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.     

Induction of HLA class II in HTLV-I infected neuronal cell lines

Prenatal methylazoxymethanol acetate (MAMac) injection disrupts cell migration in developing rats. We investigated the electrophysiological characteristics of hippocampal CA1 pyramidal neurons from young MAMac-treated animals (postnatal days 25-35). In vitro intracellular recordings from CA1 cells in MAMac-treated tissue revealed resting membrane potential (mean, -61.5 +/- 1.5 mV), action potential amplitude (mean, 69 +/- 3.1 mV), action potential duration (mean, 2.1 +/- 0.2 ms), input resistance (mean, 51.5 +/- 3.6 M omega) and time constant (mean, 33.2 +/- 1.2 ms) similar to those of CA1 cells from control tissue. However, MAMac-treated tissue could be distinguished as having a higher percentage of cells (62% vs. 10%) which fire a burst of action potentials in response to suprathreshold current injection. The synaptic responses of CA1 cells in MAMac-treated and control tissue were comparable. The CA1 field response to stimulation was also comparable at all stimulus intensities tested (50-1500 microA). Elevation of extracellular potassium concentration ([K+]o) from 3 mM to 6 mM resulted in epileptiform discharge activity in response to stratum radiatum stimulation in all MAMac-treated slices (10/10) but in only one-third of controls (3/9). Spontaneous epileptiform discharges were also observed in the majority (8/13) of MAMac-treated slices bathed in 6 mM KCl but in no controls. These data suggest that MAMac treatment during fetal development not only disrupts normal anatomical organization but also leads to alterations in electrophysiological features of the hippocampal CA1 pyramidal cell region. As such, the MAMac model may provide insights into early onset seizure syndromes associated with developmental abnormalities.     

Genetic typing of HLA class II genes in Swedish populations: application to forensic analysis

In an attempt to determine the value of DNA based typing of HLA class II loci to forensic analysis, allele and genotype frequencies at DQA1, DQB1, DPB1, and DRB1 were determined in samples from two Swedish populations using hybridization with sequence specific oligonucleotides to PCR amplified DNA. Significant allele frequency differences were observed at the DQB1 and DRB1 loci between the two populations, as well as between one of the Swedish and a Norwegian population. The average heterozygosity varies between 0.74 to 0.91 and the power of discrimination between 0.90 to 0.98, with the highest values obtained for the DRB1 locus. The probability of genotype identity by chance differs on average 2% between the populations. When applied to a paternity case with one parent deceased and a criminal case, typing of class II loci proved in both cases informative. Analyses of DR and DQ genes does not increase the power of discrimination, due to strong linkage, but offers through the reconstruction of putative haplotypes an internal control for the consistency of the typing results at several loci. Typing of the DRB1 and DPB1 loci was found to result in an approximate combined average probability of genotype identity by chance of one in a thousand.     

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.     

HLA class II genes in soybean epidemic asthma patients

From 1981 to 1987, 26 outbreaks of asthma caused by the inhalation of soybean dust, affecting a total of 688 individuals, were detected in Barcelona, Spain. Because only a small proportion of asthmatic individuals living in Barcelona expressed the epidemic phenotype, it is hypothesized that a genetically determined human leukocyte antigen (HLA) Class II factor could have played a role in the susceptible individuals. Accordingly, we studied the distribution of both HLA-DR and HLA-DQ in soybean epidemic asthmatic patients. An analysis of the HLA-DR and HLA-DQ genes for genetic polymorphisms of the beta 1 chain was done with the polymerase chain reaction (PCR) in 78 soybean epidemic asthma patients, and the findings were compared with those for 67 nonepidemic asthmatic individuals and 168 individuals from the general population. An allelic disequilibrium could be established; the risk of epidemic asthma was particularly associated with the DRB1*13 gene (p value corrected for multiple comparisons < 0.02). The association observed for the DRB1*13 gene was stronger in individuals in the lowest tertile for total IgE, with an estimated risk with a 95% confidence interval (CI), of 14.5 (1.6 to 130.8). The combination of two genes from among the DRB1*05-05, DRB1*05-06, and DRB1*06-06 genes was present in epidemic asthmatic subjects only. No association with an HLA-DQB1 allele could be observed. Genetic predisposition could contribute to the response of some asthmatic patients to exposure to soybean dust, having led to their being affected during the epidemics of asthma in Barcelona.     

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.     

Distinct associations of HLA class II genes with inflammatory bowel disease

BACKGROUND: There are relatively few studies of HLA class II association either with Crohn's disease (CD) or ulcerative colitis (UC). The few available association studies have been carried out by serological techniques, and the results from these studies are inconclusive. METHODS: The association between HLA class II genes was studied using molecular genotyping in combination with allele-specific oligonucleotide hybridization by polymerase chain reactions. RESULTS: In UC (n = 74), we observed a positive association with the HLA DR2 allele (P = 0.008) and negative associations with the DR4 (P = 0.018) and DRw6 (P = 0.028) when compared with ethnically matched controls (n = 77). No associations were observed with any DQ alleles. In contrast, in CD (n = 95) we observed a positive association with the combination of DR1 and DQw5 alleles (P = 0.021). Furthermore, stratifying DR1 and DQw5 alleles indicated that neither allele was independently associated with CD, suggesting that the association was with the haplotype rather than either of the alleles individually. A suballele of DQw5, DQB1*0501, contributed this haplotypic association (P = 0.012). CONCLUSIONS: DR and DQ molecules firmly separate UC and CD on genetic grounds, suggesting that the contribution of the HLA class II genes to the disease susceptibility is quite different for the two disorders.     

