High-resolution HLA typing for the DRB3/4/5 genes by sequence-based typing

The mechanisms underlying the circadian rhythm of methotrexate (MTX)-induced toxicity (body weight loss and leukopenia) were investigated from the viewpoints of the sensitivity of living organisms to the drug and the pharmacokinetics of the drug. ICR male mice were housed in a standardized light-dark cycle (lights on at 0700, off at 1900) with food and water ad libitum. The body weight loss after an intraperitoneal injection of MTX (400 mg/kg) was more serious in the late dark period and the early light period and milder in the late light period and the early dark period. The MTX-induced leukopenia was more serious in the late dark period and the light period and milder in the early dark period. Lower toxicity was observed when DNA synthesis, dihydrofolate reductase (DHFR) activity in bone marrow cells and folate level in plasma decreased, and higher toxicity was observed when they increased. There was a significant circadian rhythm in plasma MTX concentration, with a higher level in the light period and a lower level in the dark period. The circadian rhythm of plasma MTX concentration was associated with that of MTX-induced toxicity. The present study suggests that the circadian rhythm of MTX-induced toxicity is caused by that of the sensitivity of living organisms to the drug and the pharmacokinetics of the drug.     

Selection of unrelated bone marrow donors by PCR-SSP typing and subsequent nonradioactive sequence-based typing for HLA DRB1/3/4/5, DQB1, and DPB1 alleles

HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Here, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR-SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separation, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophorlabelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group-specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.     

A negative association of schizophrenia with an allele of the HLA DQB1 gene among African-Americans

The present study investigated the relationship between the time of nocturnal onset of urinary 6-sulfatoxymelatonin (aMT6s) secretion, and the timing of the steepest increase in nocturnal sleepiness ("sleep gate"), as determined by an ultrashort sleep-wake cycle test (7 min sleep, 13 min wake). Twenty-nine men (mean age 23.8 +/- 2.7 years) participated. The ultrashort sleep-wake paradigm started at 0700 hr after a night of sleep deprivation and continued for 24 hr until 0700 hr the next day. Electrophysiological recordings were carried out during the 7-min sleep trials, which were then scored conventionally for sleep stages. Urinary aMT6s was measured every 2 hr. The results showed that the timing of the sleep gate was significantly correlated with the onset of aMT6s secretion. These results are discussed in light of the possible role of melatonin in sleep-wake regulation.     

Preferential presentation of herpes simplex virus T-cell antigen by HLA DQA1*0501/DQB1*0201 in comparison to HLA DQA1*0201/DQB1*0201

The relation of childhood personality types, or configurations of personality traits, to adolescent development was examined. Three personality types were identified in an inverse factor analysis of California Child Q-Sort data on 128 Icelandic 7-year-olds: resilient, overcontrolled, and undercontrolled. Growth curve analyses demonstrated that in comparison to children of the other 2 types, children of the resilient personality type had higher levels of academic achievement and lower levels of concentration problems throughout adolescence; resilient children also developed sophisticated friendship reasoning and an internal locus of control more quickly. Children of the overcontrolled type were found to be more prone to social withdrawal and low levels of self-esteem during adolescence than children of the other 2 types. In contrast to the other 2 types, children classified as undercontrolled showed an increase in aggressive behavior in adolescence. Implications of the findings for research on personality development are discussed.     

Heteroduplexes for HLA DQB1 identity of family members and kidney donor-recipient pairs

DNA heteroduplex (HD) electrophoretic patterns of DQB1 alleles from 124 individuals (38 members from 7 families and 43 kidney donor-recipient pairs) were analyzed in reference to each individual's DQB1 diallelic types determined by the polymerase chain reaction-RFLP method. The assignment of DQB1 homozygosity and heterozygosity, based solely on HD patterns, was accurate and correlated well with the typing results. DQB1 homozygotes invariably gave HD patterns of a single band while heterozygotes gave HD patterns of multiple bands. Distinct HD patterns of 2 heterozygotes predict the presence of at least 1 different DQB1 type between the pair. However, pairs with identical HD patterns may have different subtypes, because HDs with 1 or 2 nucleotide differences may sometimes give an identical HD pattern. Because of its simplicity and reproducibility, this HD analysis protocol serves as an excellent alternative to screen for DQB1 homozygotes and mismatched tissue donor-recipient pairs. This protocol is also useful for confirming the correctness of DQB1 allelic type assignments in a clinical setting.     

