bluuebunny/crossref_metadata_2025 huggingface dataset:
Large sharing of T‐cell epitopes and natural ligands between HLA‐B27 subtypes (B*2702 and B*2705) associated with spondyloarthritis
Authors: Martí M. , Alvarez I. , Montserrat V. , López de Castro J.A.
Published: March 2003

Abstract: HLA‐B*2702 is an ankylosing spondylitis‐associated allotype that differs from the more common B*2705 at residues 77, 80, and 81, in the peptide‐binding site. The diversity and fine specificity of alloreactive cytolytic T‐lymphocyte (CTL) raised against B*2702 were analyzed at the clonal level. Significant crossreaction with B*2705 and B*2709 indicated that the three subtypes share numerous T‐cell epitopes. However, some epitopes shared by B*2702 and B*2705 were lost in B*2709, which correlates with weaker association of this subtype to disease. Clonal specificities were donor‐dependent, indicating that allo‐immunogenicity is variable among individuals. Anti‐B*2702 CTL were little affected by single mutations mimicking B*2702/B*2705 polymorphism, but the double mutant at positions 77 and 81 was recognized worse than B*2705, suggesting a compensatory effect of residue 80. Thus, HLA‐B27 polymorphism modulated alloreactivity through cooperative and compensatory effects on T‐cell epitope structure. Comparison of B*2705‐ and B*2702‐bound peptide repertoires revealed that they overlapped by 73% and 81%, respectively. This was larger than B*2702/B*2705 cross‐reaction, indicating that HLA‐B27 allospecificity is only partially determined by the nature of peptide repertoires. The large sharing of natural ligands and T‐cell epitopes is consistent with a pathogenetic role of B*2702 and B*2705 in spondyloarthritis based on antigen presentation.
Polymorphisms in the tumor necrosis factor (TNF) genes are associated with susceptibility to effects of ultraviolet‐B radiation on induction of contact hypersensitivity
Authors: Niizeki H. , Naruse T. , Hecker K.H. , Taylor J.R. , Kurimoto I. , Shimizu T. , Yamasaki Y. , Inoko H. , Streilein J.W.
Published: March 2003

Abstract: We investigated the allelic distributions of single nucleotide polymorphisms (SNPs) of the TNFA, TNFB and IKBL genes, 3 microsatellites within the tumor necrosis factor (TNF) region of HLA locus, and the HLA phenotypes as well as the TLR4 gene in Chromosome 9 in 26 healthy Caucasian volunteers. These individuals were also assessed as ultraviolet B (UVB)‐susceptible (S) or UVB‐resistant (R). Our results identified 12 UVB‐S and 14 UVB‐R individuals. Attempts to correlate particular HLA‐A, ‐B, ‐C, and ‐DR antigens with the UVB phenotypes failed. Similarly, attempts to correlate SNP at the NcoI‐RFLP within intron 1 of the TNFB, IKBL and TLR4 gene with UVB phenotypes also failed. However, microsatellite analyses of TNFa, TNFc, and TNFd markers revealed a significant increase in the frequencies of TNFa2 in UVB‐S individuals (P=0.00032) and of TNFd3 in UVB‐R individuals (P=0.012). Moreover, DNA sequencing analyses of 5 SNPs of the TNFA promoter region revealed a significant increase in the frequency of the allele B of the TNFA gene (TNFApB) representing the nucleotide A at position −863 and C at position −1031 (P=0.015). Since it is known that TNFa2 and TNFApB is a high TNF‐α responder, whereas TNFd3 is a TNF‐α low responder, we propose that the TNF region of HLA contains polymorphic genes that confer susceptibility and resistance to the deleterious effects of UVB radiation on the induction of contact hypersensitivity. This proposal is consistent with previous reports that a unique microsatellite region of the Tnfa gene in mice contains alleles that dictate the UVB‐dependent phenotypes in mice, and implicate TNF‐α as the primary mediator of the immune‐damaging effects of UVB radiation.
Identification of a novel HLA‐DPB1 allele, DPB1*02014, by sequence‐based typing
Authors: Curcio M. , Lapi S. , Italia S. , Rizzo G.
Published: March 2003

Abstract: In this report we describe the identification of a novel DPB1 allele, DPB1*02014, found in an Italian Caucasian individual.The new allele was detected during routine HLA sequence‐based typing (SBT) for an individual undergoing bone marrow transplantation. DPB1*02014 was identical to DPB1*02012 except for a single nucleotide substitution in codon 72 (GTG→GTT). This nucleotide change represents a synonimous mutation, as both triplets code for a valine.This new allele has been submitted to GenBank and assigned the accession number AF326565. The WHO Nomenclature Committee has officially assigned the name DPB1*02014.
Identification of two new HLA‐A alleles, A*2419 and A*3011, by sequence‐based typing
Authors: Voorter C.E.M. , Van Den Berg‐Loonen E.M.
Published: March 2003