HLA class II genes in chronic hepatitis C virus-infection and associated immunological disorders

To investigate the factors that may confer susceptibility or protection to hepatitis C virus (HCV) infection and to HCV-associated immunological disorders, we designed two studies on 420 Sardinian transfusion-dependent thalassemia patients followed in our department in Cagliari since 1974. The first one was an epidemiological survey aimed to evaluate the prevalence of HCV infection and HCV-associated immunological disorders. In the second study, the distribution of different HLA class II genes was examined by DNA analysis in 116 HCV positive patients, 30 HCV negative patients, and 606 healthy controls. Three hundred fourteen patients became infected with HCV (74.7%) after 5.6 +/- 2.8 years of regular transfusion program. Mixed cryoglobulinemia, purpura, arthritis, proteinuria, decreased complement levels, rheumatoid factor and anti-GOR, smooth muscle antibody (SMA), anti-nuclear antibody (ANA), and liver, kidney microsome (LKM) autoantibodies were significantly more represented in HCV positive patients than in negative ones (P < .05). A significant increase of HLA class II DR2 subtype (DRB1*1601,DQB1*0502) was observed in a group of 30 HCV negative patients who despite 10.3 +/- 2.2 years in a regular blood transfusion program did not show any evidence of HCV infection (Pc < .0092). Our results represent clear evidence for a relationship between HCV infection and immune extrahepatic abnormalities. A gene(s) located in the human major histocompatibility complex (MHC) region may play an important role in conferring protection against HCV infection.     

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.     

Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method

Topical application of 5-aminolevulinic acid (5-ALA), with subsequent synthesis of protoporphyrin IX (PPIX), is a novel outstanding procedure for photodynamic treatment. So far, clinical experience has been reported with creams containing 5-ALA for the therapy of skin cancer, oral application for the treatment of gastrointestinal disease and intravesical instillation of 5-ALA solutions for fluorescence detection of superficial bladder cancer. Inhalation of 5-ALA for the staining of bronchial malignancies is a preferred method in clinical pulmonology. Since no adverse reaction was observed in lung function in a canine following inhalation of 5-ALA in increasing concentrations, clinical applications were performed. Seven patients with positive or suspicious sputum cytology, but negative white light bronchoscopy, received 5-10 wt.% 5-ALA in NaCl solution by means of a medical nebulizer. No side effects were observed during and after 5-ALA inhalation. After a period of 3 h, patients underwent fluorescence bronchoscopy using violet light for fluorescence excitation and an optical multichannel analyzer for fluorescence detection in situ. The results showed fluorescence spectra which could be related to PPIX induced by 5-ALA in the bronchial mucosa. The fluorescence intensity was sufficiently high for video imaging using a target integrating color CCD camera adapted to the flexible bronchoscope. Carcinoma in situ, as well as dysplasias, showed a clear positive fluorescence. A correlation of fluorescence contrast with histology on 30 biopsies revealed a high sensitivity, but a specificity below 50%. Improvements in light and drug dosimetry will form the basis for further clinical trials.     

Efficient class II major histocompatibility complex presentation of endogenously synthesized hepatitis C virus core protein by Epstein-Barr virus-transformed B-lymphoblastoid cell lines to CD4(+) T cells

The induction of an efficient CD4(+) T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4(+) T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10(4) transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.     

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation

CD4+ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II+ but not of isogenic HLA-II- cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4+ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being "occupied" by dsDNA.     

High-resolution HLA typing for the DQB1 gene by sequence-based typing

BACKGROUND: Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation. RESULTS: TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 micrograms/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells. CONCLUSIONS: Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.     

No evidence for the influence of HLA class II in alleles in isocyanate-induced asthma

Isocyanates are one of the main causes of occupational asthma. The aim of this investigation was to study the possible genetic background of isocyanate-induced asthma under consideration of the atopy status and different lung function parameters. We investigated the human leukocyte antigen (HLA) genes DRB1,3,4,5, DQB1, and DQA1 in 55 isocyanate-exposed patients with workplace-related dyspnea (32 asthmatics, 23 nonasthmatics) and 90 nonexposed controls. In contrast to other studies, we found no significant differences for any HLA class II allele tested in our study group. Furthermore, no significant differences concerning the aspartic amino acid residue 57 of DQB1 was observed. Therefore, we are unable to confirm an involvement of a specific HLA class II allele or DQB1-Asp57 in conferring susceptibility to isocyanate asthma in our study group.     

