Influence of thermal stimulation during the late phase of incubation on hatching results and post-hatch broiler performance under commercial conditions.

Two experiments, which differed in breeder age, strain and season, were conducted to study the influence of low-intensity, short-duration thermal stimuli during the late phase of incubation on hatchability and performance. The first experiment conducted in April-June used eggs from Cobb × Ross broiler breeders at 35-41 weeks of age and the second experiment performed in February-April used eggs from Hubbard × Cobb broiler breeders at 49-53 weeks of age. Eggs in the test group had the same physical environment as eggs in the control group except that incubation temperature was increased by 1˚C for 2 h/d above the control group from 18 to 20 d of incubation (DI). The results demonstrated that thermal stimulation of 1˚C for 2 h/d above control incubation temperature during 18-21DI did not have any adverse effects on hatch and post-hatch performance of broilers. In both experiments, treatment did not significantly alter the secondary sex ratio in hatched chickens, but hatch residue showed that the proportion of unhatched male embryos was significantly lower in the test groups than in the control groups. In the first experiment, thermal stimulation improved feed conversion by 1.82% compared with the control.

IN VITRO CHEMOTHERAPY PROFILING OF WELL-DIFFERENTIATED MIDGUT NEUROENDOCRINE TUMORS (NETS) BASED ON INDIVIDUAL PATIENT TUMOR BIOMARKERS ANALYSIS.

BACKGROUND: Midgut neuroendocrine tumors (NETs) are rare malignancies with indolent clinical courses. In general, they are well-differentiated with most tumor cells in the G0 phase of the cell cycle, consistent with the poor response rate of NETs to chemotherapy in vivo. We hypothesize that insults, such as surgery, can drive NET cells from G0 into S phase and that biomarker analysis of individual patient tumors harvested and grown in the lab will provide useful practical guide for future intra and post-operative adjuvant therapy. METHODS: 97 well-differentiated midgut NET patients underwent cytoreductive surgery at our institution between May/2012 and October/2012. 148 surgical specimens were collected and submitted to a single commercial lab for processing. Primary tumors, lymph nodes and liver metastases were harvested and cultured. Their ribonucleic acids (RNA) were then extracted to analyze the expressivity, a total of 88 different biomarkers. Based on our patients' specific tumor biomarker expressivity and known correlations between 36 anti-neoplastic agents with their linked biomarkers, recommendations were reported as clinically beneficial or non-beneficial. RESULTS: A total of 148 specimens from 97 patients were tested. In four of the 97 patients, no clinically beneficial chemotherapy agent could be identified. Among the remaining 93 patients, the top three agents that are most likely to be clinically beneficial are: fluorouracil, cisplatin and carboplatin. These were reported to be clinically beneficial in 135/148 (91.2%), 103/148 (69.6%), and 103/148 (69.6%) patients respectively. CONCLUSIONS: Midgut NETs are slow growing tumors which are chemotherapeutically inert owing to the fact that most of the tumor cells are in G0 cell cycle. Surgical insult drives NET cells into active synthetic phase where they begin to express biomarkers specific to their tumor cells. Analysis of these biomarkers guides further potential beneficial therapy based on the current known associations among biomarkers and chemotherapy agents. These results must then be compared and confirmed against a direct in-vitro chemo sensitivity assessment conducted simultaneously on the same patients.

Differences in the antibiotic susceptibility of human Escherichia coli with poultry-associated and non-poultry-associated extended-spectrum beta-lactamases.

The concurrent presence of bla CTX-M-1 and bla TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla CTX-M-1 and bla TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla CTX-M-1 or bla TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p = 0.0056), gentamicin (86 % vs. 63 %; p = .0.0082), tobramycin (91 % vs. 34 %; p = 0.0001) and amikacin (98 % vs. 67 %; p = 0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.

Decontamination of the digestive tract and oropharynx in ICU patients.

