Human leukocyte antigen class-I variation is associated with atopic dermatitis: A case-control study.

Atopic dermatitis (AD) is a common immune-medicated skin disease. Previous studies have explored the relationship between Human Leukocyte Antigen (HLA) allelic variation and AD with conflicting results. The aim was to examine HLA Class I genetic variation, specifically peptide binding groove variation, and associations with AD. A case-control study was designed to evaluate HLA class I allelic variation and binding pocket polymorphisms, using next generation sequencing on 464 subjects with AD and 388 without AD. Logistic regression was used to evaluate associations with AD by estimating odds ratios (95% confidence intervals). Significant associations were noted with susceptibility to AD (B*53:01) and protection from AD (A*01:01, A*02:01, B*07:02 and C*07:02). Evaluation of polymorphic residues in Class I binding pockets revealed six amino acid residues conferring protection against AD: A9F (HLA-A, position 9, phenylalanine) [pocket B/C], A97I [pocket C/E], A152V [pocket E], A156R [pocket D/E], B163E [pocket A] and C116S [pocket F]. These findings demonstrate that specific HLA class I components are associated with susceptibility or protection from AD. Individual amino acid residues are relevant to protection from AD and set the foundation for evaluating potential HLA Class I molecules in complex with peptides/antigens that may initiate or interfere with T-cell responses.

Determining performance characteristics of an NGS-based HLA typing method for clinical applications.

This study presents performance specifications of an in-house developed human leukocyte antigen (HLA) typing assay using next-generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA-A, -B, -C, -DRB1 and -DQB1 were included in this study, which were typed at high-resolution using a combination of Sanger sequencing, sequence-specific primer (SSP) and sequence-specific oligonucleotide probe (SSOP) technologies and recorded at the two-field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African-American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA-A, -B, -C and -DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.

Towards allele-level human leucocyte antigens genotyping - assessing two next-generation sequencing platforms: Ion Torrent Personal Genome Machine and Illumina MiSeq.

Human leucocyte antigens (HLA) typing has been a challenge due to extreme polymorphism of the HLA genes and limitations of the current technologies and protocols used for their characterization. Recently, next-generation sequencing techniques have been shown to be a well-suited technology for the complete characterization of the HLA genes. However, a comprehensive assessment of the different platforms for HLA typing, describing the limitations and advantages of each of them, has not been presented. We have compared the Ion Torrent Personal Genome Machine (PGM) and Illumina MiSeq, currently the two most frequently used platforms for diagnostic applications, for a number of metrics including total output, quality score per position across the reads and error rates after alignment which can all affect the accuracy of HLA genotyping. For this purpose, we have used one homozygous and three heterozygous well-characterized samples, at HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1. The total output of bases produced by the MiSeq was higher, and they have higher quality scores and a lower overall error rate than the PGM. The MiSeq also has a higher fidelity when sequencing through homopolymer regions up to 9 bp in length. The need to set phase between distant polymorphic sites was more readily achieved with MiSeq using paired-end sequencing of fragments that are longer than those obtained with PGM. Additionally, we have assessed the workflows of the different platforms for complexity of sample preparation, sequencer operation and turnaround time. The effects of data quality and quantity can impact the genotyping results; having an adequate amount of good quality data to analyse will be imperative for confident HLA genotyping. The overall turnaround time can be very comparable between the two platforms; however, the complexity of sample preparation is higher with PGM, while the actual sequencing time is longer with MiSeq.

A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.

The genomics revolution has created a need for increased speed and generality for recombinant protein production systems as well as general methods for conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety of biochemical assays. Here we present a standardized system for purifying proteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein purification have been generated and characterized. To purify these antibodies from serum-containing medium without carrying through contaminating immunoglobulin G, a peptide-based purification process was developed. A fluorescence polarization binding assay was developed to characterize the antigen-antibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution parameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of recombinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields. The combination of the antibodies described here and 9E10 allow for almost any biochemical application to be utilized with a single simple peptide tag.

Recovery from carotid artery catheterization performed under various anesthetics in male, Sprague-Dawley rats.

This study was designed to determine the time to recovery from carotid artery catheterization using multiple criteria and to compare recovery times between three common anesthetics. Male Sprague-Dawley rats, chronically instrumented with radio-telemetry transmitters, were anesthetized with sodium pentobarbital, halothane or a mixture of ketamine, xylazine and acepromazine before an indwelling catheter was placed in the carotid artery. The procedure was completed in less than 15 min. Changes in body weight, food and water consumption, blood pressure, heart rate and activity were used to determine recovery. As judged by recovery of body weight, animals anesthetized with each of the anesthetics recovered by the 4th day after catheterization. Food and water consumption normalized by 1-2 days after surgery. Heart rates and blood pressures during the light phase of the photoperiod were significantly increased for 2 days by all anesthetics. During the dark phase of the photoperiod, heart rates and blood pressures were not significantly affected by pentobarbital or halothane anesthesia, but were significantly decreased and increased respectively on the night immediately following surgery in the ketamine / xylazine / acepromazine-anesthetized rats. Delayed elevations of heart rate were observed in pentobarbital and halothane anesthetized rats on days and/or nights 5 and 6 post surgery. Animal activity patterns during the light phase of the photoperiod were not affected by pentobarbital or halothane, but were increased by ketamine 2 days after surgery. During the dark phase, halothane transiently reduced activity whereas ketamine-anesthetized rats showed reduced activity for 4 nights post surgery. These studies show that recovery depends on the criteria selected and the anesthetic used, but, in general, 2-4 days were required for recovery from this relatively simple procedure.

