesicle and the supported bilayer.     

A helical-dipole model describes the single-channel current rectification of an uncharged peptide ion channel.

We are designing simple peptide ion channels as model systems for the study of the physical principles controlling conduction through ion-channel proteins. Here we report on an uncharged peptide, Ac-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, designed to form an aggregate of parallel, amphiphilic, membrane-spanning alpha-helices around a central water-filled pore. This peptide in planar lipid bilayers forms ion channels that show single-channel current rectification in symmetric 1 M KCl. The current at a given holding membrane potential is larger than the current measured through the same channel when the potential is reversed. Based on our hypothesized gating mechanism, the larger currents flow from the peptide carboxyl terminus toward the amino terminus. We present an ionic electrodiffusion model based on the helical-dipole potential and the dielectric interfacial polarization energy, which with reasonable values for dipole magnitude and dielectric constants, accurately replicates the current-voltage data.     

Clinical and radiological features in relation to urinary excretion of lipoarabinomannan in Ethiopian tuberculosis patients

We have previously reported on the diagnostic potential of urinary lipoarabinomannan (LAM) detection in active tuberculosis (TB). In this study, we identified clinical and radiological parameters that were significantly associated with urine LAM positivity in a clinical sample of 931 patients attending a TB control center in Addis Ababa, Ethiopa. These parameters were attributed weights and used in a diagnostic score (DS) system. Using urinary LAM as a reference, this DS system showed a sensitivity of 65.4% and a specificity of 82.9%. The positive and negative predictive values were 56.8% and 87.4%, respectively. HIV or other coinfections or deficiencies may have blurred the clinical manifestations of pulmonary TB (PTB) and thereby contributed to the relatively high number of false-positive DS results obtained. Although additional markers may be required to improve the sensitivity of the DS system, the relatively high specificity of this simple approach may be of some practical use in the field. Thus, in PTB-suspected, DS-negative cases, the likelihood of ongoing PTB is < 20%.     

Biosynthesis of mycobacterial lipoarabinomannan: role of a branching mannosyltransferase

Lipoarabinomannan (LAM), one of the few known bacterial glycosylphosphoinositides (GPIs), occurs in various structural forms in Mycobacterium species. It has been implicated in key aspects of the physiology of Mycobacterium tuberculosis and the immunology and pathogenesis of tuberculosis. Yet, little is known of the biosynthesis of LAM. A bioinformatics approach identified putative integral membrane proteins, MSMEG4250 in Mycobacterium smegmatis and Rv2181 in M. tuberculosis, with 10 predicted transmembrane domains and a glycosyltransferase (GT) motif (DID), features that are common to eukaryotic mannosyltransferases (ManTs) of the GT-C superfamily that rely on polyprenyl-linked rather than nucleotide-linked sugar donors. Inactivation of M. smegmatis MSMEG4250 by allelic exchange resulted in altered growth and inability to synthesize lipomannan (LM) but accumulation of a previously uncharacterized, truncated LAM. MALDI-TOF/MS and NMR indicated a structure lower in molecular weight than the native molecule, a preponderance of 6-linked Manp residues, and the absence of 2,6-linked and terminal Manp. Complementation of the mutant with the corresponding ortholog of M. tuberculosis H37Rv restored normal LM/LAM synthesis. The data suggest that MSMEG4250 and Rv2181 are ManTs that are responsible for the addition of alpha(1-->2) branches to the mannan core of LM/LAM and that arrest of this branching in the mutant deters formation of native LAM. The results allow for the presentation of a unique model of LM and LAM biosynthesis. The generation of mutants defective in the synthesis of LM/LAM will help define the role of these GPIs in the immunology and pathogenesis of mycobacterial infections and physiology of the organism.     

Regulation of endothelial cell migration by amphiphiles—are changes in cell membrane physical properties involved?

