bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA‐DRB1 and ‐DQB1 alleles in the Brazilian population of the northeastern region of the state of São Paulo
Authors: Louzada‐Junior P. , Smith A.G. , Hansen J.A. , Donadi E.A.
Published: March 2003

Abstract: The HLA‐DRB1 and ‐DQB1 alleles in 161 healthy unrelated individuals, including Caucasians, Blacks and Mulattos (mixed Caucasian and Black), from the Northeastern region of the state of São Paulo, Brazil were analysed. The 36 different DRB1 alleles detected included not only common Caucasian alleles, but also DRB1*0411, 0807 and 1402, typical of Amerindians, and DRB1*0302, 1503, and 0804, typical of African American Blacks.
The gene encoding the immunoregulatory signaling molecule CMRF‐35A localized to human chromosome 17 in close proximity to other members of the CMRF‐35 family
Authors: Clark G.J. , Cooper B. , Fitzpatrick S. , Green B.J. , Hart D.N.J.
Published: March 2003

Abstract: The immunoregulatory signaling (IRS) family includes several molecules, which play major roles in the regulation of the immune response. The CMRF‐35A and CMRF‐35H molecules are two new members of the IRS family of molecules, that are found on a wide variety of haemopoietic lineages. The extracellular functional interactions of these molecules is presently unknown, although CMRF‐35H can initiate an inhibitory signal and is internalized when cross‐linked. In this paper, we described the gene structure for the CMRF‐35A gene and its localization to human chromosome 17. The gene consists of four exons spanning approximately 4.5 kb. Exon 1 encodes the 5′ untranslated region and leader sequence, exon 2 encodes the immunoglobulin (Ig)‐like domain, exon 3 encodes the membrane proximal region and exon 4 encodes the transmembrane region, the cytoplasmic tail and the 3′ untranslated region. A region in the 5′ flanking sequence of the CMRF‐35A gene, that promoted expression of a reporter gene was identified. The genes for the CMRF‐35A and CMRF‐35H molecules are closely linked on chromosome 17. Similarity between the Ig‐like exons and the preceding intron of the two genes suggests exon duplication was involved in their evolution. We also identified a further member of the CMRF‐35 family, the CMRF‐35J pseudogene. This gene appears to have arisen by gene duplication of the CMRF‐35A gene. These three loci – the CMRF‐35A, CMRF‐35J and CMRF‐35H genes–form a new complex of IRS genes on chromosome 17.
Sequence, genetics and serology of a new HLA‐B allele: B*4903
Authors: Darke C. , Street J. , Guttridge M.G.
Published: March 2003

Abstract:A new HLA‐B allele – B*4903 – was detected by the polymerase chain reaction using sequence‐specific priming (PCR‐SSP), in a Caucasoid bone marrow panel donor, that differs from B*4901 by 8 nucleotides at positions 141, 142, 144, 165, 167, 193, 206 and 213 in exon 2. These substitutions all occur in HLA‐B*51 and B*52 alleles and encode 4 amino acid substitutions at positions 24 (Thr to Ala), 32 (Leu to Gln), 41 (Thr to Ala) and 45 (Lys to Thr). This suggests that B*4903 occurred following a gene conversion‐like event involving B*4901 and probably a B*51 allele. HLA‐B*4903 was identified on a haplotype with: HLA‐A*0201; Cw*07; DRB1*1302/34; DRB3*0301; DQA1*0102; DQB1*0604; BfS; C4A3; C4BQ0 and encodes a unique serological specificity which was characterised by the reactivity of 55 antisera directed towards at least four predicted epitopes. No further examples of B*4903 were found in 15,796 consecutive HLA PCR‐SSP typed donors from the Welsh Bone Marrow Donor Registry, indicating that this allele has a phenotype frequency of <0.01% and a gene frequency of <0.00004 (Note).
CD27 expression in the human splenic marginal zone: the infant marginal zone is populated by naive B cells
Authors: Zandvoort A. , Lodewijk M.E. , De Boer N.K. , Dammers P.M. , Kroese F.G.M. , Timens W.
Published: March 2003

Abstract: The splenic marginal zone of adult humans contains B cells, of which most express CD27, an antigen only recently identified as a marker for somatically mutated memory B cells. We investigated whether and to which extent the developing marginal zone in infants and children is populated by either memory (CD27+) or naive (CD27−) B cells. Frozen sections of 32 spleens of infants and children ranging in age from 6 days to 15 years and 6 adult spleens were investigated. The expression of CD27 in combination with monoclonal antibodies against CD3, CD21, IgM, IgD and ASM‐1 was analyzed by immunohistochemistry. The marginal zone was already present at 4 months after birth but CD21 expression was observed first after 2 years. CD27‐positive marginal zone B cells were observed firstly 2 years after birth and increased in number to adult levels at the age of 5 years. We demonstrated that the MZ of infants and young children is populated by naive B cells, which are replaced by memory B cells in a time frame of 2 to 5 years. Before the age of 2 years, although present, memory B cells appear to be unable to colonize the marginal zone. Because of the absence of memory B cells in the marginal zone, the immune system of a child is not capable to initiate a rapid secondary humoral immune response comparable to the adult immune response.
HLA‐DRB fluorotyping by dark quenching and automated analysis
Authors: Slateva K. , Elsner H.‐A. , Albis‐Camps M. , Blasczyk R.
Published: March 2003