A new DRB1*15 allele (DRB1*1506) identified by sequence-based typing

A new DRB1*15 allele (DRB1*1506) was detected during the studies of the 12th International Histocompatibility Workshop within the Allele and Haplotype Society #11 which studied the DR2 and DR51 antigens. The new allele was found in four unrelated Asian Indian individuals by sequence-based typing. It has a base substitution from T to C in codon 50 at a previously considered conserved position.     

Spinocerebellar ataxia and HLA linkage: risk prediction by HLA typing

To determine the possibility of genetic linkage of spinocerebellar ataxia with the histocompatibility loci, we performed HLA typing and linkage analysis on 19 members of a kindred in which spinocerebellar ataxia was segregating in an autosomal dominant inheritance pattern. The ataxia locus was located on chromosome 6 at 12-cM distance from the HLA complex with lod score of 3.15 (odds is greater than 1400:1 favoring linkage over chance findings). Thus, the presence of the ataxia gene in members of this kindred at risk can be predicted with about 90 per cent accuracy by means of HLA typing in informative matings.     

HLA DQB1*0602 is associated with cataplexy in 509 narcoleptic patients

Narcolepsy is a sleep disorder associated with HLA DR15 (DR2) and DQB1*0602. We HLA typed 509 patients enrolled in a clinical trial for the drug modafinil and analyzed the results in relation to cataplexy, a symptom of narcolepsy characterized by muscle weakness triggered by emotions. The patients were either subjects with cataplexy who had a mean sleep latency (SL) of less than 8 minutes and two or more sleep onset rapid eye movement (REM) periods (SOREMPs) during a multiple sleep latency test, or narcoleptic patients without cataplexy but with a mean SL shorter than 5 minutes and two or more SOREMPs. The respective values of DRB1*15 (DR2) and DQB1*0602 as markers for narcolepsy were first compared in different ethnic groups and in patients with and without cataplexy. DQB1*0602 was found to be a more sensitive marker for narcolepsy than DRB1*15 across all ethnic groups. DQB1*0602 frequency was strikingly higher in patients with cataplexy versus patients without cataplexy (76.1% in 421 patients versus 40.9% in 88 patients). Positivity was highest in patients with severe cataplexy (94.8%) and progressively decreased to 54.2% in patients with the mildest cataplexy. A voluntary 50-item questionnaire focusing on cataplexy was also analyzed in 212 of the 509 HLA-typed patients. Subjects with definite cataplexy as observed by an experienced clinician were more frequently HLA DQB1*0602-positive than those with doubtful cataplexy, and the manifestations of cataplexy were clinically more typical in DQB1*0602-positive patients. These results show that the HLA association is as tight as previously reported (85-95%) when cataplexy is clinically typical or severe. We also found that patients with mild, atypical, or no cataplexy have a significantly increased DQB1*0602 frequency (40-60%) in comparison with ethnically matched controls (24%). These results could be explained by increased disease heterogeneity in the noncataplexy group or by a direct effect of the HLA DQB1*0602 genotype on the clinical expression of narcolepsy.     

