Positional cloning of the APECED gene.

Autoimmune polyglandular syndrome type I (APS 1, also called APECED) is an autosomal-recessive disorder that maps to human chromosome 21q22.3 between markers D21S49 and D21S171 by linkage studies. We have isolated a novel gene from this region, AIRE (autoimmune regulator), which encodes a protein containing motifs suggestive of a transcription factor including two zinc-finger (PHD-finger) motifs, a proline-rich region and three LXXLL motifs. Two mutations, a C-->T substitution that changes the Arg 257 (CGA) to a stop codon (TGA) and an A-->G substitution that changes the Lys 83 (AAG) to a Glu codon (GAG), were found in this novel gene in Swiss and Finnish APECED patients. The Arg257stop (R257X) is the predominant mutation in Finnish APECED patients, accounting for 10/12 alleles studied. These results indicate that this gene is responsible for the pathogenesis of APECED. The identification of the gene defective in APECED should facilitate the genetic diagnosis and potential treatment of the disease and further enhance our general understanding of the mechanisms underlying autoimmune diseases.

Preparation of ortho-para ratio controlled D2 gas for muon-catalyzed fusion.

A negative muon in hydrogen targets, e.g., D2 or D-T mixture, can catalyze nuclear fusions following a series of atomic processes involving muonic hydrogen molecular formation (muon-catalyzed fusion, muCF). The ortho-para state of D2 is a crucial parameter not only for enhancing the fusion rate but also to precisely investigate various muonic atom processes. We have developed a system for controlling and measuring the ortho-para ratio of D2 gas for muCF experiments. We successfully collected para-enriched D2 without using liquid-hydrogen coolant. Ortho-enriched D2 was also obtained by using a catalytic conversion method with a mixture of chromium oxide and alumina. The ortho-para ratio of D2 gas was measured with a compact Raman spectroscopy system. We produced large volume (5-30 l at STP), high-purity (less than ppm high-Z contaminant) D2 targets with a wide range of ortho-para ratios (ortho 20%-99%). By using the ortho-para controlled D2 in muCF experiments, we observed the dependence of muCF phenomena on the ortho-para ratio.

Morphology of the inferior border of the mandible: correct relation between the lowest position of the outer and inner contours of the cortex for a successful split.

For a sagittal split ramus osteotomy to be secure, the relation between the outer and inner contours of the cortex at the inferior border of the mandible is critical. The lowest point of the outer contour is not always immediately below that of the inner contour, and the former is placed more lingually than the latter in about a third of all cases. This tendency is much more noticeable in skeletal class I and II malocclusions than class III. It is therefore important to examine the lowest point of the inferior border in every case, and to carry the inferior part of the buccal cut on to the lingual side if necessary.

Common mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients of different origins.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM *240300, also called APS 1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is generally characterized by two of the three major clinical symptoms that may be present, Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have a number of other clinical symptoms including chronic gastritis, gonadal failure, and rarely, autoimmune thyroid disease and insulin-dependent diabetes mellitus. We and others have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes, and fetal liver and encodes a protein containing motifs suggestive of a transcriptional regulator, including two zinc finger motifs (PHD finger), a proline-rich region, and three LXXLL motifs. Six mutations, in cluding R257X, the predominant Finnish APECED allele, have been defined. R257X was also observed in non-Finnish APECED patients occurring on different chromosomal haplotypes suggesting different mutational origins. Here we present mutation analyses in an extended series of patients, mainly of Northern Italian origin. We have detected 12 polymorphisms, including one amino acid substitution, and two additional mutations, R203X and X546C, in addition to the previously described mutations, R257X, 1096-1097insCCTG, and a 13-bp deletion (1094-1106del). R257X was also the common mutation in the Northern Italian patients (10 of 18 alleles), and 1094-1106del accounted for 5 of 18 Northern Italian alleles. Both R257X and 1094-1106del were both observed in patients of four different geo-ethnic origins, and both were associated with multiple different haplotypes using closely flanking polymorphic markers showing likely multiple mutation events (six and four, respectively). The identification of common AIRE mutations in different APECED patient groups will facilitate its genetic diagnosis. In addition, the polymorphisms presented provide the tools for investigation of the involvement of AIRE in other autoimmune diseases, particularly those affecting the endocrine system.