Endothelial cell (EC) migration is an integral part of angiogenesis and a prerequisite for malignant tumor growth. Recent studies suggest that amphiphilic compounds can regulate migration of bovine aortic ECs by altering the physical properties of the cell membrane lipid bilayers. A number of structurally different amphiphiles thus regulate the migration in quantitative correlation with their effects on the plasma membrane microviscosity. Many amphiphiles that affect EC migration and angiogenesis alter the physical properties of lipid bilayers, suggesting that such a regulatory mechanism may be of general importance. To investigate this notion, we studied the effects of lysophospholipids that inhibit migration of bovine aortic ECs and decrease cell membrane microviscosity, and of other amphiphiles that decrease membrane microviscosity (Triton X-100, octyl-beta-glucoside, arachidonic acid, docosahexaenoic acid, ETYA, capsaicin) on the migration of porcine aortic ECs. We further studied whether the enzyme secretory phospholipase A(2) (sPLA(2)) would affect migration in accordance with the changes in membrane microviscosity induced by its hydrolysis products lysophospholipids and polyunsaturated fatty acids. Arachidonic acid, at low concentrations, promoted cell migration by a mechanism involving metabolic products of this compound. Apart from this effect, all the amphiphiles, as well as sPLA(2), inhibited cell migration. A semi-quantitative analysis found a similar correlation between the effects on migration and on lipid bilayer stiffness measured using gramicidin channels as molecular force transducers. These results suggest that changes in cell membrane physical properties may generally contribute to the effects of amphiphiles on EC migration.     

Phospholipid bilayers are viscoelastic

Lipid bilayers provide the structural framework for cellular membranes, and their character as two-dimensional fluids enables the mobility of membrane macromolecules. Though the existence of membrane fluidity is well established, the nature of this fluidity remains poorly characterized. Three-dimensional fluids as diverse as chocolates and cytoskeletal networks show a rich variety of Newtonian and non-Newtonian dynamics that have been illuminated by contemporary rheological techniques. Applying particle-tracking microrheology to freestanding phospholipid bilayers, we find that the membranes are not simply viscous but rather exhibit viscoelasticity, with an elastic modulus that dominates the response above a characteristic frequency that diverges at the fluid-gel (L(α)?-?L(β)) phase-transition temperature. These findings fundamentally alter our picture of the nature of lipid bilayers and the mechanics of membrane environments.     

St. George's Respiratory Questionnaire Has Longitudinal Construct Validity in Lymphangioleiomyomatosis

BackgroundLymphangioleiomyomatosis (LAM) is an uncommon, progressive, cystic lung disease that causes shortness of breath, hypoxemia, and impaired health-related quality of life (HRQL). Whether St. George's Respiratory Questionnaire (SGRQ), a respiratory-specific HRQL instrument, captures longitudinal changes in HRQL in patients with LAM is unknown.MethodsUsing data from the Multicenter International Lymphangioleiomyomatosis Efficacy and Safety of Sirolimus trial, we performed analyses to examine associations between SGRQ scores and values for four external measures (anchors). Anchors included (1) FEV₁, (2) diffusing capacity of the lung for carbon monoxide, (3) distance walked during the 6-min walk test, and (4) serum vascular endothelial growth factor-D.ResultsSGRQ scores correlated with the majority of anchor values at baseline, 6 months, and 12 months. Results from longitudinal analyses demonstrated that SGRQ change scores tracked changes over time in values for each of the four anchors. At 12 months, subjects with the greatest improvement from baseline in FEV₁ experienced the greatest improvement in SGRQ scores (Symptoms domain, ?13.4 ± 14.6 points; Activity domain, ?6.46 ± 8.20 points; Impacts domain, ?6.25 ± 12.8 points; SGRQ total, ?7.53 ± 10.0 points). Plots of cumulative distribution functions further supported the longitudinal validity of the SGRQ in LAM.ConclusionsIn LAM, SGRQ scores are associated with variables used to assess LAM severity. The SGRQ is sensitive to change in LAM severity, particularly when change is defined by FEV₁, perhaps the most clinically relevant and prognostically important variable in LAM. The constellation of results here supports the validity of the SGRQ as capable of assessing longitudinal change in HRQL in LAM.     

