The common functional C(-159)T polymorphism within the promoter region of the lipopolysaccharide receptor CD14 is not associated with sepsis development or mortality

Sepsis is characterised by a systemic inflammatory response to bacterial products during infection, which interestingly both in humans and animal models is gender associated with a higher susceptibility of males than females. The CD14 receptor is involved in activation of cells by lipopolysaccharides released from Gram-negative bacteria and, as recently shown, also by products of Gram-positive bacteria (e.g., peptidoglycans and lipoteichoic acid). The functional relevance of a C(-159)T CD14 polymorphism recently has been shown based on correlation of the T allele to higher plasma levels of soluble CD14, and higher membrane expression on monocytes. We, therefore, now analysed this CD14 polymorphism in 204 patients with severe sepsis and 247 controls. No significant difference of allele frequencies was observed between sepsis patients and controls neither for males nor females. Mortality also was not associated with the polymorphism studied. This may suggest that other mechanisms for lipopolysaccharide recognition, such as the recently described Toll-like receptors are important for inflammatory cell activation in sepsis.     

Molecular detection of antimicrobial resistance

The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art.     

Impairment of a cortical event-related desynchronisation during a bimanual load-lifting task in children with autistic disorder

In autism, the abilities of communication are affected, associated with abnormalities of cognitive, sensorial and motor development. In a previous study based on a load-lifting task, we showed impairment of anticipation in children with autism as evidenced by kinematics and eletromyographic recordings [Neurosci. Lett. 348 (2003) 17]. In the present study, we assessed the cortical counterparts of the use of anticipatory postural adjustments in a group of control children and in a group of children with autism. The tasks required maintaining a stable forearm position despite imposed or voluntary lifting of an object placed either on the controlateral forearm or on a support. We investigated the differences between the two groups of children on the Event-Related Desynchronisation (ERD) which precedes movement onset in adults [Electroencephalogr. Clin. Neurophysiol. 46 (1979) 138]. Electroencephalogram (EEG) power evolution of a 6-8-Hz frequency band was averaged before and after imposed or voluntary movement onset. EEG reactivity of control and autistic children did not differ during the imposed unloading condition, but significant differences appeared in the voluntary unloading situations. Before lifting the object, control children showed an ERD above the left motor areas. An ERD also occurred above the right motor areas when the object was placed on their forearm. This indicates that the ERD can also translate the use of anticipatory postural adjustments. By contrast, children with autism did not show an ERD in the two voluntary situations. This suggests a central deficit of anticipation in both postural and motor control in children with autism.     

Age-related loss of synaptophysin immunoreactive presynaptic boutons within the hippocampus of APP751SL, PS1M146L, and APP751SL/PS1M146L transgenic mice

Neuron and synapse loss are important features of the neuropathology of Alzheimer's disease (AD). Recently, we observed substantial age-related hippocampal neuron loss in APP751SL/PS1M146L transgenic mice but not in PS1M146L mice. Here, we investigated APP751SL mice, PS1M146L mice, and APP751SL/PS1M146L mice for age-related alterations in synaptic integrity within hippocampal stratum moleculare of the dentate gyrus (SM), stratum lucidum of area CA3 (SL), and stratum radiatum of area CA1-2 (SR) by analyzing densities and numbers of synaptophysin-immunoreactive presynaptic boutons (SIPBs). Wild-type mice, APP751SL mice and PS1M146L mice showed similar amounts of age-related SIPB loss within SM, and no SIPB loss within SL. Both APP751SL mice and PS1M146L mice showed age-related SIPB loss within SR. Importantly, APP751SL/PS1M146L) mice displayed the severest age-related SIPB loss within SM, SL, and SR, even in regions free of extracellular Abeta deposits. Together, these mouse models offer a unique framework to study the impact of several molecular and cellular events caused by mutant APP and/or mutant PS1 on age-related alterations in synaptic integrity. The observation of age-related SIPB loss within SR of PS1M146L mice supports a role of mutant PS1 in neurodegeneration apart from its contribution to alterations in Abeta generation.     

The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage,cellular tropism,and neutralization

