HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing.

Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.

Serum levels of IL-6 and IL-1ß can predict the efficacy of gemcitabine in patients with advanced pancreatic cancer.

BACKGROUND: With this study, we sought to characterise the impact of pro-inflammatory cytokines on the outcomes of gemcitabine monotherapy (GEM) in patients with pancreatic cancer (PC). METHODS: Treatment-naive patients with advanced PC and no obvious infections were eligible for enrolment. All of the patients were scheduled to undergo systemic chemotherapy. Serum pro-inflammatory cytokines were measured using an electro-chemiluminescence assay method before chemotherapy. High cytokine levels were defined as values greater than the median. Clinical data were collected prospectively. RESULTS: Sixty patients who received GEM were included in the analysis. High IL-6 and IL-1ß levels were poor prognostic factors for overall survival in a multivariate analysis (P=0.011 and P=0.048, respectively). Patients with both a high IL-6 level and a high IL-1ß level exhibited shortened overall and progression-free survival, a reduction in the tumour control rate, and a high dose intensity of GEM compared with patients with low levels of both IL-6 and IL-1ß. CONCLUSION: The serum levels of IL-6 and IL-1ß predict the efficacy of GEM in patients with advanced PC.

Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

Associations between six classical HLA loci and rheumatoid arthritis: a comprehensive analysis.

Although the HLA region contributes to one-third of the genetic factors affecting rheumatoid arthritis (RA), there are few reports on the association of the disease with any of the HLA loci other than the DRB1. In this study we examined the association between RA and the alleles of the six classical HLA loci including DRB1. Six HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) of 1659 Japanese subjects (622 cases; 488 anti-cyclic citrullinated peptides (CCP) antibody (Ab) positive (82.6%); 103 anti-CCP Ab negative (17.4%); 31 not known and 1037 controls) were genotyped. Disease types and positivity/negativity for CCP autoantibodies were used to stratify the cases. Statistical and genetic assessments were performed by Fisher's exact tests, odds ratio, trend tests and haplotype estimation. None of the HLA loci were significantly associated with CCP sero-negative cases after Bonferroni correction and we therefore limited further analyses to using only the anti CCP-positive RA cases and both anti-CCP positive and anti-CCP negative controls. Some alleles of the non-DRB1 HLA loci showed significant association with RA, which could be explained by linkage disequilibrium with DRB1 alleles. However, DPB1*02:01, DPB1*04:01 and DPB1*09:01 conferred RA risk/protection independently from DRB1. DPB1*02:01 was significantly associated with the highly erosive disease type. The odds ratio of the four HLA-loci haplotypes with DRB1*04:05 and DQB1*04:01, which were the high-risk HLA alleles in Japanese, varied from 1.01 to 5.58. C*07:04, and B*15:18 showed similar P-values and odds ratios to DRB1*04:01, which was located on the same haplotype. This haplotype analysis showed that the DRB1 gene as well as five other HLA loci is required for a more comprehensive understanding of the genetic association between HLA and RA than analyzing DRB1 alone.

Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck.

Sixty-eight primary head and neck squamous cell carcinomas and nine head and neck squamous cell carcinoma cell lines were examined for mutations and homozygous deletions of the p16/CDKN2 gene. Homozygous deletions of the p16/CDKN2 gene were found in three lines, and a mutation was detected in another cell line. In contrast, none of the primary tumors showed homozygous deletions and 11 of 68 tumors had missense or nonsense base changes. Seven tumors contained somatic mutations. Five tumors, including one that also had a somatic mutation, had a probable polymorphism at codon 140 leading to an amino acid change from Ala to Thr. Three of these also contained an apparent polymorphism at codon 98, which did not lead to an amino acid change. The frequency of mutations and deletions detected differs markedly between cell lines (44%) and primary tumors (10%) suggesting that while p16/CDKN2 may play a role in tumorigenesis in some head and neck squamous cell carcinomas, inactivation of p16/CDKN2 probably occurs more frequently in cell lines as a result of adaptation to cell culture.

Correction of the HLA-DQB1*04:01:01 sequence at position 79 in exon 1.

The HLA-DQB1*04:01:01 sequence is corrected at position 79 in exon 1 (G > A, Ala > Thr).

High-resolution HLA-DQB1 typing by combination of group-specific amplification and restriction fragment length polymorphism.