Abstract: In fluorescence‐based sequence‐specific primed polymerase chain reaction (PCR), referred to as fluorotyping for HLA typing, a major problem is the overlap of the emission spectra of the single dyes. In order to increase the robustness of the previously described HLA‐DRB1,3,4,5 low‐resolution fluorotyping method, we have constructed two probes quenched by the non‐fluorescent acceptor dye DABCYL. The HLA‐DRB‐specific probe was labeled with FAM, and the internal control probe with TAMRA, respectively, as reporter fluorescent dyes. TAMRA was replaced by DABCYL as a quencher, which led to increased robustness and better discrimination between negative and positive amplification results. ROX was used as a reference to normalize the fluorescence of the reporter dyes. Moreover, as FAM and TAMRA differ strongly by their emission maxima, HLA‐DRB‐specific and internal control amplification could be clearly distinguished. To further automate data analysis, the software of the TaqMan® system 7700 was supplemented by an EXCEL‐based calculation table, which directly took over the data. Using modified fluorotyping chemistry and automated data analysis, a total of 201 DNA samples was typed correctly. In summary, HLA‐DRB fluorotyping by dark quenching and automated analysis proved to be a robust and reliable tool for research and routine purposes.
Polymorphism in the Y box controls level of cytokine‐mediated expression of HLA‐DRB1 genes
Authors: Sindwani S. , Singal D.P.
Published: March 2003

Abstract: The HLA class II molecules play an important role in immune response. The quality of immune response is dependent not only on the polymorphisms in the class II molecules, but also on the level of their cell‐surface expression. In fact, it has been demonstrated that differences in the level of expression of DRB1 and DRB3 genes restricted and activated distinct CD4+ T lymphocytes. We and others have previously described allelic polymorphisms in the upstream regulatory regions of DRB genes, which affected DNA‐protein interactions and resulted in significantly different promoter strengths. We showed that polymorphisms in both the X1 and Y box motifs affect level of constitutive expression of DRB1 genes in the DR1, DR51 and DR53 haplotype groups. In the present study, we examined the effect polymorphisms in the X1 box and the Y box on the cytokine (interferon‐γ (IFNγ), tumor necrosis factor‐α (TNFα) and granulocyte macrophage‐colony‐stimulating factor (GM‐CSF))‐mediated transcriptional activities of DRB1 promoters in these, i.e. DR1, DR51 and DR53, haplotype groups. The results demonstrate that the polymorphism in the X1 box does not affect cytokine‐mediated strength of DRB1 gene promoters. In contrast, the polymorphism in the Y box, which affects the inverted CCAAT sequence, plays a dominant role on the cytokine‐mediated transcriptional activity of DRB1 promoters.
Establishment of a quantitative ELISA capable of determining peptide – MHC class I interaction
Authors: Sylvester‐Hvid C. , Kristensen N. , Blicher T. , Ferré H. , Lauemøller S.L. , Wolf X.A. , Lamberth K. , Nissen M.H. , Pedersen L.Ø. , Buus S.
Published: March 2003

Abstract:Many different assays for measuring peptide–MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC‐I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC‐I molecules are functional equivalents of “empty molecules”. When diluted into aqueous buffer containing beta‐2 microglobulin (β2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA–based assay capable of measuring the affinity of the interaction between peptide and MHC‐I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.
HLA class II markers and clinical heterogeneity in Swedish patients with primary biliary cirrhosis
Authors: Wassmuth R. , Depner F. , Danielsson Å. , Hultcrantz R. , Lööf L. , Olson R. , Prytz H. , Sandberg‐Gertzen H. , Wallerstedt S. , Lindgren S.
Published: March 2003

Abstract: Genetic susceptibility to PBC can, at least in part, be ascribed to the major histocompatibility complex. The relevance of immunogenetic markers for the clinical presentation and course, however, is unclear. Thus, the aim of this study was to investigate the contribution of HLA class II genes to susceptibility, clinical presentation and course of disease in PBC patients. HLA genotyping for HLA‐DRB1, ‐DQB1 and ‐DPB1 was carried out in a total of 99 Swedish PBC patients and 158 controls. Clinical parameters including epidemiologic variables, signs and symptoms of PBC‐related liver disease and histologic data were collected and analyzed in 92 patients at study entry and at follow‐up five years later. Significant clinical heterogeneity was seen among PBC patients upon study entry. Although a significant disease association was seen for HLA DRB1*08 and DQB1*0402, immunogenetic markers identified neither a particular subset of patients nor an association with the clinical course of the disease. HLA‐DRB1*08 and DQB1*0402 provide the strongest immunogenetic influence in PBC. However, this association is not restricted to any particular, clinically defined subgroup of patients and it is not predictive for the course of the disease.
HLA B27 polymorphism in Western India
Authors: Shankarkumar U. , Ghosh K. , Mohanty D.
Published: March 2003