Abstract: In this report, we describe two new HLA‐A alleles, A*2419 and A* 3011, that were initially recognized by an aberrant serological pattern. Sequence‐based typing revealed sequence differences with other known HLA‐A alleles. Allele A*2419 showed 4 nucleotide differences with A*2404, resulting in 4 amino acid differences at codons 70, 76, 77 and 90. Compared with other A*24 alleles, A*2419 lacks the Bw4 motif, as do A*2404 and A*2428. The A*3011 allele showed 2 mismatches with A*3001, resulting in one amino acid difference at codon 80.
Characterization of a novel MICA allele, MICA*047
Authors: Zhang Y. , Lázaro A.M. , Mirbaha F. , Lavingia B. , Vorhaben R. , Stastny P.
Published: March 2003

Abstract: We report the identification of a novel MICA allele, MICA*047. It was initially detected because of an unusual hybridization pattern with sequence‐specific oligonucleotides (SSOP) in a normal subject of Caucasian origin. Cloning and sequencing of both strands, and comparison of the sequence with previously defined MICA alleles, revealed that the new allele is similar to MICA*041 except for one nucleotide substitution at position 811 (C→G). It appears that this new allele could have been generated by an interallelic sequence exchange between MICA*011 and MICA*0411.
Identification of two new alleles in a single Korean individual, HLA‐B*1568 and HLA‐DRB1*1208
Authors: Sheldon M.H. , Bunce M. , Dunn P.P.J. , Day S. , Lee G.D. , Park Y‐J. , Bang B.K. , Kim B.K. , Oh E‐J.
Published: March 2003

Abstract: We have identified a new HLA‐B*15 allele and a new HLA‐DRB1*12 allele, named B*1568 and DRB1*1208, respectively. The alleles were identified using a combination of sequence specific primers, reverse line sequence specific oligonucleotide probing and sequence‐based typing. Both alleles were identified in a single individual of Korean origin. HLA‐B*1568 appears to be an HLA‐B*4801/B*1507 hybrid combining the exon 2 sequence of B*4801 and the exon 3 and 4 sequences of B*1507. Exon 2 of DRB1*1208 was most similar to DRB1*1201 or 1206, with a single mismatch at nucleotide position 165 (A to C). At the protein level, this substitution results in a phenylalanine substitution at position 26 that creates an identical amino acid sequence to DRB3*0202 between amino acid positions 17 and 36.
Evidence for extensive DLA polymorphism in different dog populations
Authors: Kennedy L.J. , Barnes A. , Happ G.M. , Quinnell R.J. , Courtenay O. , Carter S.D. , Ollier W.E.R. , Thomson W.
Published: March 2003

Abstract: Many of the genes within the Canine Major Histocompatibility Complex are highly polymorphic. Most of the alleles defined to date for DLA‐DRB1, DQA1 and DQB1 come from the analysis of European or North American pure bred dogs. Little is known about DLA gene polymorphisms in other dog populations. We have studied Alaskan Husky dogs and Brazilian mongrel dogs and compared them with a panel of 568 European dogs and 40 Alaskan gray wolves. DNA sequence based typing was used to characterize a series of 12 Alaskan Huskies and 115 Brazilian mongrels for their DLA‐DRB1, DQA1 and DQB1 alleles. Within these dogs, 22 previously undescribed DLA class II alleles were identified: 10 DRB1, 5 DQA1 and 7 DQB1 alleles. All these alleles were found in more than one animal, and, in some cases, as a homozygote. Several alleles initially observed in Alaskan gray wolves were found in these dogs. Each new allele was found in specific haplotypic combinations. Many new DLA class II haplotypes were identified. Several of the new alleles and haplotypes were also identified in the European dogs used for comparison. One new haplotype, containing a previously unknown DLA‐DRB1 allele together with DQA1 and DQB1 alleles only seen before in gray wolves, was found in 20 Brazilian dogs, including three homozygous animals. It appears likely that the extent of polymorphism of the DLA genes will increase substantially as dogs from a wider geographic distribution are studied. This has major implications for the study of disease susceptibility and immune responsiveness in dogs.
A novel HLA‐DQB1 allele, DQB1*05022, isolated from the Jing ethnic group in South‐west China
Authors: Fu Y. , Chen W. , Liu Z. , Lin J. , Jia Z. , Pan D. , Xu A.
Published: March 2003

Abstract: A novel DQB1 allele, DQB1*05022, has been identified from an individual of the Jing ethnic group in South‐west China. The sequence was confirmed by cloning and sequencing. The allele differs from DQB1*05021 at codon 47 (TAC to TAT) and from DQB1*05031 at codon 57 (GAC to AGC).
High resolution MIC genotyping: Design and application to the investigation of inflammatory bowel disease susceptibility
Authors: Ahmad T. , Marshall S.E. , Mulcahy‐Hawes K. , Orchard T. , Crawshaw J. , Armuzzi A. , Neville M. , Van Heel D. , Barnardo M. , Welsh K.I. , Jewell D.P. , Bunce M.
Published: March 2003