Modulation of CTP:phosphocholine cytidylyltransferase by membrane curvature elastic stress

The activity of CTP:phosphocholine cytidylyltransferase, a rate-limiting enzyme in phosphatidylcholine biosynthesis, is modulated by its interaction with lipid bilayers [Kent, C. (1997) Biochim. Biophys. Acta 1348, 79-90]. Its regulation is of central importance in the maintenance of membrane lipid homeostasis. Here we show evidence that the stored curvature elastic stress in the lipid membrane's monolayer modulates the activity of CTP:phosphocholine cytidylyltransferase. Our results show how a purely physical feedback signal could play a key role in the control of membrane lipid synthesis.     

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.     

Evolution of the extensively drug-resistant F15/LAM4/KZN strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa.

BACKGROUND: Although several hot spots of multidrug-resistant tuberculosis have been identified on the African continent, extensive drug resistance (XDR) has not been reported until recently, when a large number of XDR cases were identified in KwaZulu-Natal. The majority of the patients involved were infected with the same strain of Mycobacterium tuberculosis (F15/LAM4/KZN). We report this strain's development from multidrug resistance to XDR. METHODS: We searched databases for studies performed during the period 1994-2005 that involved the resistance patterns of isolates of M. tuberculosis with the F15/LAM4/KZN strain fingerprint. RESULTS: As early as 1994, the F15/LAM4/KZN strain was responsible for a number of cases of multidrug-resistant tuberculosis, indicating the ability of the strain to cause cases of primary resistant tuberculosis. Some of the isolates were also resistant to streptomycin. From 1994 onwards, multidrug-resistant isolates with resistance to additional drugs were found, and the first XDR isolate was discovered in 2001. CONCLUSIONS: Drug resistance to as many as 7 drugs developed in a local strain of M. tuberculosis in slightly more than a decade. This coincided with the introduction of the directly observed therapy-based and directly observed therapy-plus-based tuberculosis-control programs. It is postulated that the introduction of these programs in the absence of susceptibility testing or drug resistance surveillance has been instrumental in the development of XDR in this highly transmissible F15/LAM4/KZN strain. The expanding pool of human immunodeficiency virus-infected, tuberculosis-susceptible individuals has likely contributed to this development.     

Slaved diffusion in phospholipid bilayers

The translational diffusion of phospholipids in supported fluid bilayers splits into two populations when polyelectrolytes adsorb at incomplete surface coverage. Spatially resolved measurements using fluorescence correlation spectroscopy show that a slow mode, whose magnitude scales inversely with the degree of polymerization of the adsorbate, coexists with a fast mode characteristic of naked lipid diffusion. Inner and outer leaflets of the bilayer are affected nearly equally. Mobility may vary from spot to spot on the membrane surface, despite the lipid composition being the same. This work offers a mechanism to explain how nanosized domains with reduced mobility arise in lipid membranes.     

Structure,function, and biogenesis of the cell wall of Mycobacterium tuberculosis

Much of the early structural definition of the cell wall of Mycobacterium spp. was initiated in the 1960s and 1970s. There was a long period of inactivity, but more recent developments in NMR and mass spectral analysis and definition of the M. tuberculosis genome have resulted in a thorough understanding, not only of the structure of the mycobacterial cell wall and its lipids but also the basic genetics and biosynthesis. Our understanding nowadays of cell-wall architecture amounts to a massive "core" comprised of peptidoglycan covalently attached via a linker unit (L-Rha-D-GlcNAc-P) to a linear galactofuran, in turn attached to several strands of a highly branched arabinofuran, in turn attached to mycolic acids. The mycolic acids are oriented perpendicular to the plane of the membrane and provide a truly special lipid barrier responsible for many of the physiological and disease-inducing aspects of M. tuberculosis. Intercalated within this lipid environment are the lipids that have intrigued researchers for over five decades: the phthiocerol dimycocerosate, cord factor/dimycolyltrehalose, the sulfolipids, the phosphatidylinositol mannosides, etc. Knowledge of their roles in "signaling" events, in pathogenesis, and in the immune response is now emerging, sometimes piecemeal and sometimes in an organized fashion. Some of the more intriguing observations are those demonstrating that mycolic acids are recognized by CD1-restricted T-cells, that antigen 85, one of the most powerful protective antigens of M. tuberculosis, is a mycolyltransferase, and that lipoarabinomannan (LAM), when "capped" with short mannose oligosaccharides, is involved in phagocytosis of M. tuberculosis. Definition of the genome of M. tuberculosis has greatly aided efforts to define the biosynthetic pathways for all of these exotic molecules: the mycolic acids, the mycocerosates, phthiocerol, LAM, and the polyprenyl phosphates. For example, we know that synthesis of the entire core is initiated on a decaprenyl-P with synthesis of the linker unit, and then there is concomitant extension of the galactan and arabinan chains while this intermediate is transported through the cytoplasmic membrane. The final steps in these events, the attachment of mycolic acids and ligation to peptidoglycan, await definition and will prove to be excellent targets for a new generation of anti-tuberculosis drugs.     