Abstract: We have characterized HLA B27 alleles in a sample population of Maharastra, Western Indians (n = 51), with the aim to investigate the different subtypes present among this population. The study was carried out using polymerase chain reaction with sequence‐specific primers (PCR‐SSP) and reverse line strip (RLS) techniques. Significant new findings have arisen from this study: B*2704, B*2705, B*2707, B*2708 and B*2714 alleles were found to be present, and two novel B27 alleles, B*2708 and B*2714, were found in this Indian population. In addition, B*2714 was observed in a patient with ankylosing spondylitis. This association has not been previously reported in ethnic groups from India.
Definitive high resolution typing of HLA‐E allelic polymorphisms: Identifying potential errors in existing allele data
Authors: Grimsley C. , Kawasaki A. , Gassner C. , Sageshima N. , Nose Y. , Hatake K. , Geraghty D.E. , Ishitani A.
Published: March 2003

Abstract: A set of robust PCR‐SSP reactions were developed for each of the five polymorphic sites that define the five alleles of the HLA class Ib gene, HLA‐E. This method was developed using 28 homozygous cell lines and further tested in a sample of African‐Americans, a sample of Japanese, and a core panel of cell lines compiled for the 13th International Histocompatibility Workshop. Three alleles were found in each of these four sample groups, HLA‐E*0101 (64.29, 50.00, 32.00 and 56.58%, respectively), *01031 (5.36, 20.65, 39.00 and 18.42%) and *01032 (30.35, 29.35, 29.00, and 25.00%). HLA‐E*0102 was not detected in any of these samples nor in the cell line, LCL 722.221, in which this allele was originally described. HLA‐E*0104 was not found either. This latter allele was originally reported in Japanese at a frequency of 1/22 (4.5%), which should have been high enough to have resulted in multiple occurrences of the *0104 allele in the samples tested in this study. We propose that the existence of the HLA‐E*0102 and E*0104 alleles should be questioned.
HLA‐A and ‐B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais
Authors: Stephens H.A.F. , Klaythong R. , Sirikong M. , Vaughn D.W. , Green S. , Kalayanarooj S. , Endy T.P. , Libraty D.H. , Nisalak A. , Innis B.L. , Rothman A.L. , Ennis F.A. , Chandanayingyong D.
Published: March 2003

Abstract: Little is known of the role of classical HLA‐A and ‐B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case–control study of 263 ethnic Thai patients infected with either DEN‐1, ‐2, ‐3 or ‐4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA‐A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA‐A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN‐1 and DEN‐2 infections only. Conversely, HLA‐B*51 was associated with the development of DHF in patients with secondary infections, and HLA‐B*52 was associated with DF in patients with secondary DEN‐1 and DEN‐2 infections. Moreover, HLA‐B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.
Synthesis and biological evaluation of destruxin A and related analogs
Authors: Ast T. , Schmidt M. , Germeroth L. , Wenschuh H. , Barron E. , Kinne L. , Simmons K.
Published: March 2003

Abstract:This report describes the development of an efficient solid‐phase synthesis protocol and adaptation of reported solution phase procedures for the synthesis of the cyclic depsihexapeptide destruxin A and related analogs. The solid‐phase method described is based on standard Fmoc peptide chemistry, including a new synthetic method for the assembly of the depsi bond‐containing unit. In order to select analogs of destruxin A for synthesis and evaluation of insecticidal activity, the work of Hellberget al., describing a set ofZ‐descriptors for amino acid side‐chains comparing their physicochemical properties, was utilized. Destruxin A and 27 different analogs with structural variations in four residues were synthesized and insecticidal activity was evaluated via injections into tobacco budworm (Heliothis virescens) larvae. Several destruxin A analogs were found to be at least as potent as the native compound.
Syntheses, characterization and application of cross‐linked polystyrene−ethyleneglycol acrylate resin (CLPSER) as a novel polymer support for polypeptide syntheses
Authors: Leena S. , Kumar K.S.
Published: March 2003