Abstract:The highly polymorphic nonclassical MHC class I chain‐related (MIC) genesMICAandMICBencode stress inducible glycoproteins expressed on a variety of epithelial cells including intestinal cells. Interaction with the receptor NKG2D is likely to provide an important costimulatory signal for activation and proliferation of NK cells, activated macrophages and CD8 αβ and γδ T cells. Fifty‐fourMICAand 17MICBalleles have been described to date. Although the functional significance of this polymorphism is not known, the high degree of nonconservative substitution, concentration to the putative ligand‐binding site and recent observation that differentMICAalleles bind to NKG2D with varying affinity has generated much interest. TheMICgenes are attractive functional and positional candidate genes for inflammatory bowel disease susceptibility as a consequence of their position in the HLA region and expression on the gastrointestinal epithelium. We developed a robust, high‐resolution PCR‐SSP genotyping method that can be incorporated into the standard ‘Phototyping’ system and which effectively identifies 46 of 54MICAalleles, and all 17MICBalleles. We applied this system in combination with microsatellite genotyping of the exon 5 variable number of tandem repeats (VNTR) to the investigation of genetic susceptibility to the inflammatory bowel diseases, ulcerative colitis and Crohn's disease. We studied 248 patients with Crohn's disease, 329 with ulcerative colitis and 354 ethnically matched controls. Linkage disequilibrium patterns between HLA‐B,MICAandMICBare presented. Analysis by individual allele or by multilocus haplotype failed to identify any significant disease associations.
Population of the HLA Ligand Database
Authors: Sathiamurthy M. , Hickman H.D. , Cavett J.W. , Zahoor A. , Prilliman K. , Metcalf S. , Fernandez‐Vina M. , Hildebrand W.H.
Published: March 2003

Abstract: We have established an HLA ligand database to provide scientists and clinicians with access to Major Histocompatibility Complex (MHC) class I and II motif and ligand data. The HLA Ligand Database is available on the world wide web at http://hlaligand.ouhsc.edu and contains ligands that have been published in peer‐reviewed journals. HLA peptide datasets prove useful in several areas: ligands are important as targets for various immune responses while algorithms built upon ligand datasets allow identification of new peptides without time‐consuming experimental procedures. A review of the HLA class I ligands in the database identifies strengths and deficiencies in the database and, therefore, the utility of the dataset for identifying new peptides. For instance, 212 HLA‐A phenotypes exist of which 23 have a motif determined and 43 have peptides characterized. In terms of number of ligands, HLA‐A*0201 has 258 characterized ligands, A*1101 has 25 peptides, while the remaining two‐thirds of the HLA‐A phenotypes have less than 10 associated peptide sequences. Characterization of ligands and motifs remains roughly the same at the HLA‐B locus while the peptides of the HLA‐C locus tend to be less characterized. These data show that 74% of HLA class I molecules do not have ligands represented in the database and thus algorithms based on the dataset could not predict ligands for a majority of the US population. Building upon this dataset and knowledge of HLA allelic frequencies, it is possible to plan a systematic expansion of the HLA class I ligand database to better identify ligands useful throughout the population.
Synthesis and binding potencies of cyclic hexapeptide analogs of somatostatin incorporating acidic and basic peptoid residues
Authors: Tran T.‐A. , Mattern R.‐H. , Goodman M. , Morgan A. , Taylor J.E.
Published: March 2003

The synthesis, binding affinity, and structure–activity relationships of compounds related to the cyclic hexapeptide, c[Pro6‐Phe7‐D‐Trp8‐Lys9‐Thr10‐Phe11], L‐363,301 (the numbering in the sequence refers to the position of the residue in native somatostatin) is reported. The Pro residue in this compound is replaced with the peptoid residues Nasp [N‐(2‐carboxyethyl) glycine], Ndab [N‐(2‐aminoethyl) glycine] and Nlys [N‐(4‐aminobutyl) glycine]. This series of compounds enables us to draw conclusions about the influence of positively or negatively charged residues in the bridging region on the binding affinity towards the isolated human somatostatin receptors. A loss of binding to the recombinant human somatostatin (hsst) receptors in the Nasp analog compared with L‐363,301 and compared with the Ndab and Nlys analogs clearly demonstrates that the presence of an acidic residue in the bridging region is unfavorable for binding to the hsst receptors. Comparison between the Ndab analog and the Nlys analog suggests that the presence of a basic residue in the bridging region might be advantageous for binding to the hsst5 receptor provided that the residue bearing the basic group extends far enough to allow for interaction with the receptor, while the length of the basic peptoid residue does not influence binding to the hsst2 receptor. These results are useful for the design of hsst5 selective somatostatin analogs.
Conformational studies of irreversible HIV‐1 protease inhibitors containing cis‐epoxide as an amide isostere
Authors: Ro S. , Baek S. ‐G. , Lee B. , Ok, J.H.
Published: March 2003

Abstract: We have carried out NMR and molecular modeling studies of peptidomimetic HIV‐1 protease inhibitors, LB71116: Qc‐Asn‐Pheψ[(1R,2S)‐cis‐epoxide]Gly‐NH‐CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc‐(SMe)Pen(O)2‐Pheψ[(1R,2S)‐cis‐epoxide]Gly‐NH‐CH(isopropyl)2 where (SMe)Pen(O)2 stands for S‐methyl‐S‐dioxo‐penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis‐epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the ψ of Phe(cep) is maintained about 130° and the φ angle of (cep)Gly prefers ± 150°[where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe‐Gly peptide bond with cis‐epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule (‘flap water’), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X‐ray studies of the complex between LB71116 and HIV‐1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV‐1 protease by the similarity between the preferred conformations in solution and the binding conformation.
Grafting an RGD motif onto an epidermal growth factor‐like module: chemical synthesis and functional characterization of the chimeric molecule
Authors: Vella F. , Hernandez J.F. , Arlaud G.J. , Molla A. , Block M.R.
Published: March 2003