BACKGROUND: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) are infection-prevention measures used in the treatment of some patients in intensive care, but reported effects on patient outcome are conflicting. METHODS: We evaluated the effectiveness of SDD and SOD in a crossover study using cluster randomization in 13 intensive care units (ICUs), all in The Netherlands. Patients with an expected duration of intubation of more than 48 hours or an expected ICU stay of more than 72 hours were eligible. In each ICU, three regimens (SDD, SOD, and standard care) were applied in random order over the course of 6 months. Mortality at day 28 was the primary end point. SDD consisted of 4 days of intravenous cefotaxime and topical application of tobramycin, colistin, and amphotericin B in the oropharynx and stomach. SOD consisted of oropharyngeal application only of the same antibiotics. Monthly point-prevalence studies were performed to analyze antibiotic resistance. RESULTS: A total of 5939 patients were enrolled in the study, with 1990 assigned to standard care, 1904 to SOD, and 2045 to SDD; crude mortality in the groups at day 28 was 27.5%, 26.6%, and 26.9%, respectively. In a random-effects logistic-regression model with age, sex, Acute Physiology and Chronic Health Evaluation (APACHE II) score, intubation status, and medical specialty used as covariates, odds ratios for death at day 28 in the SOD and SDD groups, as compared with the standard-care group, were 0.86 (95% confidence interval [CI], 0.74 to 0.99) and 0.83 (95% CI, 0.72 to 0.97), respectively. CONCLUSIONS: In an ICU population in which the mortality rate associated with standard care was 27.5% at day 28, the rate was reduced by an estimated 3.5 percentage points with SDD and by 2.9 percentage points with SOD. (Controlled Clinical Trials number, ISRCTN35176830.)

Evaluation of the DiversiLab system for detection of hospital outbreaks of infections by different bacterial species.

Many bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMérieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter (n = 26) and Stenotrophomonas maltophilia (n = 13) isolates. With two exceptions, DL typing of Klebsiella isolates (n = 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter (n = 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n = 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli (n = 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa (n = 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonas isolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureus generally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spa typing, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and esp gene typing was poor. All of the results are reflected in Simpson's index of diversity and adjusted Rand's and Wallace's coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp., S. maltophilia, the Enterobacter cloacae complex, Klebsiella spp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.

Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.

OBJECTIVES: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

The demographic profile of sero-discordant couples enrolled in clinical research in Rwanda and Zambia.

This paper examines the demographic profile of two cohorts of sero-discordant couples enrolled in research activities at two clinical research sites in Kigali, Rwanda and Lusaka, Zambia and compares their background characteristics by country, gender and sero-status. Differences between the two cohorts represent economic and cultural differences between the two countries. Recruitment procedures appear to be successful in reaching the intended audience - couples from poor urban communities - and we suggest that similar recruitment strategies could be adopted to reach other population groups in other settings. The profiles of sero-discordant couples highlight several potential intervention points, and call for attention to be focused towards prevention efforts aimed at young women and their male partners.

Dynamics of colistin and tobramycin resistance among Enterobacter cloacae during prolonged use of selective decontamination of the digestive tract.

Background: A high prevalence of colistin resistance among E. cloacae isolates in two intensive care units (ICU) (of 16 and 6 beds) using selective digestive decontamination (SDD) since 1990 instigated a retrospective and prospective investigation to quantify the role of clonal transmission. SDD is topical application of colistin and tobramycin and systemic use of cefotaxime during the first days of ICU-admission. Methods: Multi-resistant E. cloacae (MREb) was defined as ESBL production and/or tobramycin non-susceptibility and/or colistin non-susceptibility. Incidence of acquisition and prevalence of carriage with MREb was determined from microbiological culture results. Results: Colistin-resistant E. cloacae was first detected in November 2009 and carriage was demonstrated in 141 patients until October 2014. Mean incidence of MREb acquisition was 4.61 and 1.86 per 1000 days at risk in ICUs 1 and 2, respectively, and the mean monthly prevalence of MREb in both ICUs was 7.0 and 3.1%, respectively, without a discernible trend in time. Conversion rates from carriage of colistin-susceptible to resistant E. cloacae were 0.20 and 0.13 per 1000 patient days, respectively. Whole genome sequencing of 149 isolates revealed eight clusters, with the number of SNPs of the largest two clusters ranging between 0 and 116 for cluster 1 (n = 49 isolates), and 0 and 27 for cluster 2 (n = 36 isolates), among isolates derived between 2009 and 2014. Conclusions: This study demonstrates a stable low-level endemicity of MREb in two Dutch ICUs with prolonged use of SDD, which was characterized by the persistent presence of two clusters, suggesting incidental clonal transmission.