Abstract:Cross‐linked polystyrene−ethyleneglycol acrylate resin (CLPSER) was developed for the solid‐phase synthesis of peptide by introducing a cross‐linker,O,O′‐bis(2‐acrylamidopropyl)polyethylene glycol1900(Acr2PEG), into polystyrene. The cross‐linker was prepared by treating acryloyl chloride withO,O′‐bis(2‐aminopropyl) polyethylene glycol1900[(NH2)2PEG] in the presence of diisopropylethylamine. The copolymer was prepared either by bulk or inverse suspension copolymerization of Acr2PEG1900and styrene using sorbitan monolaurate as the suspension stabilizer, and a mixture of ammonium peroxodisulfate and benzoyl peroxide as the radical initiators. The resin was characterized using gel‐phase13C NMR, infrared (KBr) spectroscopic techniques and the morphological features of the resin were investigated using scanning electron microscopy photographs. CLPSER showed excellent swelling in a broad range of solvents and was found to be chemically inert to various reagents and solvents used in solid‐phase peptide synthesis. To demonstrate the usefulness of the new resin in polypeptide synthesis, the support was derivatized with an ‘internal reference’ amino acid (norleucine) and a handle 4‐(4‐hydroxymethyl‐3‐methoxy)butyric acid. The new resin was compared with commercial supports such as Merrifield and Sheppard resins by synthesizing an acyl carrier protein (65−74) fragment under the same experimental conditions. HPLC profiles revealed the high efficiency of the newly developed support. Resin capability in peptide synthesis was further demonstrated by the solid phase synthesis of a 25‐residue peptide from the E2/NS1 region hepatitis C viral polyprotein.
Structure−function studies on the amphibian peptide brevinin 1E: translocating the cationic segment from the C‐terminal end to a central position favors selective antibacterial activity
Authors: Kumari V.K. , Nagaraj R.
Published: March 2003

Abstract: Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC, is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide‐bridged forms have relatively broad‐spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKCAANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.
Solid‐phase synthesis of peptides containing the spin‐labeled 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)
Authors: Martin L. , Vita C. , Ivancich A. , Formaggio F. , Toniolo C.
Published: March 2003

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a nitroxide spin‐labeled, achiral Cα‐tetrasubstituted amino acid recently shown to be not only an effective β‐turn and 310/α‐helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid‐phase synthesis in an automated apparatus.
Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2receptor
Authors: Ahmed S.I. , Wibowo F. , Gembitsky D.S. , Bozsó Z. , Murphy R.F. , Lovas S.
Published: March 2003

Abstract:The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2receptor were assessed using analogs of [Leu15]G(11−17). The following peptides were synthesized, Ac[Leu15]G(11−17), Ac[Leu15]G(11−16)NH2, [Leu15]G(11−17), [Leu15,Ala17]G(11−17), [Leu15,Abu17]G(11−17), [Leu15,Val17]G(11−17), [Leu15,Leu17]G(11−17), [Leu15,Cha17]G(11−17), [Leu15,Trp17]G(11−17), [Leu15,Tic17]G(11−17), [Leu15,d‐Phe17]G(11−17) and [Leu15,p‐X‐Phe17]G(11−17), where X = F, Cl, Br, I, OH, CH3, NH2and NO2.Competition binding experiments with [3H]CCK‐8 were performed using human CCK2receptors stably expressed in CHO cells. Phe17was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in theg(+) org(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation ofd‐Phe decreases binding. The size and electron withdrawing/donating properties of theparasubstituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2receptor.
Role of N‐t‐Boc group in helix initiation in a novel tetrapeptide
Authors: Ganesh S. , Jayakumar R.
Published: March 2003

Abstract: Protecting groups in N‐ and C‐terminal positions play a decisive role in the conformational preference of smaller peptides. Conformational analysis of tetrapeptide derivatives containing Ala, Ile and Gly residues was performed. Peptide 1, Boc‐Ala‐Ile‐Ile‐Gly‐OMe (Boc: tert‐butyloxycarbonyl) has a predominantly helical turn conformation in all the alcoholic solvents studied, whereas in the solid state it has a β‐sheet conformation. In contrast, peptide 2, Ac‐Ala‐Ile‐Ile‐Gly‐OMe (Ac: acetyl) has a random coil conformation in solution. The FTIR spectrum of peptide 1 shows a lower frequency of urethane carbonyl, indicating involvement of the carbonyl group in hydrogen bonding in the helical turn.
Function of anti–Porphyromonas gingivalis immunoglobulin classes in immunized Macaca fascicularis
Authors: Houston L. S. , Lukehart S. A. , Persson G. R. , Page R. C.
Published: March 2003

