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.     

Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia

Male genital tract obstructions may result from infections, previous inguinal and scrotal surgery (vasectomy) and congenital bilateral absence of the vas deferens (CBAVD). Microsurgery can sometimes be successful in treating the obstruction. In other cases and in cases of failed surgical intervention, the patient can be treated by microsurgical or percutaneous epididymal sperm aspiration (MESA, PESA) or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). We present the results of 39 ICSI procedures for obstructive azoospermia in 24 couples. The aetiology of the obstruction was failed microsurgery in 11 patients, CBAVD in nine and genital infections in four. Sperm retrieval was accomplished via MESA in four cases, PESA in 18 cases and via TESE in 11 cases. TESE was only applied when PESA failed to produce enough spermatozoa for simultaneous ICSI. In six patients, the ICSI procedure was performed with cryopreserved spermatozoa after an initial PESA procedure. Fertilization occurred in 47% of the metaphase II oocytes; embryo transfer was performed in 92% of procedures and resulted in a clinical pregnancy in 13/39 procedures. Ongoing pregnancy was achieved in 10/39 procedures. One pregnancy was terminated early after prenatal investigation showed a cytogenetic abnormality (47,XX+18, Edwards syndrome). The other nine pregnancies resulted in the live birth of 10 children, without any congenital abnormalities. Epididymal and testicular retrieved spermatozoa were successfully used for ICSI to treat obstructive azoospermia, and resulted in an ongoing pregnancy in 10 of 24 couples (41.6%) after 39 ICSI procedures, a success rate of 25.6% per treatment cycle and of 27.7% per embryo transfer.      

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.     

Differential organization of touch and pain in human primary somatosensory cortex

Processing of tactile stimuli within somatosensory cortices has been shown to be complex and hierarchically organized. However, the precise organization of nociceptive processing within these cortices has remained largely unknown. We used whole-head magnetoencephalography to directly compare cortical responses to stimulation of tactile and nociceptive afferents of the dorsum of the hand in humans. Within the primary somatosensory cortex (SI), nociceptive stimuli activated a single source whereas tactile stimuli activated two sequentially peaking sources. Along the postcentral gyrus, the nociceptive SI source was located 10 mm more medially than the early tactile SI response arising from cytoarchitectonical area 3b and corresponded spatially to the later tactile SI response. Considering a mediolateral location difference between the hand representations of cytoarchitectonical areas 3b and 1, the present results suggest generation of the single nociceptive response in area 1, whereas tactile stimuli activate sequentially peaking sources in areas 3b and 1. Thus nociceptive processing apparently does not share the complex and hierarchical organization of tactile processing subserving elaborated sensory capacities. This difference in the organization of both modalities may reflect that pain perception rather requires reactions to and avoidance of harmful stimuli than sophisticated sensory capacities.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.