HLA class II DRB1, DQA1 and DQB1 polymorphisms in the Polish population from Wielkopolska

HLA DRB1, DQA1 and DQB1 alleles were determined by DNA PCR-SSO typing in a sample of 99 individuals originating from Wielkopolska (midwestern Poland). A high number of alleles (38 DRB1, 8 DQA1 and 14 DQB1) was detected at each locus, many of them presenting notable frequencies in this population. The three HLA loci are thus characterized by very high heterozygosity levels (93% for DRB1, 85% for DQA1, and 88% for DQB1), which confirms the results found for other European populations. A total of 6 DRB1-DQA1-DQB1 haplotypes are detected with an estimated frequency higher than 5%, namely, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*0701-DQA1*0201-DQB1*0201, DRB1*0101-DQA1*0101-DQB1*0501, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*03011-DQA1*0501-DQB1*0201, and DRB1*1301-DQA1*0103-DQB1*0603. A genetic distance analysis between the Polish and other world populations tested for HLA class II indicates that the Wielkopolska community is close to geographically close, rather than linguistically related populations from Europe. More generally, a good agreement between genetics and geography is found for DRB1 and DQB1 polymorphisms in Europe, suggesting that these two loci are highly informative for assessing historical relationships among humans.     

High-resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.     

DNA typing of HLA class II genes in B-lymphoblastoid cell lines homozygous for HLA

The HLA class II genotypes were determined in the B-lymphoblastoid cell lines selected for the Tenth International Histocompatibility Workshop. The HLA class II genes were determined by the PCR-SSOP method using the reagents provided by the Eleventh Histocompatibility Workshop. Additional studies have been performed for further characterization of HLA class II polymorphism on these cell lines. It is observed that several cell lines have HLA class II haplotypes with the same DRB1, DQA1 and DQB1 alleles on both haplotypes but different alleles at the other class II loci, confirming that these cell lines are not truly HLA class II-homozygous. Other cell lines carried HLA class II haplotypes which were only different at the DRB1 gene. These results suggest double recombination events or gene conversion-like events in generation of HLA DR, DQ haplotypes. These cell lines provide an important tool as references for HLA DNA typing.     

Characterization of a new DQB1 allele (DQB1*0610) which differs from DQB1*0602 at the highly polymorphic 57-codon

A prospective, randomized double-blind study was made of topical ciprofloxacillin (0.5%) compared with topical gentamicin (0.3%) in the treatment of simple chronic otitis media (COM) and diffuse external otitis (DEO). The study included 47 patients with COM and 54 patients with DEO. Success rates in the COM subgroup were 95% for ciprofloxacillin and 96% for gentamicin (p = 0.082), and in the DEO subgroup, 87% for ciprofloxacillin and 79% for gentamicin (p = 0.19). Both drugs were well tolerated and there was no significant change in audiometric measurements with either medication in either group. Therefore, ciprofloxacillin is at least as effective as gentamicin in such ear infections and has no potential ototoxic effect.     

HLA class I and class II DNA typing and the origin of Basques

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5DeltaSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5DeltaSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5DeltaSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5DeltaSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype.     

Mutational analysis of the hMLH1 gene using an automated two-dimensional DNA typing system

Directiveness and nondirectiveness are considered here as psychological phenomena and separated from the issue of giving or withholding advice. The former is a form of persuasive communication involving various combinations of deception, coercion, and threat, whereas the latter describes procedures that promote and enhance the autonomy and self-directedness of clients. Examples are given showing that professionals have considerable difficulty dealing with relatively simple, common issues arising in genetic counseling. It is suggested that many, if not most, problems involving the issue of nondirectiveness arise because of inadequacies in applying basic counseling skills. Several examples are given of nondirective counseling in situations involving direct questions and the proffering of "advice." The need to raise standards in counseling training is underscored if the field of genetic counseling is to remain nondirective.     