We previously reported that Macaca fascicularis immunized with formalin‐killed Porphyromonas gingivalis were protected against the bone loss of periodontitis. To examine mechanisms of protection, we determined specific immunoglobulin G (IgG), IgM and IgA titers and opsonic capacities of sera from immunized and control animals. Serum IgG and IgA titers to P. gingivalis appeared early and persisted throughout the 36‐week observation period. IgM titers were elevated until 6 to 12 weeks and then decreased through week 36. A significant association was observed between peak IgM titers prior to ligature placement and protection against bone loss (measured at week 30). In control monkeys, no significant IgG, IgA or IgM titers were seen. In sera from immunized animals, significant opsonic capacity was seen by 6–12 weeks and persisted throughout the study. In contrast, control sera showed only low opsonization capacity. Anti–P. gingivalis antibody titers in purified IgG, IgA and IgM fractions were determined by enzyme‐linked immunosorbent assay, and opsonic activity was demonstrated only in the IgG fraction.
Actinobacillus actinomycetemcomitans serotype e – biotypes, genetic diversity and distribution in relation to periodontal status
Authors: Doğan B. , Saarela M. H. , Jousimies‐Somer H. , Alaluusua S. , Asikainen S.
Published: March 2003

Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1–6 isolates per subject (n=61) were genotyped using arbitrarily primed–polymerase chain reaction (AP‐PCR) and apaH gene polymerase chain reaction–restriction fragment‐length polymorphism (PCR‐RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n=219), localized juvenile periodontitis (n=55) and other forms of early‐onset periodontitis (n=18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non‐periodontitis subjects (n=64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early‐onset periodontitis patients and in 5 (8%) non‐periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP‐PCR distinguished 3 and apaH gene PCR‐RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP‐PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP‐PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.
Oral and systemic immunoglobulin G‐subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin
Authors: Engström P.‐E. , George M. , Larsson P. , Lally E. T. , Taichman N. S. , Norhagen E. G.
Published: March 2003

Salivary, gingival crevicular fluid and serum‐specific immunoglobulin G (IgG)‐ subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin were quantified by enzyme‐linked immunosorbent assay. Samples were taken from six patients with periodontal pockets ≥5 mm, harboring A. actinomycetemcomitans in subgingival plaque and from six healthy, sex‐ and age‐matched controls, who did not harbor A. actinomycetemcomitans. In individuals suffering from periodontitis, the median values of specific IgG1‐ and IgG2‐subclass antibodies in saliva, gingival crevicular fluid and serum were, respectively IgG1 147 ng/ml, 5226 ng/ml and 7318 ng/ml and IgG2 4.8 ng/ml, 934 ng/ml and 860 ng/ml. In the patients, specific IgG3 antibodies were detected in one out of six individuals in saliva, in two individuals in gingival crevicular fluid and in five out of six patients in serum with a median value of 561 ng/ml. The median values of specific IgG4 antibodies in saliva, gingival crevicular fluid and serum were below detectable levels. The median values of the total IgG subclasses in saliva and serum were 14622 ng/ml and 10.3 g/l respectively. Individuals with periodontitis had, compared with controls, a higher ratio of specific IgG1 antibodies to total IgG1 in saliva (P<0.05) and in serum (P<0.05) and a higher ratio of specific IgG antibodies to total IgG in saliva (P<0.05) and in serum (P<0.01). The results show an elevation of both oral and systemic specific antibodies to A. actinomycetemcomitans leukotoxin.
Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains
Authors: Beem J. E. , Nesbitt W. E. , Leung K.‐P.
Published: March 2003

A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli. The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6‐kb insert from Sau 3 AI–digested P. intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18. EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release. Cell suspensions of EB34 demonstrated a dose‐dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants. Cell‐free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity. Sub‐cloning experiments localized the hemolytic region of the insert to a 3.9‐kb fragment under direction of the lac promoter. Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay. Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay. Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates. Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.
Herpesvirus infection of inflammatory cells in human periodontitis
Authors: Contreras A. , Zadeh H. H. , Nowzari H. , Slots J.
Published: March 2003

Human cytomegalovirus (HCMV) and Epstein‐Barr virus type 1 (EBV‐1) are frequently detected in human periodontitis lesions. However, no information is available on the types of gingival cells infected by herpesviruses. The present study determined the presence of herpesviruses in polymorphonuclear neutrophils, monocytes, macrophages and T and B lymphocytes in biopsies of periodontitis lesions from 20 adults. A nested polymerase chain reaction method was employed to detect HCMV, EBV‐1, EBV‐2, human herpes virus‐6 and herpes simplex virus (HSV) in periodontal tissue biopsy and in gingival cell fractions separated by immunomagnetic cell sorting. Tissue specimens from 18 (90%) and cell fractions from 14 (70%) patients demonstrated herpesviruses. Periodontitis‐derived monocytes and macrophages revealed HCMV in cell fractions from 11 (55%) patients and HSV in cells from 1 (5%) patient. T lymphocytes harbored HCMV in cell fractions from 4 (20%) patients and HSV in cell fractions from 4 (20%) patients. B lymphocytes showed EBV‐1 in cell fractions from 9 (45%) patients. Periodontal polymorphonuclear neutrophils demonstrated no herpesviruses. This study suggests that HCMV mainly infects periodontal monocytes, macrophages and less frequently T lymphocytes and that EBV‐1 infects periodontal B lymphocytes. The possible etio‐pathologic significance of periodontal herpesvirus infection is discussed.
A DNA probe specific to Streptococcus sobrinus
Authors: Ida H. , Igarashi T. , Yamamoto A. , Goto N. , Sasa R.
Published: March 2003