We have studied infectivity and neutralization of X4, R5, and R5X4 tropic HIV-1 mutants, which are lacking N-linked glycosylation sites for glycans g13, g14, g15, and g17 in the V3 loop region of gp120. X4-tropic NL4-3 mutants lacking combinations of g14/15 or g15/17 showed markedly higher infectivity in CXCR4-specific infection. The role of g15 in CCR5-specific infection was investigated using viruses with high (NL-918, R5-monotropic), medium (NL-991, R5-monotropic), and low (NL-952, R5X4-dualtropic) CCR5-specific infectivity. For NL-991, a reduction of infectivity on GHOST-CCR5 cells was observed for a mutant lacking g15. For NL-952 mutants all lacking g15, a complete loss of CCR5-specificity was observed and NL-952 was shifted from R5X4 to X4 tropism. For all mutants of NL4-3, NL-991, and NL-952, where the lack of g15 markedly influenced infectivity or coreceptor usage, neutralization was enhanced. In contrast, NL-918 mutants with or without g15 showed no difference in neutralization and no difference in GHOST-CCR5 infection rates. Thus, for viruses with a low or medium CCR5-specificity the role of g15 for changing CCR5-usage and sensitivity to neutralization was more significant than for viruses with high infection rates on GHOST-CCR5 cells. Our data demonstrate that V3 glycans play an important role in the usage of CXCR4 and CCR5. The lack of g15 was relevant for a more efficient use of CXCR4, whereas interaction with CCR5 was facilitated in the presence of g15. This study also demonstrates that glycan g15 is involved in blocking of neutralizing antibodies and shifting HIV tropism from R5X4 to X4.     

A mouse model for alpha-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids

alpha-Methylacyl-CoA racemase (Amacr) deficiency in humans leads to sensory motor neuronal and liver abnormalities. The disorder is recessively inherited and caused by mutations in the AMACR gene, which encodes Amacr, an enzyme presumed to be essential for bile acid synthesis and to participate in the degradation of methyl-branched fatty acids. To generate a model to study the pathophysiology in Amacr deficiency we inactivated the mouse Amacr gene. As per human Amacr deficiency, the Amacr(-/-) mice showed accumulation (44-fold) of C27 bile acid precursors and decreased (over 50%) primary (C24) bile acids in bile, serum and liver, however the Amacr(-/-) mice were clinically symptomless. Real-time quantitative PCR analysis showed that, among other responses, the level of mRNA for peroxisomal multifunctional enzyme type 1 (pMFE-1) was increased 3-fold in Amacr(-/-) mice. This enzyme can be placed, together with CYP3A11 and CYP46A1, to make an Amacr-independent pathway for the generation of C24 bile acids. Exposure of Amacr(-/-) mice to a diet supplemented with phytol, a source for branched-chain fatty acids, triggered the development of a disease state with liver manifestations, redefining the physiological significance of Amacr. Amacr is indispensable for the detoxification of dietary methyl-branched lipids and, although it contributes normally to bile acid synthesis from cholesterol, the putative pMFE-1-mediated cholesterol degradation can provide for generation of bile acids, allowing survival without Amacr. Based upon our mouse model, we propose elimination of phytol from the diet of patients suffering from Amacr deficiency.     

Practical Evaluation of Methods for Detection and Specificity of Autoantibodies to Extractable Nuclear Antigens

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient''s clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.A diagnosis of autoimmune disease in patients is based upon clinical history, physical examination, and laboratory detection of antinuclear antibodies (ANAs). A particular class of ANAs specific for extractable nuclear antigens (ENA) was initially described in 1959 (3). Since that time, many different anti-ENA antibodies have been described. The detection of these autoantibodies and identification of their specificity have become well-established tools for the laboratory diagnosis of several autoimmune diseases. Studies of patients with ENA antibodies have shown that detection of these autoantibodies may have both diagnostic and prognostic significance, and the detection of anti-ENA antibodies has assumed an important role in the management of these patients (5, 16, 22). In most cases, ENA testing is ordered after an initial ANA screen. The indications for use are to establish a diagnosis in patients with suggestive clinical symptoms, to exclude a diagnosis of autoimmune disease in patients with few or uncertain clinical signs, to subclassify patients with a known diagnosis, and to monitor disease activity.Testing for anti-ENA antibodies has historically relied on gel-based immunoprecipitation techniques such as double immunodiffusion (DID) and counterimmunoelectrophoresis (2, 14). The associations of specific types of ENA autoantibodies with rheumatological diseases were established by using these gel-based immunoassay techniques (15). In the last decade, enzyme-linked immunoassay (ELISA) systems have been developed to detect and determine the specificity of anti-ENA antibodies. ELISA systems permit more rapid processing of more specimens with a faster turnaround time than gel-based assays. ELISA-based methods may also have increased sensitivity for detection of ENA antibodies. However, the increased sensitivity of these ELISAs may influence the clinical relevance of their detection because diagnostic specificity may be reduced (10, 12, 17, 24). As yet, a set of reference standards with known antibody specificities against defined antigen preparations is not available for evaluation of various methods or kits. Serum reference panels are available from the Association of Medical Laboratory Immunologists (4), but the specificities of these sera were determined by consensus results from multiple laboratories. The purpose of this study was to address the relationship between DID and ELISA methods for the detection and identification of anti-ENA antibodies by evaluating and comparing two DID kits and three ELISA kits. We evaluated both screening ELISAs and monospecific antigen ELISAs to determine anti-ENA specificity.