Abstract: A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14‐residue loop of the EGF‐like module from human complement protease C1r. The resulting chimeric EGF‐RGD module (52 residues, three disulfide bridges) was assembled by automated solid‐phase synthesis using the t‐Boc strategy. Using reduced/oxidized glutathione, the EGF‐RGD module was folded as efficiently as the natural C1r‐EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N‐terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF‐RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the α5 subunit of α5β1 integrin had no effect. The ability of EGF‐RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.
Identification of the molecular interaction site of amyloid β peptide by using a fluorescence assay
Authors: Watanabe K. , Segawa T. , Nakamura K. , Kodaka M. , Okuno H. , Konakahara T.
Published: March 2003

Abstract: β‐Amyloid peptides (Aβ) are the main protein components of neuritic plaques and are important in the pathogenesis of Alzheimer's disease. It is reported that Aβ itself is not toxic; however, it becomes toxic to neuronal cells once it has aggregated into amyloid fibrils by peptide–peptide interactions. In this study, to specify the molecular mechanism of aggregation, a novel fluorescence assay was designed. For this purpose, possible partial peptides (38 types of 5‐mer) were synthesized on solid‐phase. The molecular interactions were examined by a fluorescence probe possessing Lys‐Leu‐Val‐Phe‐Phe (KLVFF) as a molecular recognition site. KLVFF is known to be a minimum sequence for formation of the Aβ aggregate. A specific interaction was observed between labeled and immobilized KLVFF. It suggests that the aggregation of Aβ was controlled by the recognition of KLVFF itself by hydrophobic and electrostatic interactions.
Designing heterodimeric two‐stranded α‐helical coiled‐coils: the effect of chain length on protein folding, stability and specificity
Authors: Litowski J.R. , Hodges R.S.
Published: March 2003

Abstract:The E/K coil, a heterodimeric coiled‐coil, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and affinity chromatography. In this design, heterodimer formation is specified through the placement of charged residues at theeandgpositions of the heptad repeat. The affinity and stability of the E/K coil has been modified in order to allow a greater range of conditions for association and dissociation by varying the chain length to obtain three, four and five heptad coiled‐coils (21, 28 and 35 residues per polypeptide chain). The effect of chain length on stability and folding was examined by circular dichroism spectroscopy, guanidine hydrochloride denaturation, and redox equilibrium experiments. It was found that increases in chain length produced increases in the stability of heterodimeric coiled‐coils, but in a nonlinear fashion. The resulting disulfide‐bridged heterostranded molecules and reduced heterodimers span a wide range of stabilities (ΔG = 3.3–11.9 kcal/mol), greatly expanding their scope for use in protein engineering and biomedical applications.
Solution structure of a cathelicidin‐derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy
Authors: Yu K. , Park K. , Kim Y. , Kang S.‐W. , Shin S.Y. , Hahm K.‐S.
Published: March 2003

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.
Isolation and characterization of the rml gene homologs from Porphyromonas gingivalis
Authors: Shibata Y. , Yamashita Y. , Nakano Y. , Koga T.
Published: March 2003

We cloned four genes from the Porphyromonas gingivalis chromosome, the gene products of which catalyze the anabolism of dTDP‐
l‐rhamnose from
d‐glucose‐1‐phosphate when they were expressed in Escherichia coli. The amino acid sequences deduced from these genes showed significant homology to proteins encoded by the rml genes involved in dTDP‐
l‐rhamnose biosynthesis in other gram‐negative bacteria. Reverse transcriptase polymerase chain reaction analysis revealed that these four genes are expressed as a single transcript in P. gingivalis. To clarify the role of the rml gene homologs in this organism, construction of mutants defective in the rml gene homologs was attempted by allelic exchange. Unexpectedly, any mutants defective in the rml gene homologs were unable to be isolated, and the allelic exchange was possible only if the wild‐type rml gene homologs were present on the chromosome. These results suggest that the rml gene homologs might be essential for the viability of P. gingivalis under the culture conditions used in this study.
Genotyping of Actinobacillus actinomycetemcomitans serotype d isolates based on polymerase chain reaction
Authors: Dogˇan B. , Saarela M. , Asikainen S.
Published: March 2003