Localization of 16 exons to a 450-kb region involved in the autoimmune polyglandular disease type I (APECED) on human chromosome 21q22.3.

As a step toward identifying the pathogenic genes for autoimmune polyglandular disease type I (APECED) and other disorders mapped to the PFKL locus on chromosome 21q22.3, we have constructed a cosmid/BAC (bacterial artificial chromosome) contig of 450 kb covering markers D21S1460-D21S25-PFKL-D21S154 and performed exon trapping. We isolated 22 distinct exons including 6 exons derived from two known genes (PFKL and EHOC-1). Among 16 novel exons, 2 exons matched with human expressed sequence tags (EST) and 7 exons showed homology at predicted amino acid sequence level with proteins from other species. These 16 exons were mapped back to the cosmid contigs, 12 of which were confirmed for their expression by polymerase chain reaction (PCR) screening of human cDNA libraries of various tissues. These exon sequences and a transcript map will aid for isolation of corresponding genes which will be identified as candidate genes involved in the pathogenesis of disorders mapped to the 21q22.3 region.

Characterization of elevated neutrophil-associated IgG in various autoimmune disorders: not anti-neutrophil autoantibodies, but possibly immune complexes, bind to neutrophils.

Neutropenia is frequently observed in a variety of autoimmune disorders. As the mechanism of neutropenia in these disorders, the destruction of neutrophils by anti-neutrophil autoantibodies has been believed since elevated levels of neutrophil-associated IgG (NAIgG) have been described. However, no data exists to characterize the nature of NAIgG and show NAIgG is an anti-neutrophil autoantibodies. We investigated whether the elevated NAIgG in these patients consists of anti-neutrophil autoantibodies. The NAIgGs of 91 patients with autoimmune disorders including 50 patients with idiopathic thrombocytopenic purpura, 13 patients with systemic lupus erythematosus, 11 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease were analyzed. The level of NAIgG was high in 36 of 91 patients. Elution studies were performed to determine whether NAIgG has a nature of autoantibodies. In model experiments, the ether eluate from neutrophils sensitized with neutrophil-specific alloantibody (anti-NA2) reacted with donor neutrophils, whereas the eluates from those with model immune complexes (ICs) failed. These data indicated that the ether elution technique is useful to determine whether NAIgG consists of anti-neutrophil autoantibodies. The NAIgG on patient's neutrophils was eluted with ether and the reactivity of the eluate with normal neutrophils was investigated. The eluates from 34 of 36 patients with various autoimmune disorders with elevated NAIgG level failed to react with donor neutrophils. These data indicated that the elevated NAIgG in the majority of these patients did not consist of anti-neutrophil autoantibodies, but possibly of ICs.

A simple solid-phase method for the identification of red cell-bound antibodies without elution.

The authors have developed a solid-phase direct hemadherence assay (SPDHA) to identify red cell-bound antibody without elution. The procedure of SPDHA is as follows: (1) commercially available panel cells were immobilized on the well of microplane; (2) 22% polymerized albumin and 0.3% test red cells were added, and the plate was centrifuged at low speed, and incubated; (3) finally the plate was centrifuged, and the results were read macroscopically. SPDHA could detect antibodies against D, C, c, E, e, Fya, Fyb, K, k, A and B antigens. The sensitivity of SPDHA was high in Rh antibodies as compared with that in the other antibodies. SPDHA failed to detect anti-Jka, -Jkb, -S, -s and -Dia antibodies. In cases of suspected hemolytic disease of newborn, Rh antibodies could be identified using very small volume of red cells. In conclusion, SPDHA is a useful and simple method to identify red cell-bound antibodies, especially when only a small volume of red cell sample is available, such as the sample from fetus or newborn.