Quantifying within-household transmission of extended-spectrum ß-lactamase-producing bacteria.

OBJECTIVES: Patients can acquire extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae during hospitalization, and colonized patients may transmit these bacteria after discharge, most likely to household contacts. In this study, ESBL transmission was quantified in households. METHODS: Faecal samples were longitudinally collected from hospitalized patients colonized with ESBL-producing bacteria and from their household members during hospitalization of the index patient and at 3, 6, 12 and 18 months. A mathematical household model was developed, which allowed for person-to-person transmission, acquisition from other sources (background transmission), and losing carriage. Next, a deterministic population model with a household structure was created, informed by parameter values found in the household model. RESULTS: In all, 74 index patients and 84 household members were included. In more than half of the household members ESBL-producing bacteria were demonstrated at some time during follow up. Person-to-person transmission occurred at a rate of 0.0053/colonized person/day (0.0025-0.011), background transmission at 0.00015/day (95% CI 0.00002-0.00039), and decolonization at 0.0026/day (0.0016-0.0040) for index patients and 0.0090/day (0.0046-0.018) for household members. The estimated probability of transmission from an index patient to a household contact was 67% and 37% vice versa. CONCLUSION: There is frequent transmission of ESBL-producing bacteria in households, which may contribute to the observed endemicity of ESBL carriage in the Netherlands. However, the population model suggests that there is not a single dominant acquisition route in the community.

Effects of packaging atmospheres on shelf-life quality of ground ostrich meat.

Fresh ground ostrich meat was packaged under high oxygen (O2), high nitrogen (N2), vacuum (VAC) and ambient air (AIR) atmospheres, stored at 4±1°C and displayed under 1700±100lux of fluorescent lighting for 9 days. The meat was evaluated for changes in typical shelf-life characteristics consisting of pH, color properties (CIE L(∗), a(∗), b(∗), and total color difference, ΔE), oxidative changes (thiobarbituric acid value and hexanal content) and bacterial counts (total viable cell, coliform, lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp.) Initial meat pH was 6.16 and declined slightly during storage. TBA values and hexanal content were highest in O2 and lowest (P⩽0.05) in VAC and N2 atmospheres. Surface lightness (L(∗)) and redness (a(∗)) were highest in O2 packaging initially, decreasing (P⩽0.05) by day 9. ΔE of the ground ostrich increased during storage in only O(2) and AIR packaging. All packaging methods had generally similar effects on microbial outgrowth. Total aerobic bacteria attained >10(6) CFU/g meat between day 3 and day 6. Ground ostrich meat was below saleable quality in less than 6 days based on all of the meat attributes. For O2 packaging however, quality based on lipid oxidation and color properties indicated a shelf-life of less than 3 days. Oxidation is likely the limiting factor for shelf-life of ground ostrich meat.

[Optimalisation of the antibiotic policy in The Netherlands. XI. The national electronic antibiotic guide'SWAB-ID' for use in hospitals]. / Optimaliseren van het antibioticabeleid in Nederland. XI. Het nationale elektronische antibioticaboekje SWAB-ID voor ziekenhuizen.

The 'Stichting Werkgroep Antibioticabeleid' (Dutch Working Party on Antibiotic Policy) has developed an electronic national antibiotic guide for the antibiotic treatment and prophylaxis of common infectious diseases in hospitals. This guide also contains information on the most important characteristics of antimicrobial drugs. Advice on antibiotic treatment is based on existing national evidence-based guidelines, where available. Where no guideline is available, the advice is based on an inventory of the antibiotic policies of the 12 Dutch centres with an infectious disease or medical microbiology training programme. The national antibiotic guide can be accessed through the SWAB website (www.swab.nl) and can also be downloaded on PDA/PocketPC, free of charge. Every hospital antibiotic formulary committee in the Netherlands will be offered the opportunity to edit The national version for local use.

Predicting carriage with extended-spectrum beta-lactamase-producing bacteria at hospital admission: a cross-sectional study.