Three DNA fragments (SSB‐1, ‐2 and ‐3) in the dextranase gene (dex) of Streptococcus sobrinus were amplified by polymerase chain reaction and used as DNA probes. The probes were examined for the specificity and the sensitivity of hybridization with DNA of oral streptococcal species. While probes SSB‐1 and SSB‐2 were specific to both S. sobrinus and Streptococcus downei, SSB‐3 was specific only to S. sobrinus. SSB‐3 was able to detect 5 ng of chromosomal DNA purified from S. sobrinus NIDR6715 and DNA extracted from 1×105 cells of the strain. In addition, SSB‐3 could differentiate clinical isolates of S. sobrinus from Streptococcus mutans. These results suggest that SSB‐3 is an effective DNA‐probe to detect and to identify S. sobrinus.
Prevalence of β‐lactamase‐producing strains among 149 anaerobic gram‐negative rods isolated from periodontal pockets
Authors: Fosse T. , Madinier I. , Hitzig C. , Charbit Y.
Published: March 2003

In a prospective study, 47 adults presenting a rapidly progressive periodontitis were selected in order to evaluate the prevalence of β‐lactamase‐producing strains among oral anaerobic gram‐negative rods. Predominant anaerobes were identified from two of the deepest periodontal pockets. β‐Lactamase‐positive strains fulfilled to at least two of three criteria: positive nitrocefin test, penicillin Etest minimal inhibitory concentration>1 μg/ml, and disk diffusion synergy between amoxycillin and clavulanic acid>10 mm. At least one β‐lactamase‐producing strain was found in 53.2% of patients and 39.4% of the periodontal pockets investigated. Prominent β‐lactamase‐positive species were Prevotella buccae and Prevotella intermedia (respectively 16 of 38: 42% and 18 of 52: 35% positive strains), followed by Prevotella bivia, Prevotella disiens, Prevotella denticola and Fusobacterium nucleatum (respectively 1 of 6: 17%, 1 of 10: 10%, 1 of 10: 10%, and 1 of 13: 8% positive strains). No β‐lactamase producer could be evidenced in Porphyromonas gingivalis (10 strains tested). All the β‐lactamase‐positive strains with the nitrocefin test had penicillin minimal inhibitory concentrations>1 μg/ml with the Etest, and a strong synergy between amoxicillin and clavulanic acid was always observed.
Adhesion of oral Candida species to human buccal epithelial cells following brief exposure to nystatin
Authors: Ellepola A. N. B. , Panagoda G. J. , Samaranayake L. P.
Published: March 2003

Opportunistic oral infections caused by Candida albicans and non‐albicans Candida species are particularly common in compromised patients. Nystatin, which belongs to the polyene group of antimycotics, is frequently used as a topical agent in the treatment of oro‐pharyngeal candidosis. It is recognized that due to the delivery mode of nystatin (i.e. topical, intermittent), as well as the cleansing effect of saliva within the oral environment, the yeasts undergo a relatively brief exposure to this drug during treatment. Nevertheless, there is a sparsity of data on the effect of such brief exposure to nystatin on the pathogenic attributes of Candida such as their adherence to host surfaces. The adhesion of microbes to host mucosal surfaces is a major determinant of successful colonization and infection. Thus the main aim of our investigation was to compare the in vitro adhesion of 30 oral isolates of Candida belonging to six different species (comprising Candida albicans, Candida tropicalis, Candida glabrata, Candida guilliermondii, Candida krusei and Candida parapsilosis) to human buccal epithelial cells, following their brief exposure (1 h) to minimum inhibitory concentration of nystatin, and subsequent removal of the drug. The adhesion of these isolates to buccal epithelial cells was assessed by a previously described adhesion assay. Compared with the controls, there was a significant reduction in buccal epithelial cell adhesion of all six Candida species after drug exposure (54%–68%). However the adhesion of C. albicans isolates was the least affected by nystatin exposure, which was significantly different from that of the non‐albicans species. These findings imply that sub‐therapeutic levels of nystatin, which are likely to persist in the oral cavity during dosing intervals, may also be beneficial, as they inhibit candidal colonization. The significant difference in nystatin‐induced suppression of adhesion between C. albicans and the non‐albicans species investigated is a further testimonial for the pre‐eminent virulence of the former species.
Factors affecting the formation of spherical bodies in the spirochete Treponema denticola
Authors: De Ciccio A. , McLaughlin R. , Chan E. C. S.
Published: March 2003