Non-destructive elemental analysis of vertebral body trabecular bone using muonic X-rays.

Non-destructive elemental analysis with muonic X-rays was performed on human vertebral bone and lumbar torso phantoms. It can provide quantitative information on all elements in small deep-seated localized volumes. The experiment was carried out using the superconducting muon channel at TRIUMF in Vancouver, Canada and a lithium drifted germanium detector with an active area of 18.5 cm2. The muon channel produced backward-decayed negative muons with wide kinetic energy range from 0.5 to 54.2 MeV. The muon beam was collimated to a diameter of 18 mm. The number of incoming muons was about 4 x 10(6) approximately 5 x 10(7) per data point. In the measurements with human vertebral bones fixed with neutralized formaldehyde, the correlation coefficient between calcium content measured by muons and by atomic absorption analysis was 0.99 and the level of significance was 0.0003. In the measurements with lumbar torso phantoms, the correlation coefficient between calcium content measured by muons and by atomic absorption analysis was 0.99 and the level of significance was 0.02. The results suggest that elemental analysis in vertebral body trabecular bone using muonic X-rays closely correlates with measurements by atomic absorption analysis.

Functional posterior rhizotomy for severely disabled children with mixed type cerebral palsy.

The authors evaluated the impact of functional posterior rhizotomy (FPR) for children with severely disabled mixed type cerebral palsy (CP). Three quadriplegic children at the age of 3, 4, and 10 years underwent FPR. They were classified as mixed type CP based on the clinical presentation of marked spasticity with dystonic posture. Preoperative Ashworth score of the lower extremity was 3.5, 4.5, 4.8 respectively. Two children showed prominent opisthotonus and all showed severe subluxation of the hip joint. Advanced scoliosis was associated in two children. FPR was performed from L2 to S1 in one child, L2 to S2 in one and L2 to S1/S2 in one based on the result of pudendal mapping. Rootlet cutting rate ranged from 66 to 75%. Postoperatively, Ashworth score dropped to 1.4, 1.2, 1.3, respectively. Functional improvement of the upper extremity and urination were confirmed in two children. Hip subluxation was reduced in one child and remained stable in two. A one-year follow-up review confirmed no relapse of spasticity among them. FPR achieved highly satisfactory surgical effects in children with severe mixed type CP. Although long-term follow-up is mandatory since there was a report of relapsed spasticity after FPR in this particular population of CP, FPR could be a choice of surgery in severely disabled children with mixed type CP.

[Detection of anti-neutrophil antibodies by enzyme linked immunosorbent assay--comparison of results obtained by ELISA with those by leukocyte agglutination test and granulocyte cytotoxicity test].

We examined anti-neutrophil antibodies in 12 patients with various disorders using enzyme-linked immunosorbent assay (ELISA), and the results obtained by ELISA were compared with those obtained by leukocyte agglutination test (LAT) and granulocyte cytotoxicity test (GCT). IgG anti-neutrophil antibody was positive in 7 of 12 patients, and IgM type antibody was positive in 6 patients. There was a significant correlation between IgG and IgM anti-neutrophil antibodies. The results obtained by ELISA were not in accord with those obtained by LAT or GCT. In addition, we examined anti-neutrophil antibodies in serially collected serum samples from two patients with immune neutropenia. The results obtained by ELISA correlated with their neutrophil counts, suggesting that anti-neutrophil antibodies detected by ELISA have pathological relevance.

[Evaluation of IgM anti human cytomegalovirus antibody].

An anti-human cytomegalovirus (HCMV) antibody in 330 sera from patients and normal subjects was examined by ELISA. The test results were compared with the results of CF test and there was 99.1% agreement. Specific anti-HCMV IgM antibody was searched for and only 4 sera were positive. This makes it unlikely that screening for IgM anti-HCMV will effectively prevent posttransfusion HCMV infections.