The prevalence of patients colonized with extended-spectrum beta-lactamase (ESBL)-producing bacteria increases, especially in long-term-care facilities (LTCFs). Identification of ESBL carriers at hospital admission is relevant for infection control measures and antibiotic therapy for nosocomial infections. We aimed to develop a prediction rule for ESBL carriage at hospital admission for patients admitted from home and LTCFs, and to quantify incidences of nosocomial infections caused by ESBL-producing bacteria. The ESBL-carrier status was determined of patients admitted from LTCFs and from home settings in four hospitals in the Netherlands using perianal swabs obtained within 48 hours of admission. Risk factors for ESBL carriage were assessed. Infections caused by ESBL-producing bacteria were identified retrospectively. Among 1351 patients, 111 (8.2%) were ESBL carriers at admission: 50/579 (8.6%) admitted from LTCFs and 61/772 (7.9%) from home settings (p 0.63). Previous ESBL carriage and previous hospital admission were risk factors for ESBL carriage in multivariable analysis. The area under the curve of the receiver operating characteristic curve of the model was 0.64 (95% CI 0.58-0.71). Presence of ≥1 risk factor (n = 803; 59%) had sensitivity of 72%. Incidences of nosocomial infections caused by ESBL-producing bacteria were 45.5/10,000 and 2.1/10,000 admission days for ESBL carriers and non-carriers, respectively (p <0.05). In conclusion, prevalence of ESBL carriage at hospital admission was 8.2%, and was comparable among patients admitted from LTCF and home. A clinically useful prediction rule for ESBL carriage at admission could not be developed. The absolute incidence of nosocomial infections by ESBL-producing bacteria was low, but higher among patients carrying ESBL-producing bacteria at the time of hospital admission.

The influence of infusion rate on the hemodynamic effects of felodipine.

The hemodynamic effects of the calcium entry blocker felodipine were studied during and after different infusion rates. Eight healthy normotensive volunteers had their individual pharmacokinetics of felodipine determined, and they subsequently entered a double-blind, randomized, crossover study. Individualized infusions of felodipine were given by a computerized infusion pump to reach plasma concentrations of 6 ng/ml (15.6 nmol/L) after 20 minutes, to be sustained for 8 hours (fast infusion) or the same plasma concentration after 8 hours (slow infusion). Control infusions with saline and vehicle were given. Blood pressure, heart rate, ECG conduction times, and baroreceptor sensitivity by the Valsalva test were measured, as well as the plasma concentrations of felodipine. The infusion system used produced the expected plasma concentration-time profiles with higher plasma concentrations after the fast infusion until 8 hours. Both slow and fast infusion increased heart rate (p less than 0.05) and produced a similar decrease in diastolic blood pressure (p less than 0.05). Slow infusion therefore reduced blood pressure more effectively. The tachycardia after the fast infusion was more pronounced during the first hour of the infusion but was indistinguishable from the slow infusion later, when plasma concentrations were still significantly different. Baroreceptor responsiveness was diminished by both felodipine treatments. There was no obvious difference in side effects caused by the two infusion regimes. The initial tachycardia after felodipine can be diminished by a slow rate of administration of the drug with a similar effect on blood pressure.

The effect of ethylene on MAPKinase-like activity in Arabidopsis thaliana.

Protein kinase activity was studied in cytosolic extracts from leaves of wild type Arabidopsis thaliana, the ethylene-insensitive mutant, etr1, and the constitutive triple-response mutant, ctr1. Treatment of wild type with ethylene resulted in increased myelin basic protein (MBP) phosphorylation. In etr1, constitutive protein kinase activity was lower than in wild type, but in ctr1, activity was enhanced. A protein of M(r) approximately 47 kDa associated with MBP-phosphorylating activity was detected using in gel protein kinase assays and phosphorylation of this protein was promoted by ethylene treatment in wild type while activity in the mutants reflected that of MBP phosphorylation. Both MAPKinase (ERK 1) and phosphotyrosine antibodies immunoprecipitated MBP-phosphorylating activity and detected a polypeptide band at M(r) approximately 47 kDa. Immunoprecipitated MBP-phosphorylating activity was again much lower in etr1 compared to wild type but much higher in ctr1. Antibodies to phosphorylated MAPKinase recognised proteins at approximately 47 kDa and the signal was upregulated in response to ethylene. The data obtained suggest that the detected protein(s) is a MAPKinase and provide further evidence confirming that a MAPKinase cascade(s) is involved in ethylene signal transduction.

Healthy human T-cell receptor beta-chain repertoire. Quantitative analysis and evidence for J beta-related effects on CDR3 structure and diversity.