The aims of the study were to determine the genetic diversity of the rare Actinobacillus actinomycetemcomitans serotype d and to compare the ability of the repetitive extragenic palindromic element (REP)–based polymerase chain reaction (PCR) with that of the arbitrarily primed (AP)‐PCR to discriminate between and within A. actinomycetemcomitans serotypes. The material included 26 A. actinomycetemcomitans serotype d isolates, 3 reference strains, and 21 A. actinomycetemcomitans isolates, representing the previously described 17 AP‐PCR genotypes from 4 serotypes (a, b, c and e). Among A. actinomycetemcomitans serotype d isolates (n=26), the AP‐PCR primer distinguished 2 genotypes, whereas the REP‐primer pair (REP1R‐I and REP2‐I) and the (GACA)4 primer each produced one genotype. Among the total of 50 A. actinomycetemcomitans isolates, REP‐primer pair distinguished 6 genotypes, the primer (GACA)4 7 genotypes, and the AP‐PCR 19 genotypes. Among A. actinomycetemcomitans serotype a isolates (n=6), REP‐primer pair yielded 3 genotypes and (GACA)4 and AP‐PCR primer 4 genotypes, and among serotype e isolates (n=6) 3 genotypes. All serotype b isolates (n=7), representing the AP‐PCR genotypes 2, 9, 8, 12, 13, 16 and serotype c isolates (n=5), AP‐PCR genotypes 3, 4, 14, 15, belonged to the (REP1R‐I and REP2‐I)‐PCR genotype 4 and to the (GACA)4‐PCR genotype 4. In conclusion, based on both the AP‐PCR method and the less discriminative REP‐PCR methods, the present genotyping results indicated limited genetic diversity among serotype d isolates of A. actinomycetemcomitans.
Herpesviruses in periodontal pocket and gingival tissue specimens
Authors: Contreras A. , Nowzari H. , Slots J.
Published: March 2003

Human cytomegalovirus (HCMV) and Epstein‐Barr virus type 1 (EBV‐1) are frequently detected in crevicular fluid of deep periodontal pockets, but little or no information is available on occurence of herpesviruses in gingival tissue. This investigation studied the presence of herpesviruses in periodontal pockets and the corresponding gingival tissues from 11 periodontally healthy and 14 periodontitis sites. A nested‐polymerase chain reaction was employed to identify the presence of HCMV, EBV‐1, EBV‐2, herpes simplex virus, human herpesvirus (HHV)‐6, HHV‐7 and HHV‐8 in each test sample. In healthy periodontal sites, HCMV was detected in 1 (9%) and EBV‐1 in 2 (18%) pocket samples, and HCMV was detected in 2 (18%) and EBV‐1 in 3 (27%) gingival tissue samples. In periodontitis lesions, HCMV was detected in 9 (64%) pocket samples and in 12 (86%) gingival tissue samples, and EBV‐1 was detected in 6 (43%) pocket samples and in 11 (79%) gingival tissue samples. HHV‐6 and HHV‐8 were detected exclusively in gingival tissue samples. The present findings confirm the frequent presence of HCMV and EBV‐1 in periodontitis lesions and suggest using gingival tissue specimens for detecting periodontal HHV‐6, HHV‐7 and HHV‐8.
Molecular cloning, sequence and characterization of a novel streptococcal phosphoglycerate dehydrogenase gene
Authors: Kawabata S. , Terao Y. , Hamada S.
Published: March 2003

The nucleotide sequence of a Streptococcus mutans serA gene that encodes
d‐3‐phosphoglycerate dehydrogenase has been determined. The gene consisted of 1308‐bp nucleotides coding for a 436‐amino‐acid polypeptide (48,546 Da). The deduced amino acid sequence showed a 66% identity with SerA from Bacillus subtilis and possessed specific residues (G‐R‐P‐N‐V‐G) in the coenzyme‐binding domain, αB helix. Recombinant streptococcal SerA was expressed using pMAL‐c2 expression vector and purified by amylose resin affinity chromatography and DEAE‐Sephacel column chromatography. This SerA enzyme catalyzed detectable reduction of α‐ketoglutarate to 2‐hydroxyglutaric acid. These findings indicate that the novel streptococcal phosphoglycerate dehydrogenase, SerA, is a member of a
d‐isomer‐specific family of 2‐hydroxyacid dehydrogenases.
Salivary and serum antibody response to Streptococcus mutans antigens in humans
Authors: Chia J.‐S. , Chang W.‐C. , Yang C.‐S. , Chen J.‐Y.
Published: March 2003

Humoral immunity against Streptococcus mutans infection was analyzed in caries‐active and caries‐free young adults by immunoblotting. All volunteers from both groups had detectable salivary immunoglobulin A (IgA) and serum IgG antibodies, with similar profiles. They could be classified on the basis of relative intensity of the immunoblot bands into categories of high or low responders. Common protein antigens with molecular weight ranging from approximately 45 to 190 kDa could be found either extracellularly or associated with the cell wall of S. mutans cultured in vitro. The predominant reactive antigens recognized by both IgA and IgG were of proteins around 63 and 60 kDa. Detection of IgA antibodies to the various antigens of S. mutans in individual saliva samples did not always correlate with serum IgG antibody profiles. In addition, distinct bands, which reacted preferentially with either IgA or IgG, could be detected by antibodies from specific subjects. Differential reactivities of salivary IgA and serum IgG antibodies to two, cell‐wall associated protein antigens around 33 and 36 kDa were found in caries‐active and caries‐free young adults; 30.8% of caries‐free subjects and 12% of caries‐active subjects (P<0.01) exhibited detectable antibody response to these antigens. This difference was not attributable to variations in antibody levels, since antibody response to these proteins were still detectable in some caries‐free but not caries‐active individuals whose levels of antibodies to other antigens were low. Thus, a new antibody profile which correlates with dental caries disease activity has been identified in a selected population. Differences in mucosal and systemic immune responses to S. mutans seem to be both antigen and host dependent.
Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction
Authors: Oho T. , Yamashita Y. , Shimazaki Y. , Kushiyama M. , Koga T.
Published: March 2003