The effects of routine cage-changing on cardiovascular and behavioral parameters in male Sprague-Dawley rats.

The objective of this study was to determine whether the blood pressure and heart rate of adult male Sprague-Dawley rats are affected by the routine animal husbandry procedure of moving animals to clean cages. Cardiovascular parameters were obtained by using radiotelemetry; behavior in the home cage also was evaluated. Each rat had a radiotelemetry transmitter implanted in the peritoneal cavity, with the attached catheter placed in the femoral artery. After a 7- to 9-day recovery period, half of the rats were moved to clean cages with fresh wood-chip bedding; the other animals were left undisturbed. Systolic, diastolic, and mean arterial blood pressures; heart rate; and cage behavior (movement, rearing, grooming) increased promptly and significantly when animals were placed in clean cages. These cardiovascular and behavioral responses lasted for 45 to 60 min. Those animals not moved to clean cages but present in the animal room when this procedure was done did not show significant increases in blood pressure, heart rate, or activity. When rats were moved to clean cages that contained new bedding plus a small quantity of the soiled bedding from their previous cage, the cardiovascular and behavioral responses were similar to those of animals exposed to completely fresh bedding. The responses of rats being moved to new cages did not diminish between the first and fourth weekly cage change. Rats whose cages were not changed for 2 weeks showed small, but significant, increases in cardiovascular and behavioral responses above the responses in animals with weekly cage changes. We conclude that ordinary animal husbandry procedures such as moving rats to a clean cage can induce transient, but significant, cardiovascular and behavioral changes. Investigators and animal care staff should recognize that such routine procedures could confound experiments conducted shortly thereafter.

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Molecular recognition of cyclic urea HIV-1 protease inhibitors.

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.

Molecular basis of HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with cyclic urea inhibitors.

In cell cultures, the key residues associated with HIV-1 resistance to cyclic urea-based HIV-1 protease (PR) inhibitors are Val82 and Ile84 of HIV-1 PR. To gain an understanding of how these two residues modulate inhibitor binding, we have measured the Ki values of three recombinant mutant proteases, I84V, V82F, and V82F/I84V, for DMP323 and DMP450, and determined the three-dimensional structures of their complexes to 2.1-1.9 A resolution with R factors of 18.7-19.6%. The Ki values of these mutants increased by 25-, 0.5-, and 1000-fold compared to the wild-type values of 0.8 and 0.4 nM for DMP323 and DMP450, respectively. The wild-type and mutant complexes overall are very similar (rms deviations of 0.2-0.3 A) except for differences in the patterns of their van der Waals (vdw) interactions, which appear to modulate the Ki values of the mutants. The loss of the CD1 atom of Ile84, in the I84V mutant complexes, creates a hole in the S1 subsite, reducing the number of vdw contacts and increasing the Ki values. The V82F mutant binds DMP323 more tightly than wild type because the side chain of Phe82 forms additional vdw and edge-to-face interactions with the P1 group of DMP323. The Ki values of the single mutants are not additive because the side chain of Phe82 rotates out of the S1 subsite in the double mutant (the chi 1 angles of Phe82 and -182 in the V82F and V82F/I84V mutants differ by 90 and 185 degrees, respectively), further reducing the vdw interactions. Finally, compensatory shifts in the I84V and V82F/ I84V complexes pick up a small number of new contacts, but too few to offset the initial loss of interactions caused by the mutations. Therefore, our data suggest that variants persist in the presence of DMP323 and DMP450 because of a decrease in vdw interactions between the mutant proteases and inhibitors.

Quantitative determination of recombinant fibroblast growth factor receptor in baculovirus-infected insect cell cultures.

Members of the fibroblast growth factor protein family are involved in several biologically important processes, including angiogenesis, wound healing, and tumor growth and metastasis. Interactions of basic fibroblast growth factor (bFGF) and its receptors are of considerable pharmacological importance. Attempts were made to produce gram quantities of a soluble extracellular form of basic fibroblast growth factor receptor type 1 (bFGFR1) in order to study the energetics of its interaction with bFGF. The aim of the present study was to develop a method for monitoring changes in concentration of bFGFR1 during its production by large-scale baculovirus-infected insect cell culture. A simple reverse-phase high-performance liquid chromatographic assay was developed for direct determination of the soluble receptor secreted into insect cell-culture media. The method permitted cell-culture samples containing varying amounts of fetal calf serum and bFGFR1 (10-30 mg/L) to be analyzed without prior purification. The assay was linear for added receptor in the range of 1-7 micrograms.

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli.

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.