DNA-based HLA class II postmortem typing: evaluation of different techniques for prospective corneal allografting

BACKGROUND: Two new types of lasers, the pulsed dye laser and the Q-switched ruby laser, have shown good to excellent results in the treatment of vascular malformations and benign pigmented lesions of the skin. A new and very effective alternative to pulsed dye laser is the recently introduced Photoderm VL. This device is based on the use of a wide-band non-coherent intense pulsed light source which emits a continuous spectrum in the range of 515 nm to 1200 nm. PATIENTS AND METHODS: More than a 1000 patients with a variety of lesions of the skin were treated by these new laser systems and the Photoderm VL. The Q-switched ruby laser (wavelength 694 nm, pulse duration 25 ns) is suitable for the treatment of benign lentigines, café-au-lait macules, seborrhoic ceratosis, tattoos, and traumatic tattoos. The pulsed dye laser (585 nm, 0,3-0,45 ms) treats nevi flammei, capillary hemangiomas, telangiectasias, xanthelasma, hypertrophic scarring, and adenoma sebaceum. In addition we present the facilities of the new Photoderm VL (515 nm-1200 nm, 0,5-20 ms) for the treatment of nevi flammei, benign hemangiomatous malformations, telangiectasias, erythrosis interfollicularis colli, hypertrophic scarring, and hypertrichosis. RESULTS AND CONCLUSIONS: the Q-switched ruby laser, the pulsed dye laser, and the Photoderm VL show excellent results in the treatment of lesions of the skin, which otherwise would have been difficult to treat of untreatable. The efficiency of the laser types presented is based on the theory of selective photothermolysis. Scarring is almost never seen and hypo- or hyperpigmentation is in most cases transient.     

Improvement of HLA class I and class II PCR-SSP typing by using timed-release activity of DNA polymerase

These are hard times for medical school deans--high turnover among deans, the fiscal distress of many medical schools, the gap between what deans expect the job will be and what is required of them, the stark differences between what the job of dean is today and what it was in the past, and the threats to the academic missions of education and research. Using stories, anecdotes, and parables, the authors illustrates how these very difficulties might be an opportunity to rethink the role of deans and to re-examine the attributes and skills required of successful deans today. The ultimate goals of medical education have not changed, but the drastic nature of the changes taking place all around, and within, medical education make it more critical than ever to keep in mind what is really important. Deans must be exquisitely attuned to what is really important and they must make sure that the academic medical community never loses sight of what that is. To do that, deans must be deeply rooted personally in the enduring values and commitments that inform medicine as a profession and a vocation and in the fundamental values of medical education and scholarship; they must personify and embody these values; and they must remind us of these values and inspire us to embrace them and be guided by them. This is the sense in which deans must be "spiritual" leaders--that is, through their personal example, they must rekindle and engage the spirit of those working on behalf of the academic mission. While the need for fiscal expertise, management skills, and diplomatic and interpersonal skills in deans is widely acknowledged, the need for sensitivity to the spiritual dimensions of the work of deans has not received the attention it deserves.     

Improvement of sensitivity in HLA-DRB1 typing by semi-nested PCR-RFLP

A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10 pg of genomic DNA extracted from lymphocytes and from 0.5 microliter of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.     

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)-this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.     

Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediate early genes

The immediate early (IE) genes of human cytomegalovirus (HCMV) are expressed in lymphocytes and are known to transactivate both viral and cellular promoters. The mechanism by which IE gene products of HCMV transactivate expression of the HLA A2 gene promoter in Jurkat cells, a T-lymphocyte cell line, was investigated. Transient expression assays were performed using plasmids containing the HLA A2 promoter-regulatory region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE genes. The upregulation of the HLA A2 promoter by HCVM IE gene products was shown not to be secondary to either interferon-gamma or -alpha. Previously described MHC class I regulatory or enhancer elements such as the interferon-stimulated response element (ISRE), NF-kappa B and H2TF1 binding sequences, and the interferon consensus sequence (ICS) were not required for transactivation of the A2 promoter. Rather, the only known regulatory elements in the HLA A2 promoter necessary for both basal expression and transactivation by HCVM IE gene products are the CCAAT box and TATA box motifs. These results support a model in which HCVM IE gene products act through the minimal HLA A2 promoter elements to increase gene expression.