A reliable method for high-resolution HLA-DQB1 typing using the combination of group-specific amplification and RFLP analysis is described. Group-specific amplification was carried out for the alleles of two groups using the two primer pairs under the same PCR conditions. One group contains DQ5 and DQ6 specificities and the other DQ2, DQ3, and DQ4 specificities. Computer analysis on cleavage patterns for 19 alleles of the DQB1 gene showed that the 11 alleles of the former group could be distinguished with five restriction enzymes and the eight alleles of the latter group could be distinguished with four enzymes. We could reduce the number of restriction endonucleases required compared with the number used in previous studies because we selected appropriate restriction enzymes which had at least one recognition site in almost all DQB1 alleles as a form of internal control. Moreover, DQB1*0602 and 0603, which were indistinguishable using the previously reported PCR-RFLP methods, could be distinguished by the present method. The results of typing of 100 samples from Japanese individuals by this method showed no discrepancy with the results obtained by serologic methods. The calculated allele frequencies showed good agreement with those reported at the 11th International Histocompatibility Workshop.

Family study on HLA-DPB1 polymorphism: linkage analysis with HLA-DR/DQ and two "new" alleles.

An extensive family study on HLA-DPB1 was performed in 105 families living in northeastern Japan. In a linkage study between HLA-DPB1 and other HLA loci, five apparent recombinations between DPB1 and DR/DQ loci were observed. The recombination frequency (theta) with maximum probability was estimated to be 0.017 by the lod score method. DPB1 allele and haplotype frequencies in unrelated parents were determined by direct counting. The most common allele was DPB1*0501 with the frequency of 41.2% and the second was DPB1*0201 with 24.0%. Nine DPB1-DR and six DPB1-DQ haplotypes were in significant linkage disequilibrium. Seven kinds of extended haplotypes were observed to be over 1%, in which the most common haplotype A24-B52-DR15-DQ6-DPB1*0901 occurred at 6.0%. Moreover, we found two "new" DPB1 alleles in this study. The first one possesses a single base substitution from DPB1*0501 resulting in an amino acid change. The other is most likely to be formed by an intraexonic recombination between DPB1*0301 and DPB1*0501.

3,5-Dihydroxyphenyl-glycine: a potent agonist of metabotropic glutamate receptors.

An amino acid, 3,5-dihydroxyphenylglycine (DHPG) induced current responses in Xenopus oocytes expressing a metabotropic glutamate receptor clone mGluR1. Apparent EC50 of DHPG for mGluR1 was slightly lower than that of (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD). DHPG responses were partially inhibited by 2-amino-3-phosphonopropionic acid (AP-3). DHPG had no effect on ionotropic glutamate receptors whose expression was induced in the oocytes following injection of poly(A)+ mRNA of rat brains. In hippocampal slices, DHPG produced slow excitation of pyramidal cells, resulting from a depression of Ca(2+)-dependent K+ current and a voltage-dependent K+ current. These results indicate that DHPG is a specific and potent agonist of mGluRs.

Detection and quantitation of HBV DNA by semi-nested PCR in donated blood: comparison with HBV serological markers.

To detect and quantitate hepatitis B virus (HBV) DNA, semi-nested polymerase chain reaction (PCR) method was designed for amplifying the HBV core region DNA. Cloned HBV core region DNA was used as a quantitation control, and upon electrophoresis of the semi-nested PCR product, one, two, or three bands of amplified DNA were observed using a small (< 50 mol), moderate (around 200 mol), or large (> or = 1250 mol) quantity of the template DNA, respectively. Using this semi-nested PCR method, HBV DNA was quantitated in donated blood and tested for HBV serological markers. Most of the HBV surface antigen (HBsAg) high titer samples showed three bands on the electrophoresis, indicating a high level of HBV DNA, while most of the HBsAg low titer samples showed one band, indicating a low level of HBV DNA. HBV DNA was detected in 7 out of 36 HBsAg-undetectable and anti-HBc-positive samples (19.4%) but all 7 showed one band, indicating a low level of HBV DNA. In almost all of the HBV e antigen-positive samples the HBsAg titer was high, and three bands were observed indicating a high level of HBV DNA.

A lectin from mycelia of the fungus Ganoderma lucidum.

A lectin (GLL-M) was isolated from mycelia of Ganoderma lucidum using affinity chromatography on BSM-Toyopearl. GLL-M is a monomer in its native form with a M(r) of 18,000. Another lectin was also purified from fruiting bodies of the same fungus. The two lectins were partially compared with each other.

Videokeratography for quantitative surface analysis of used soft contact lenses.