Streptococcus mutans and Streptococcus sobrinus are major pathogens causing dental caries in humans. A simple and rapid method to detect these species in human saliva simultaneously was developed using the polymerase chain reaction (PCR). Chromosomal DNA was extracted by boiling bacterial cells in lysis solution containing 1% Triton X‐100. Oligonucleotide primers specific for portions of the glucosyltransferase genes (gtfB of S. mutans and gtfI of S. sobrinus) were designed. After PCR using two sets of these primers, S. mutans and S. sobrinus were specifically identified. The method was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 1×103 cells, or from 10 μl of clinical saliva samples containing 1×103 colony‐forming units of either streptococcal species. A second PCR, using the first PCR product as a template with newly designed internal primers, made it possible to detect 1×102 colony‐forming units of either streptococcal species in 10 μl of saliva samples. These results indicate that the PCR method developed in this study is useful for detecting S. mutans and S. sobrinus in saliva and that it can be used in epidemiological studies to evaluate the prevalence level of these organisms.
A Porphyromonas gingivalis genetic locus encoding a heme transport system
Authors: Slakeski N. , Dashper S. G. , Cook P. , Poon C. , Moore C. , Reynolds E. C.
Published: March 2003

Porphyromonas gingivalis has been implicated in the onset and progression of periodontitis and the availability of hemin for in vitro growth has been associated with virulence of the bacterium in animal models. We report here the cloning and sequence analysis of a P. gingivalis TonB‐linked outer membrane receptor gene tlr. This gene was previously identified as a TonB‐linked adhesin gene tla and shown to be essential for growth at low concentrations of hemin. The tlr gene is immediately downstream of four open reading frames (htrABCD) that encode a putative ATP binding cassette transport system with sequence similarlity to heme transport systems of other bacteria. Analysis of P. gingivalis W50 mRNA revealed that the htrABCD genes are cotranscribed similar to hemin transport genes of other bacteria.
A link between parotid salivary Ig level and recurrent respiratory infections in young Down's syndrome patients
Authors: Chaushu S. , Yefenof E. , Becker A. , Shapira J. , Chaushu G.
Published: March 2003

The purpose of the present investigation was to assess the correlation between recurrent respiratory infections and the levels of salivary Ig in a group of young Down's syndrome (DS) individuals. Twenty‐three DS and 10 age‐ and sex‐matched healthy individuals were included. DS individuals who had experienced three or more upper respiratory tract infections (n = 10) in the previous 12 months were compared to DS individuals who had not experienced recurrent respiratory infections (n = 13) and to healthy controls (n = 10). A statistically significant reduction in the Ig salivary secretion rate was recorded in the subgroup with recurrent respiratory infections. No significant differences were seen between the subgroup without recurrent respiratory infections and controls. It is suggested that the secretory immune system provides local immune protection against pathogens in the respiratory tract. Detection of salivary Ig levels may serve as a predictor of the susceptibility of DS individuals to recurrent respiratory tract infections.
Identification of the peptide motifs that interact with HLA‐DR8 (DRB1*0802) in Streptococcus mutans proteins
Authors: Nomura Y. , Eto A. , Hanada N. , Senpuku H.
Published: March 2003

A glucosyltransferase (GTF) and a surface protein antigen (PAc) of Streptococcus mutans have been suggested as possible components of an effective dental caries vaccine. To identify antigenic peptides in GTF and PAc that bind to MHC class II (HLA‐DR8, DRB1*0802) molecules, we investigated binding activities to DR8 molecules of overlapping synthetic peptides at several sites in GTF and in the alanine‐rich repeating region of PAc using an ELISA‐inhibition competitive binding assay for the interaction between the HLA‐DR molecule and the PAc (316–334) peptide. Six GTF peptides and 10 PAc peptides strongly bound to the HLA‐DR8 molecule. In a homology analysis of the amino acid sequences of the six GTF peptides, two binding motifs were found in L/Y– –Y/L–A/N and Y/L– –N/G/E– –Y–V/L/P. Moreover, a new binding motif in PAc was found in L‐ –Y‐A. It is suggested that these binding motifs could be useful in designing a dental caries vaccine in humans.
Sequence conservation and distribution of the fusobacterial immunosuppressive protein gene, fipA
Authors: Hunt Gerardo S. , Yoder S. C. , Citron D. M. , Goldstein E. J. C. , Haake S. Kinder
Published: March 2003

Fusobacterium nucleatum is a gram‐negative anaerobe involved in various diseases, including periodontitis. Recently, other investigators isolated the

 F. nucleatum FDC 364 fusobacterial immunosuppressive protein (FIP). One subunit, FipA, impairs T‐cell activation in vitro and shows homology with beta‐ketothiolases. However, its distribution and variability among fusobacteria was not reported. Cloned fipA gene sequences from F. nucleatum ssp. polymorphum (ATCC 10953) and F. nucleatum ssp. nucleatum (ATCC 23726) shared 89 and 92% identity, respectively, with FDC 364 fipA, and 90 and 94% identity, respectively, with the FDC 364 FipA predicted amino acid sequence. Southern blot analyses of chromosomal DNA from fusobacterial strains, including