Membrane lipid phase as catalyst for peptide-receptor interactions.

Catalysis of ligand-receptor interactions is proposed as an important function of the lipid phase of the cell membrane. The catalytic mechanism is deduced from observed specific interactions of amphiphilic peptides with artificial lipid bilayers. In our model a direct ligand-receptor reaction is replaced by multiple sequential steps including surface accumulation of charged ligands, ligand-membrane interactions, and ultimately binding to the receptor itself. By dividing the total free energy of binding among several steps, the energy per step, including the intrinsic receptor interaction energy, is kept to moderate values. The model thereby yields simple explanations for the large apparent association constants, the high association and dissociation rates, and the heterogeneity of binding sites so frequently found with pharmacological and biochemical ligand-receptor interactions. Furthermore, the measured apparent association constant is a function of the whole system rather than just the receptor. The same, fully functional receptor may show different binding characteristics in different surroundings, such as in another tissue or in a reconstituted system.     

Sequential monitoring of leprosy patients with serum antibody levels to phenolic glycolipid-I, a synthetic analog of phenolic glycolipid-I, and mycobacterial lipoarabinomannan

Sequential serum samples from leprosy patients at various stages of antibacterial treatment were tested by an ELISA for antibodies to phenolic glycolipid I (PGL-I), a synthetic PGL-I analog (ND-BSA), and lipoarabinomannan (LAM) from Mycobacterium tuberculosis to determine if these antibodies could be useful in monitoring response to therapy. Among patients with positive initial anti-PGL-I IgM, a significant decrease in this antibody was seen over time (p less than 0.01), whether assayed by PGL-I or ND-BSA. The two antigens showed good agreement in the detection of decrease in anti-PGL-I IgM. The greatest decrease was seen in patients with a high initial anti-PGL-I IgM and a high bacterial index (BI). Patients with a declining BI were seen to have generally declining antibody levels to PGL-I and to LAM; in those patients with a fluctuating BI, antibody levels were less predictable. We conclude that antibodies to PGL-I and LAM can be useful in following response to therapy in leprosy patients and that either the native PGL-I or ND-BSA can serve as antigen for the ELISA.     

Magnitude of the solvation pressure depends on dipole potential.

As polar surfaces in solvent are brought together, they experience a large repulsive interaction, termed the solvation pressure. The solvation pressure between rough surfaces, such as lipid bilayers, has been shown previously to decay exponentially with distance between surfaces. In this paper, we compare measured values of the solvation pressure between bilayers and the dipole potential for monolayers in equilibrium with bilayers. For a variety of polar solvents and lipid phases, we find a correlation between the measured solvation pressures and dipole potentials. Analysis of the data indicates that the magnitude of the solvation pressure is proportional to the square of the dipole potential. Our experiments also show that the oriented dipoles in the lipid head-group region, including those of both the lipid and solvent molecules, contribute to the dipole potential. We argue that (i) the field produced by these interfacial dipoles polarizes the interbilayer solvent molecules giving rise to the solvation pressure and (ii) both the solvation pressure and the dipole potential decay exponentially with distance from the bilayer surface, with a decay constant that depends on the packing density of the interbilayer solvent molecules (1-2 A in water). These results may have importance in cell adhesion, adsorption of proteins to membranes, characteristics of channel permeability, and the interpretation of electrokinetic experiments.