The oral spirochete Treponema denticola typically is a helically shaped, motile bacterial cell. However, morphological variations of T. denticola cells in the form of “spherical bodies” are sometimes seen. Little is known about the environmental factors that cause their formation. The effects of oxygen, growth temperature, nutrient depletion and the addition of metabolic end‐products were tested to determine their role in the morphogenesis of the spherical bodies. It was found that the age of the culture, the omission of individual components (yeast extract, rabbit serum, volatile fatty acids or thiamine pyrophosphate) from the medium and the addition of the metabolic end product lactic acid enhanced the formation of these bodies. However, their formation was decreased upon omission of the medium components asparagine and sodium bicarbonate.
Antimicrobial effect of acidified nitrite on cariogenic bacteria
Authors: Silva Mendez L. S. , Allaker R. P. , Hardie J. M. , Benjamin N.
Published: March 2003

The antimicrobial agent nitric oxide (NO) is formed in the mouth and its concentration is directly related to salivary nitrite, which in turn is related to dietary nitrate intake. The aim of this study was to determine whether nitrite under acidic conditions will have an inhibitory effect, possibly ocurring through NO production, on Streptococcus mutans, Lactobacillus casei and Actinomyces naeslundii. Whereas the growth of S. mutans was inhibited by a more acid pH, the addition of nitrite caused a marked, further dose‐dependent reduction in bacterial numbers after 24 h of exposure. Similar effects were observed with A. naeslundii and L. casei. The ability of these bacteria to recover from nitrite exposure was also markedly affected by nitrite concentration. At acidity levels below pH 7, low concentrations of nitrite (0.2 mM) caused effective complete killing of S. mutans, with similar effects on the other organisms tested. These results demonstrate the possibility that nitrite in saliva has an effect on the growth and survival of cariogenic bacteria.
Enhanced interleukin 1β, interleukin 6 and tumor necrosis factor α in gingival crevicular fluid from periodontal pockets of patients infected with human immunodeficiency virus 1
Authors: Baqui A. A. M. A. , Meiller T. F. , Jabra‐Rizk M. A. , Zhang M. , Kelley J. I. , Falkler W. A.
Published: March 2003

Loss of periodontal support and eventually tooth loss is a common finding among acquired immunodeficiency syndrome (AIDS) patients. The cause of this destruction may be an increase in periodontal disease activity at sites within the same individual and also may be related to an increase in the pro‐inflammatory cytokines, diffused through the gingival crevicular sulcus in AIDS patients. A study was undertaken to determine the relative levels of the pro‐inflammatory cytokines, interleukin 1β (IL‐1β), IL‐6, and tumor necrosis factor α (TNF‐α), in gingival crevicular fluid collected from the deep (>5 mm periodontal pocket depth) and shallow (≤3 mm periodontal pocket depth) periodontal pockets of 39 HIV‐1‐infected patients and 20 age‐, race‐ and sex‐matched uninfected controls. Complete medical history including risk factors such as intravenous drug abuse was taken. Gingival crevicular fluid samples were collected on periopaper strips. Cytokines were estimated by solid‐phase enzyme‐linked immunosorbent assay. To assess the degree of HIV activity, the viral load of these patients was determined by an Amplicor HIV‐1 monitor kit using reverse transcriptase polymerase chain reaction. Gingival crevicular fluid from HIV‐1‐infected patients showed a two‐fold increase in both IL‐1β and TNF‐α in deep periodontal pockets in comparison to shallow pockets, whereas IL‐6 increased 1.8‐fold. There was a significant (P<0.05) increase in IL‐1β, IL‐6 and TNF‐α in gingival crevicular fluid (both shallow and deep pockets) from HIV‐1‐infected patients in comparison to uninfected controls and also significantly elevated in deep versus shallow pockets in these patients. Although IL‐1β, IL‐6 and TNF‐α levels among HIV‐1‐infected patients with a high viral load (>10,000 copies/ml) were higher than those from patients with a low viral load (<400 copies/ml), only the increase in IL‐1β level associated with deep pockets was significant (P<0.05). There was also a trend of an increase in all the three cytokines among intravenous drug–abusing HIV‐1‐infected patients in comparison to non‐intravenous drug abusers, but only the difference in IL‐1β levels from deep pockets reached significance (P<0.05). These enhanced pro‐inflammatory cytokine levels in the gingival crevicular fluid of HIV‐positive patients may be an important factor in causing the advanced periodontal lesions sometimes observed in HIV‐positive patients.
Subclinical reactivation of herpes simplex virus type 1 in the oral cavity
Authors: Knaup B. , Schünemann S. , Wolff M. H.
Published: March 2003