[Analysis of immunoglobulin classes of five anti-phospholipid antibodies in sera from patients with SLE, ITP and recurrent fetal abortion by enzyme linked immunosorbent assay].

Using a panel of phospholipid(PL) antigens, we have established an enzyme linked immunosorbent assay (ELISA) for measuring both IgG and IgM type anti-phospholipid antibodies (APA) in sera from patients with systemic lupus erythematosus (SLE), idiopathic thrombocytopenic purpura (ITP) and recurrent fetal abortion (RFA). The percentage of anticardiolipin antibody (aCL) positive patients was increased in SLE (73% for IgG, 74% for IgM), ITP (24% for IgG, 8% for IgM) and RFA (20% for IgG, 52% for IgM) as compared with normal controls. The percentage of other APAs in each disease was significantly different from one another, suggesting the existence of disease-specific APA in these autoimmune disorders. We consider that it is important to analyze these APAs for the investigation of pathogenesis of autoimmune disorders.

[The implication of hyperglucagonemia and -insulinemia in liver cirrhotics].

Glucagon and insulin metabolism was studied in 9 cirrhotic patients and 4 non-cirrhotic controls. Net output of glucagon and insulin into portal circulation was calculated from difference between portal venous and systemic arterial concentrations multiplied by portal plasma flow. Metabolic clearance rate was also calculated as the ratio of the output to systemic concentration. Portal blood flow was measured by the continuous local thermodilution method. The results were as follows: 1) Arterial glucagon concentration was elevated in liver cirrhotics compared with non-cirrhotic-controls. Glucagon output in cirrhotics was higher than in controls [46.3 +/- 11.8 vs. 13.2 +/- 2.5 ng/min (mean +/- SEM), p less than 0.05]. Metabolic clearance rate of glucagon was not significantly different between the two groups. 2) There was a significant correlation between glucagon output and portal venous concentration of norepinephrine (r = 0.625, p less than 0.05). 3) Insulin levels in systemic arterial blood were higher in cirrhotic patients than non-cirrhotic subjects. Insulin output was not significantly different between the two groups, however, metabolic clearance rate of insulin in cirrhotics was reduced in comparison with the controls (274.5 +/- 44.3 vs. 557.7 +/- 108.6 ml/min, p less than 0.05).

Nocturnal blood glucose and IGFBP-1 changes in type 1 diabetes: Differences in the dawn phenomenon between insulin regimens.

OBJECTIVE: Insulin-like growth factor binding protein-1 (IGFBP-1) is known to regulate the bioavailability of insulin-like growth factor (IGF) and the levels of IGFBP-1 are increased in the morning in patients with type 1 diabetes mellitus. We investigated the nocturnal fluctuations of glucose, IGFBP-1, and free IGF-1 levels with three insulin regimens. RESEARCH DESIGN AND METHODS: Forty-eight type 1 diabetes patients were divided into three groups according to their basal insulin therapy (continuous subcutaneous insulin infusion [CSII], insulin glargine, NPH insulin). Blood samples were obtained every 2 h between 2 300 h and 0700 h to measure plasma glucose, IGFBP-1 and free IGF-1 levels. RESULTS: The dawn phenomenon was more frequent with NPH (62.1%) than with glargine (16.6%, p<0.05) and CSII (14.3%, p<0.05). In the NPH group, the serum IGFBP-1 levels were markedly increased from 21.0+/-3.6 ng/ml at 2 300 h to 200.3+/-21.8 ng/ml at 0700 h and free IGF-1 levels were inversely decreased; these changes were partially suppressed in the CSII and glargine groups. CONCLUSIONS: The use of insulin regimens that provide sufficient insulin levels in the early morning can suppress the dawn phenomenon, leading to improved glycemic control. The increase in circulating IGFBP-1 in the morning, as a result of waning of insulin action, lowers free IGF-1 levels and may cause insulin resistance.