 F. nucleatum and other Fusobacterium species, were performed using partial fipA sequences as probes. The results indicate that fipA is highly conserved among the F. nucleatum strains examined and that fipA homologues are widely distributed among fusobacteria. A clear relationship between immune suppression, metabolism and the FipA protein remains to be determined.
Characterization of in vitro oral bacterial biofilms by traditional and molecular methods
Authors: Pratten J. , Wilson M. , Spratt D. A.
Published: March 2003

The aim of this study was to compare culture‐based bacterial isolation methods with direct amplification and cloning of 16S rRNA genes from oral biofilms grown in an in vitro model. The model used was a constant depth film fermentor which was inoculated with pooled human saliva. The use of culture techniques and cloning resulted in the identification of 36 different bacterial species from the saliva inoculum and from the biofilms. Of these, only five were detected solely by molecular methods. Three taxa were detected which, according to the databases, were unidentified. Using the molecular methods of detection, differences in the number of species observed were found using different 16S rRNA gene primers and numbers of PCR cycles. We have shown that microcosm supragingival plaque biofilms grown in a fermentor consisted of a community most of the members of which could be cultivated on laboratory media.
Anti‐Streptococcus mutans antibodies in saliva of children with different degrees of dental caries
Authors: Naspitz Giselle Maria Collesi Carneiro , Nagao Aparecida Tiemi , Mayer Marcia Pinto Alves , Carneiro‐Sampaio Magda Maria Sales
Published: March 2003

The aim of this study was to evaluate the relationship between the secretory immune system and dental caries. Forty‐nine 3–5‐year‐old children with primary dentition were classified into three groups according to their caries indices: no caries (group I), one or two surfaces with caries lesions (group II) and rampant caries (group III). Lower numbers of mutans streptococci were found in group I in relation to groups II and III. Secretory IgA and anti‐S. mutans IgA, IgM and IgG antibody levels were not significantly different among the groups. Western blotting analysis showed that some S. mutans proteins, including the 39, 59, 97 and 150 kDa molecular mass bands, were recognized by almost all the saliva samples. Antibodies against the 185 kDa band, known as antigen I/II, were present in all adults’ saliva and in only one child in group III. The absence of antibodies to the 185 kDa band in children’s saliva suggest a specific immunologic immaturity. Further prospective studies will be necessary to establish the possible effect of reactivity to this antigen on the S. mutans colonization in this age group.
Clinical management of hereditary angio‐oedema in children
Authors: Farkas Henriette , Harmat George , Füst George , Varga Lilian , Visy Beáta
Published: March 2003

Hereditary angio‐oedema (HAE) results from the deficiency of C1‐esterase inhibitor (C1‐INH). The clinical picture of this autosomal dominant disorder is characterized by recurrent attacks of subcutaneous oedema and/or potentially life‐threatening swelling of the submucosa. This review discusses the authors' decade‐long experience obtained in the treatment and follow‐up of pediatric patients with HAE. Twenty‐six children with HAE were reviewed. Pedigree analysis was performed in all cases to identify afflicted relatives. C1‐INH concentrate was reserved for the emergency treatment of acute oedematous attacks, whereas tranexamic acid and danazol were administered for short‐ or long‐term prophylaxis. Follow‐up care included laboratory tests and abdominal ultrasound, which was repeated at regular intervals. Twenty‐one children had Type I HAE and five suffered from Type II HAE. Clinical manifestations of the disease first occured in children when 2.5–12 years of age. Oedema formation primarily afflicted subcutaneous tissues. Mechanical trauma was identified as a precipitating factor in 20 patients. Pedigree analysis revealed 24 patients with relatives who suffered from HAE. Long‐term prophylaxis with tranexamic acid or danazol was initiated in 11 patients; two children required short‐term prophylaxis. No drug‐related adverse effects were observed, except for one case of delayed menarche. Therapy improved serum complement parameters significantly and substantially reduced the frequency and severity of clinical episodes. Adequate prophylaxis and follow‐up care can spare pediatric patients from oedematous attacks caused by HAE. Undesirable adverse effects can be avoided and the patient's quality of life enhanced considerably by administering the lowest effective drug dose.
Thromboxane A2 receptor gene polymorphism is associated with the serum concentration of cat‐specific immunoglobulin E as well as the development and severity of asthma in Chinese children
Authors: Leung Ting Fan , Tang Nelson L. S. , Lam Christopher W. K. , Li Albert M. , Chan Iris H. S. , Ha G.
Published: March 2003