The greater utilization of soft contact lenses calls for the development of new and objective methods of evaluating the optical performance of the different lens types, in situ. We used videokeratography to examine the surface topography of soft contact lenses that had been worn for more than 1 year on 27 eyes of 23 patients, and compared the resulting color-coded maps and topographic indices as well as contact lens-corrected visual acuities, with those obtained with new replacement lenses on the same eyes. Visual inspection of the color-coded maps revealed differences in the surface characteristics of the used and new lenses. Comparison of quantitative indices including the SAI (Surface Asymmetry Index), IAI (Irregular Astigmatism Index), SRI (Surface Regularity Index), and SDP (Standard Deviation of Powers) showed that asymmetry, surface irregularity, potential visual acuity (derived from the SRI), and corneal power distribution were significantly poorer with the used lenses than with the new (P = 0.0001). The videokeratoscope is useful for evaluating the soft contact lens surface in situ, as an indicator of optical quality; it also permits the objective evaluation of lens cleaning techniques.

Effects of contact lens wear on mitosis of corneal epithelium and lactate content in aqueous humor of rabbit.

Various types of contact lenses, i.e., poly-methyl-methacrylate hard lenses, poly-2-hydroxyethyl-methacrylate soft lenses and two kinds of gas-permeable hard lenses with different permeabilities to oxygen, were placed on one eye of the albino rabbit for varying lengths of time. Using the fellow eye as the control, studies were carried out of the effects of the lens wear on the mitosis in the corneal epithelium and the lactate concentration in the aqueous humor. The contact lens wear suppressed the mitosis and increased the concentration of aqueous lactate, the degree of changes being greater as the permeability of the lens to oxygen decreased and the period of wear became longer.

[Surgical emergency induced by chest tumors].

Mechanisms and therapeutic procedures for emergencies induced by chest tumors are reviewed. 1) Carcinomatous pleuritis is the most frequently encountered situation for clinicians. At first, pleural effusion should be punctured for diagnosis and chest drainage should be performed consequently. Intrathoracic chemotherapy and pleurodasis (OK-432 is usually used) are then used if necessary. 2) Airway stenosis is the most critical state. Conventionally, violent incubation throughout the stenotic portion of the airway has been used with poor results. Recently, however, irradiation of Nd:YAG laser can be utilized, and the prognosis of the airway stenosis is much improved. Another effective procedure is the insertion of several kinds of airway stent. Silicon stents like T-tube, Dumon tube and Dynamic stent or metallic stents like EMS (Expandable Metallic Stent) are generally used. The advantages of EMS are easy installation and little disturbance of sputum excretion. The disadvantages of EMS are difficult removal and re-stenosis of the airway. On the other hand, silicon tubes are easy to be removed, re-insertion is possible and re-stenosis is rare, but the insertion should be performed under general anesthesia and sputum excretion may pose problems. A case of laser irradiation and two cases of stent insertion are presented. 3) Airway bleeding is also emergent. Bronchofiberscopic ethanol injection (BEI) is effective against continuous bleeding of the central airway. We performed BEI for 33 lung cancer cases, and the method was effective for all cases. 4) Cardiac tamponade, SVC syndrome, esophago-bronchial fistula, bronchial stump fistula are also important emergencies induced by chest tumors. Standard therapeutic procedures are explained for all oncology surgeons and physicians.

Cervicofacial subcutaneous emphysema associated with dental laser treatment.

Cervicofacial subcutaneous emphysema is a rare complication of dental procedures. Although most cases of emphysema occur incidentally with the use of a high-speed air turbine handpiece, there have been some reports over the past decade of cases caused by dental laser treatment. Emphysema as a complication caused by the air cooling spray of a dental laser is not well known, even though dental lasers utilize compressed air just as air turbines and syringes do. In this study, we comprehensively reviewed cases of emphysema attributed to dental laser treatment that appeared in the literature between January 2001 and September 2012, and we included three such cases referred to us. Among 13 cases identified in total, nine had cervicofacial subcutaneous and mediastinal emphysema. Compared with past reviews, the incidence of mediastinal emphysema caused by dental laser treatment was higher than emphysema caused by dental procedure without dental laser use. Eight patients underwent CO2 laser treatment and two underwent Er:YAG laser treatment. Nine patients had emphysema following laser irradiation for soft tissue incision. Dentists and oral surgeons should be cognizant of the potential risk for iatrogenic emphysema caused by the air cooling spray during dental laser treatment and ensure proper usage of lasers.