Reactivation in the oral cavity either symptomatically (recrudescence) or without symptoms (recurrence) may contribute to the transmission of herpes simplex virus type 1 (HSV‐1), especially in critical areas of exposure such as dentistry. In order to measure the frequency of HSV‐1 reactivation, nested polymerase chain reaction (PCR) was performed on oral swabs collected from 30 healthy people over a period of 58–161 days. In total 19 of 25 (76%) seropositive people were PCR‐positive at least once, 6 of these 19 (32%) had recrudescence and 13 (68%) had only asymptomatic reactivation. Frequencies of additional recurrences were higher in people showing symptomatic reactivation than in those who had only recurrences. Recrudescence is a risk factor for elevated levels of asymptomatic HSV‐shedding. In most cases HSV‐1 was detected only by nested PCR investigated by early onset of therapy or time span before sampling.
Genetic transformation in Streptococcus mutans requires a peptide secretion–like apparatus
Authors: Petersen F. C. , Scheie A. AA.
Published: March 2003

Competence for genetic transformation in Streptococcus pneumoniae and Streptococcus gordonii involves the ComAB secretion apparatus, which is thought to export the competence‐stimulating peptide. Homologous secretory systems are also used for the export of certain bacteriocins and bacteriocin‐like peptides. In this study, a similar secretory apparatus was found in the Streptococcus mutans genome, and its role in transformation was investigated. Gene inactivation resulted in a mutant deficient in transformability. We suggest that secretion of a peptide, possibly the competence‐stimulating peptide itself, is involved in competence induction also in S. mutans.
Antimicrobial effect of acidified nitrite on periodontal bacteria
Authors: Allaker R. P. , Silva Mendez L. S. , Hardie J. M. , Benjamin N.
Published: March 2003

The antimicrobial agent nitric oxide (NO) is formed in the mouth and its concentration is directly related to salivary nitrite, which in turn is related to dietary nitrate intake. The aim of this study was to determine whether nitrite under acidic conditions will have an inhibitory effect, possibly occurring through NO production, on the periodontal disease pathogens Fusobacterium nucleatum, Eikenella corrodens and Porphyromonas gingivalis. Whereas the growth of these organisms was inhibited by a more acid pH, the addition of nitrite caused a marked, further dose‐dependent reduction in bacterial numbers after exposure. The ability of these bacteria to recover from nitrite exposure was also affected by pH and nitrite concentration. At acidity levels below pH 5.0, low concentrations of nitrite (0.2 mM) caused effective complete killing of the periodontal bacteria. Addition of sodium thiocyanate did not increase the bacteriostatic or bacteriocidal activity of acidified nitrite against any of the 3 bacteria. These results demonstrate the possibility that nitrite in saliva, under appropriate conditions, may have an effect on the growth and survival of the bacteria implicated in periodontal disease.
Adhesion of viridans group streptococci to sialic acid‐, galactose‐ and N‐acetylgalactosamine‐containing receptors
Authors: Takahashi Y. , Ruhl S. , Yoon J.‐W. , Sandberg A. L. , Cisar J. O.
Published: March 2003

The binding of 10 viridans group streptococci to sialic acid‐, galactose (Gal)‐ and N‐acetylgalactosamine (GalNAc)‐containing receptors was defined by analysis of the interactions between these bacteria and structurally defined glycoconjugates, host cells and other streptococci. All interactions with sialic acid‐containing receptors were Ca2+‐independent as they were not affected by ethyleneglycoltetraacetic acid (EGTA), whereas all interactions with Gal‐ and GalNAc‐containing receptors were Ca2+‐dependent. Recognition of sialic acid‐, Gal‐ and GalNAc‐containing receptors varied widely among the strains examined, in a manner consistent with the association of each of the three lectin‐like activities with a different bacterial cell surface component.
The relationship between the levels of SigA, lactoferrin and α1 proteinase inhibitor in saliva and permanent dentition caries in 15‐year‐olds
Authors: Sikorska M. H. J. , Mielnik‐Blaszczak M. , Kapeć E.
Published: March 2003

A group of 15‐year‐olds were clinically examined for general state of dentition and level of dental caries. Unstimulated whole saliva from the subjects was laboratory tested to determine the levels of lactoferrin, Secretory IgA (SIgA) and α1 proteinase inhibitor. A significant relationship was found between the decayed surface index and the levels of lactoferrin, SIgA and α1 proteinase inhibitor in the unstimulated saliva. The decayed surface index rose with increases in the levels of lactoferrin, SIgA and α1 proteinase inhibitor, the relationship with α1 proteinase inhibitor being strongest.
Immunohistochemical localization of Toll‐like receptors 2 and 4 in gingival tissue from patients with periodontitis
Authors: Mori Y. , Yoshimura A. , Ukai T. , Lien E. , Espevik T. , Hara Y.
Published: March 2003

The present study investigated the expression of Toll‐like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2‐, TLR4‐, CD14‐ and CD1a‐positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2‐positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4‐positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14‐positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down‐regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.