Thromboxane A2 and its receptor (TBXA2R) are involved in the constriction of vascular and respiratory smooth muscles. The T924C polymorphism in the TBXA2R gene was recently found to be associated with asthma in Japanese adults but not in children. Its relationship with atopy or asthma severity in children has not been defined. To investigate this further, we first assessed the severity of asthma in Chinese children using a standardized questionnaire modified from the Disease Severity Score and spirometric evaluation. Then, peripheral blood was analyzed for serum total and aeroallergen‐specific immunoglobulin E (IgE) levels, and TBXA2R T924C genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. One‐hundred and fifty three asthmatic patients and 57 control children were recruited, of respective mean ages 9.9 and 11.0 years (p = 0.07). The mean logarithmic serum total IgE concentration was 2.57 and 2.09, respectively, for the asthmatic group and control group (p < 0.0001). Atopy was detected in 132 (86%) asthmatics and 33 (58%) controls. A significant association was observed between T924C and the diagnosis of atopic asthma (p = 0.044; odds ratio: 1.84). In addition, those asthmatics homozygous for the mutant allele in T924C had a lower forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) (p = 0.032 and 0.002, respectively). Among our asthmatic patients, the TBXA2R T924C polymorphism correlated with the concentration of cat‐specific IgE in serum (p = 0.046). Nonetheless, this gene marker did not show an association with the serum total IgE concentration or any clinical indicator of asthma severity. In conclusion, our results suggest that the T924C marker in the TBXA2R gene is associated, in Chinese children, with an increased susceptibility of developing atopic asthma. This marker is also associated with the extent of allergic sensitization to cat, as well as with reduced FEV1 and FVC values.
Hepatic artery thrombosis in pediatric liver transplantation: Graft salvage after thrombectomy
Authors: Garcia‐gallont R. , Bar‐nathan N. , Shaharabani E. , Yussim A. , Shapira R. , Zahavi I. , Shapira Z. , Dinari G. , Mor E.
Published: March 2003

Abstract: Hepatic artery thrombosis (HAT) is a devastating complication that may occur after orthotopic liver transplantation (OLT). A higher incidence has been reported in children. Salvage of the graft by thrombectomy has been suggested as an alternative to re‐transplantation. In this study we report the outcome of three children who underwent thrombectomy for HAT. Between January 1992 and June 1998, 14 children (< 17 yrs of age) underwent liver transplantation. Three developed HAT (one a whole‐liver graft recipient, age 17; two living‐related graft recipients, ages 4 and 4.5 yr). In the first patient, thrombosis of the hepatic artery was associated with scattered areas of parenchymal necrosis on computed tomography. In the two living‐related patients, HAT was found incidentally during re‐exploration for bleeding (day 2 and day 10). Thrombectomy was performed in all three patients. At 18–24 months after thrombectomy, all three children had normal graft function. In the first patient, complete regeneration of the liver has been documented by computed tomography and a late asymptomatic recurrent thrombosis is suggested by absence of arterial flow on Doppler examination. The hepatic artery is patent in the two living‐related recipients. One of these living‐related recipients developed ischemic bile duct stricture and underwent successful percutaneous balloon dilatation. We conclude that long‐term normal graft function can be achieved by thrombectomy in pediatric liver recipients with HAT, even in the presence of limited parenchymal damage.
Living related liver transplantation from heterozygote genetic carriers to children with Wilson’s disease
Authors: Asonuma K. , Inomata Y. , Kasahara M. , Uemoto S. , Egawa H. , Fujita S. , Kiuchi T. , Hayashi M. , Tanaka K.
Published: March 2003

Abstract:We reviewed the outcome of children undergoing living related liver transplantation (LRLT) for Wilson’s disease (WD), and specifically addressed the potential risk associated with the use of donors who were heterozygous for the Wilson genetic defect. LRLTs were carried out in 11 children with WD, nine of whom presented with fulminant hepatic failure and two with end‐stage hepatic insufficiency. The age of the patients ranged from 6 to 16 yr. Eight patients had hepatic encephalopathy and were plasmapheresed preoperatively. The donors (all parents: six fathers and five mothers) were all one‐haplotype matched with their respective recipients, and were all therefore heterozygote carriers of the WD genetic defect. The serum ceruloplasmin levels were within normal limits in all donors (mean: 20.0 ± 2.85 mg/dL). All recipients but one had low serum ceruloplasmin levels with a mean value of 11.6 ± 7.36 mg/dL before transplantation. The serum ceruloplasmin levels had increased to an average of 21. 0 ± 3.76 mg/dL after LRLT at the latest evaluation, which ranged between 7 and 75 months after transplantation. A marked reduction in urinary copper excretion was observed in all recipients after transplantation. Of eight recipients presenting preoperatively with Kayser‐Fleischer (K‐F) rings, this abnormality resolved completely after LRLT in five patients and partially in three. All recipients are alive and remain well, and none have developed signs of recurrent WD after a mean follow‐up period of 31 months (range 7–75 months). In conclusion, LRLT is an excellent choice for effective treatment of WD, and grafts chosen from heterozygote carriers of the condition do not appear to confer any risk of recurrence in the recipients.
Mycophenolate mofetil – is it worth the cost? The in‐favor opinion
Authors: Seikaly M. G.
Published: March 2003

Abstract:Since its introduction, mycophenolate mofetil (MMF) has rapidly gained acceptance among renal transplant physicians. Three large multicenter studies have shown that MMF decreased the incidence of acute rejection episodes by 50%. While clinical data supporting the long‐term benefits of MMF are not available, there is some experimental evidence which demonstrates that MMF may play a role in the prevention of chronic allograft rejection. Furthermore, MMF is a potent immunosuppressive agent, which could result in a reduction of the dose of cyclosporin A (CsA) required and subsequently reduce its associated toxicity. Despite the fact that MMF is six to seven times more expensive than azathioprine (AZA), its introduction into maintenance immunosuppressive protocols may not increase renal transplant charges during the first year after renal transplant as its cost can be offset by the reduced number of acute rejection episodes (ARE). The long‐term societal impact of MMF will need to be evaluated as the data related to graft and patient survival, as well as chronic rejection, become available.