Association of HLA class I and class II alleles with myositis in Japanese patients

OBJECTIVE: To investigate the correlation of HLA class I and class II antigens and alleles with various forms of myositis in Japanese patients. METHODS: Eighty-four Japanese patients with myositis [22 with polymyositis (PM), 46 with dermatomyositis (DM), 16 with myositis overlapping with other collagen vascular diseases] were typed serologically for HLA-A, B, C antigens. HLA-DRB1, DQA1, and DQB1 alleles were determined by polymerase chain reaction dependent DNA typing methods. Fifty-eight Japanese controls were typed serologically while HLA-DRB1, DQA1, and DQB1 allele typing was carried out in 175, 95, and 104 controls, respectively. RESULTS: HLA-B7 was higher in patients than controls [20.2 vs 6.9% in controls: p=0.02, odds ratio (OR)=3.4]. The increase of HLA-B7 was largely dependent on the increase in overlap patients (37.5%; p=0.005, OR=8.1). HLA-A24 and B52 were significantly decreased in PM as compared to DM, while CW3 was significantly increased in PM versus DM. DRB1*08 alleles were significantly increased in patients (36.9 vs 20.5% in controls; p=0.004, OR=2.3), especially in PM and DM. DQA1*0501 and DQB1*0301 were significantly decreased in patients [4.8 vs 13.7% in controls; p=0.04, OR=0.32, and 8.3 vs 20.2% in controls; p=0.02, OR=0.36, respectively]. CONCLUSION: HLA-class I and class II alleles associated with Japanese patients with myositis may be different from those associated with Caucasian patients.     

HLA class II genotyping of sarcoidosis patients in Hokkaido by PCR-RFLP

To confirm the significant association of sarcoidosis with HLA-DR5, -DR6, and -DR8 associated DRB1 alleles, in sarcoidosis patients from the eastern Japan (Kanto) area found in our previous study, we used HLA class II genotyping of patients in another region-Hokkaido, in northern Japan. The annual incidence of sarcoidosis in Hokkaido is about three times that of eastern Japan, and Hokkaido has one of the world's highest incidences of this disease. For the HLA class II (HLA-DRB1, -DRB3, -DQA1, -DQB1) genotyping, we used the polymerase chain reaction restriction fragment polymorphism (PCR-RFLP) method with 150 subjects: 40 sarcoidosis patients and 110 healthy controls. The frequencies of DRB1*12, DRB1*14, DRB1*08, DQA1*0501, and DQB1*0301 were significantly increased in the patients, compared with the controls. Our finding of a high frequency of DRB1*08 (which lacks the DRB3 gene encoding the DR52 antigen) in patients living in both eastern Japan and in Hokkaido, confirms that it is the HLA-DRB1 locus, rather than that of the HLA-DRB3, -DQA1, or -DQB1, which determines the susceptibility to sarcoidosis.     

Differential expression of DNA topoisomerase II alpha and II beta genes between small cell and non-small cell lung cancer

The purpose of this study is to describe the appearance of bowel-related abscesses on magnetic resonance (MR) images. Sixteen consecutive patients who had bowel-related abscesses underwent MR examination at 1.5T. MR sequences included T1-weighted fat-suppressed imaging pre- and post-intravenous gadolinium chelate administration (all patients) and breathing-independent single-shot T2-weighted half Fourier turbo (fast) spin echo (6 patients). Patients with pelvic abscesses also underwent sagittal imaging with post-gadolinium T1-weighted images (9 patients) and T2-weighted turbo (fast) spin echo (8 patients). Abscesses were confirmed by open surgery or surgical drainage (6 patients), percutaneous drainage (8 patients), or combined physical examination, fluoroscopic fistulogram, and clinical follow-up (2 patients). Oval-shaped fluid collections were identified in all of the patients, which ranged in diameter from 2 cm to 18 cm, mean: 8 cm. Abscesses were low to intermediate in signal on T1-weighted images, heterogenous and moderately high signal on T2-weighted images, and low signal on post-gadolinium images. A layering effect of lower signal material in the dependent portion of the abscess was noted in abscesses in 6 of 14 patients on T2-weighted images. Post-gadolinium images demonstrated a definable 3- to 7-mm thick abscess wall, which enhanced substantially with contrast. Definition of the wall was best shown on fat-suppressed images post-gadolinium. Substantial enhancement of surrounding periabscess tissues was demonstrated in all cases and was most clearly defined on fat-suppressed images. Image acquisition in two orthogonal planes was of value to demonstrate that fluid collections were oval, and separate from bowel. Image acquisition in the sagittal plane was useful in the evaluation of pelvic abscesses. The results from this preliminary study show that bowel-related abscesses are demonstrable on MR images using gadolinium-enhanced fat-suppressed T1-weighted and turbo (fast) spin-echo T2-weighted sequences. The presence of a thickened, enhancing lesion wall and enhancement of perilesional tissues on T1-weighted fat-suppressed images were observed in all abscesses. A layering effect of low signal intensity material in the dependent portion of the abscess was an important ancillary feature.