Analysis of TCR repertoire usage and clonality is of potential value in understanding the pathogenesis of a number of human immune-mediated diseases. In diseases that are likely to be dependent on antigen-driven T cells, it has been suggested that particular TCR junctional region or CDR3 sequences may be critical. Rigorous methods for TCR analysis which are both quantitative and qualitative are therefore required. Of those commonly available, only anchor and inverse PCR are capable of giving high-quality information on V, D, N, and J region usage, but it has not been established whether both methods are quantitatively or analytically equivalent. We show here that both methods detected considerable variability in the usage of V beta and J beta segments in the peripheral blood repertoire of a normal individual. No preferential V-J pairing could be demonstrated. An excess usage of J beta 2 family members was indicated by both methods, although the relative usage of different J beta 2 families differed between the two techniques. The predominantly used V beta usage showed that for some families, estimates of their frequency in the repertoire differed significantly between the anchor and inverse libraries. When sampling relatively few clones the variation between V beta families estimated using the two methods can be considerable. This is likely to be a result of sampling error from a large gene family. Large-scale screening of several thousand clones is recommended to confirm the absolute values obtained from sequencing. Variation in CDR3 length appeared to be normally distributed, suggesting that a statistically optimal junctional region length may have been selected for contact with antigen. CDR3 length distribution differs significantly between receptors, which have rearranged to J beta 1 versus J beta 2 families, with the J beta 2-associated CDR3 on average between 0.5 and 1.2 of an amino acid longer. Thus the TCR beta junctional region repertoire of humans is subject to structural constraints associated with J beta usage. It will be important to establish whether variation in CDR3 length and J beta usage exists between subsets of human T cells in order to interpret TCR repertoire data from disease and control tissues.

A family study confirms that the HLA-DP associations with celiac disease are the result of an extended HLA-DR3 haplotype.

Genotyping for HLA-DR, -DQ, and -DP antigens by restriction fragment length polymorphism (RFLP) analysis along with identification of restriction fragments associated with celiac disease (CD) were undertaken in 13 families in which more than one member had CD. Major histocompatibility complex class II haplotypes for the family members were constructed which included both genotypes and RFLP markers. In 12 of the families all the affected members shared an HLA haplotype which included HLA-DR3a, DQw2 and a BglII 4.0-kb DQA fragment. Eight of these 12 haplotypes also included HLA-DPw1 and both a RsaI 4.0-kb DPB fragment and an XbaI 16.0-kb DPA fragment. In one family, the two affected members shared an HLA-DR7, DQw2 haplotype, although both their second haplotypes included HLA-DR3a and -DQw2. The results suggest that HLA-DP genes do not play an independent predisposing role in the etiology of CD but do mark a disease-associated extended haplotype. This haplotype contains genes coding for specific HLA products which may be necessary for the disease to develop. The findings support the hypothesis that the presence of a specific DQ alpha/DQ beta heterodimer, encoded in a cis arrangement on HLA-DR3a haplotypes, predisposes to celiac disease.

Celiac disease among Ashkenazi Jews from Israel. A study of the HLA class II alleles and their associations with disease susceptibility.

The immunogenetics of celiac disease demonstrates a highly significant association with the HLA class II alleles DQA1*0501 DQB1*0201 encoded in either a cis- or trans-configuration. In Northern Europe, these alleles are found in linkage disequilibrium with DRB1*0301 while in Southern Europe an additional secondary association through linkage disequilibrium is seen with the combination DRB1*1101/0701. This study examines 34 Ashkenazi Jews with celiac disease and 36 ethnically matched controls to determine alleles at the DRB, DQA1, DQB1, and DPB1 loci using SSO probes in conjunction with gene amplification by the PCR. The results confirm a highly significant association with the DQA1*0501 DQB1*0201 allelic combination (71% celiac vs 8% control individuals; p = 0.00005; chi 2 = 21.4). Of celiac subjects, 29% were negative for the proposed DQ susceptibility alleles, the majority of whom were DRB1*0402 positive (20% overall celiac group). No additional susceptibility was associated at the DRB3 and DPB loci. This study confirms that the MHC-linked celiac disease susceptibility among Ashkenazi Jews is closely associated with the presence of the combination of alleles DQA1*0501 DQB1*0201. However, within this population of relatively high-prevalence celiac disease, 30% of celiac patients do not carry these alleles and are therefore not covered by a single "unifying" hypothesis.

HLA-DQ2 second-domain polymorphisms may explain increased trans-associated risk in celiac disease and dermatitis herpetiformis.

Sequence polymorphism in the DQ2 beta chain was investigated in 80 Caucasoid patients with CD, 23 patients with DH, and 64 healthy controls. A set of amplification primers were designed to amplify a 281-bp region between amino acids 95 and 135 encoding the second domain of the DQ beta chain gene. A polymorphism at amino acid 135 was shown to distinguish DR3 and DR7 haplotypes. Two SSO probes were designed to identify amino acid sequences (133-135) RND (DR3-DQ2) and RNG (DR7-DQ2). To establish whether polymorphism existed elsewhere in the second-domain sequence, which could explain the migratory characteristics of the CD-associated DR3-DQ2 beta-chain reported by Roep et al. DQB1 second-domain PCR products were sequenced from the genomic DNA of three CD patients. The results showed that the polymorphism at amino acid 135 distinguishing DR3 and DR7 haplotypes was present in CD, DH patients, or normal controls of the appropriate DR and DQ genotypes by oligonucleotide hybridization. Cloning and sequencing of DQB1 second domains of three CD patients (two DR3,3 and one DR3,7) gave normal sequences expected from their genotypes. No specific polymorphism of DQB1 second domains on CD-associated DR3 haplotypes distinguishes them from normal DR3 haplotypes. We conclude that individuals positive for the DR3,7 genotype have the potential to express a unique trans-encoded heterodimer with enhanced ability to predispose people to CD.

Sequence analysis of HLA-DR4B1 subtypes: additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing.

The stimulating capacity of HLA-DR4 variants in mixed leukocyte culture correlates with variation in the polymorphic regions of the first domains of their DR beta 1 chains. Variability between amino acids 67 and 86 appears largely to determine HLA-DR4,Dw type. We have used a combination of a DR4B1-specific primer set in the polymerase chain reaction and specific oligonucleotide probes to examine DR4,Dw-associated nucleotide polymorphisms. Phenotype and gene frequencies are reported among 44 normal DR4 Caucasoids. Oligonucleotide probes were selected which enabled definition of Dw4-, w14-, w10-, w13-, and w15-associated sequences. Unexpectedly, several subjects were positive for Dw15 sequences which are usually characteristic of Oriental populations. Dw15 assignment was confirmed by nucleotide sequencing of DR4B1 polymerase chain reaction products. A pair of oligonucleotides informative for the glycine or valine dimorphism at position 86 was used to identify two novel DR4B1 alleles, designated 13.2 and 14.2. Nucleotide sequencing shows that these represent recombinants between third hypervariable regions associated with Dw13 and Dw14 and a codon for glycine at position 86 which is usually found associated with Dw4 and Dw15 sequences.

Celiac disease is associated with an extended HLA-DR3 haplotype which includes HLA-DPw1.

HLA-DP polymorphism was examined among celiac disease patients and controls. Restriction fragment length polymorphism (RFLP) genotyping showed a significant association of DPw1 in celiac disease (32/80) compared with controls (6/53, p = 0.0002). DPw1 typing by RFLP was verified using DPB1-amplified DNA and an oligonucleotide probe specific for the DPw1-associated DPB1 gene. The RFLP-assigned DPw1 genotype corresponded closely to the binding pattern of the sequence-specific oligonucleotide probe, although discrepancies did occur. The association between celiac disease and DPw1, however, remained. Oligonucleotide probe specificity was confirmed by sequencing DPB1-amplified DNA from four DPw1-genotyped celiacs. DPw1 is only present in celiacs who genotype DR3a-positive. Of DR3a controls 24% are DPw1-positive compared with 5% of non-DR3a controls (p = 0.03), suggesting that an extended DR3a, DPw1 haplotype occurs in the control population. This haplotype forms a large proportion of the DR3a haplotypes predisposing to celiac disease. Alternatively, DPw1 may represent an independent risk factor inherited in linkage with HLA-DR3 and -DQw2. Although predisposition to celiac disease is likely to be mediated by a specific DQ alpha/DQ beta heterodimer, a direct role for the DPw1 antigen cannot be discounted.