bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐10 expressed at early tumour sites induces subsequent generation of CD4+ T‐regulatory cells and systemic collapse of antitumour immunity
Authors: Seo Naohiro , Hayakawa Satoshi , Takigawa Masahiro , Tokura Yoshiki
Published: March 2003

We investigated the relationship between transforming growth factor‐β (TGF‐β)‐secreting T‐regulatory (Tr) cells and anti‐B16 melanoma immunity, and studied the association of early cytokines expressed at tumour sites with the generation of Tr cells. A large number of CD4+ Tr cells producing interleukin (IL)‐4, IL‐10 and TGF‐β accumulated with functionally depressed CD8+ cytotoxic T lymphocytes (CTLs) at tumour sites on day 20 after subcutaneous (s.c.) inoculation of B16 tumour cells. Tr cells consisted of two populations, which were termed T helper 3 (Th3) and Tr1 cells. B16‐infiltrating Tr cells strongly inhibited the generation of B16‐specific T helper 1 (Th1) cells in a TGF‐β‐dependent manner and were assumed to suppress effective generation of CTLs. In addition, B16 cells markedly progressed in mice transferred adoptively by the cultured B16‐infiltrating Tr cells compared with untreated mice. The capacity of these Tr cells to produce TGF‐β was hampered by neutralizing anti‐IL‐10 and partly anti‐IL‐4 monoclonal antibodies (mAbs) injected intralesionally during the early development of B16 tumours, and this treatment markedly attenuated B16 growth. Furthermore, a lesional injection of recombinant mouse IL‐10 at an early tumour site resulted in the vigorous progression of B16 tumours. These results provide evidence that Tr cells, belonging to the T helper 3/T‐regulatory 1 (Th3/Tr1) type, are activated in B16‐bearing hosts under the influence of T helper 2 (Th2) cytokines, mainly IL‐10 (produced at early tumour lesions), and that this regulatory T‐cell population functions as a suppressor of anti‐B16 immunity.
In vitro stimulation with a non‐peptidic alkylphosphate expands cells expressing Vγ2‐Jγ1.2/Vδ2 T‐cell receptors
Authors: Evans Peter S. , Enders Patrick J. , Yin Cheng , Ruckwardt Tracy J. , Malkovsky Marek , Pauza C. David
Published: March 2003

The majority of peripheral blood γδ T cells in healthy adult humans express the Vγ2/Vδ2 T‐cell receptor (TCR) and generate TCR‐mediated, major histocompatibility complex (MHC)‐unrestricted proliferative responses to low molecular weight alkylphosphates. Vγ2/Vδ2 populations after antigen proliferation maintained diversity in the CDR3s of Vγ2 mRNA, indicating that the response was polyclonal or oligoclonal, and were enriched for Vγ2 TCR chains containing the Jγ1.2 segment. Alkylphosphate stimulation further skewed an already biased peripheral blood γδ T‐cell population and increased the abundance of Vγ2‐Jγ1.2/Vδ2 T cell receptors, suggesting similarities between the alkylphosphate response and peripheral selection mechanisms shaping this repertoire in human beings.
Schiff base‐mediated co‐stimulation primes the T‐cell‐receptor‐dependent calcium signalling pathway in CD4 T cells
Authors: Hall Simon R. , Rhodes John
Published: March 2003

In addition to macromolecular interactions that provide co‐stimulation during antigen‐presenting cell (APC) and CD4+ T‐cell conjugation, covalent chemical events between specialized ligands have been implicated in T‐cell co‐stimulation. These take the form of transient Schiff base formation between carbonyls and amines expressed on APC and T‐cell surfaces. Small Schiff base‐forming molecules, such as tucaresol, can substitute for the physiological donor of carbonyl groups and provide co‐stimulation to T cells, thereby functioning as orally active immunopotentiatory drugs. The Schiff base co‐stimulatory pathway in T cells has been partially characterized in terms of changes in Na+ and K+ transport, and activation of the mitogen activated protein kinase (MAPK) ERK2. In the present study, the effects of Schiff base co‐stimulation by tucaresol on the T‐cell receptor (TCR)‐dependent pathway leading to Ca2+ release were investigated. Schiff base co‐stimulation by tucaresol was found to prime for enhanced TCR‐dependent phospholipase C‐γ phosphorylation, inositol 1,4,5‐triphosphate production, and Ca2+ mobilization that correlated with functional enhancement of interleukin‐2 production in primary T cells. The effects on Ca2+ occurred comparably in Jurkat and primary CD4+ T cells responding to anti‐CD3 monoclonal antibody. Enhancement of the Ca2+ response required a 10‐min priming period and was prevented by prior covalent ligation of cell‐surface free amino groups by sulpho‐N‐hydroxy succinimido‐biotin; clofilium‐mediated inhibition of tucaresol‐induced changes in intracellular K+; and selective inhibition of the MAPK pathway. The data are consistent with a priming mechanism in which late co‐stimulation‐triggered events exert a positive influence on early TCR‐triggered events. In additional studies of murine T cells expressing trans‐gene TCRs, tucaresol was likewise shown to prime for enhanced Ca2+ mobilization in response to physiological TCR‐engagement by MHC–peptide complexes.
Administration of plasmids expressing interleukin‐4 and interleukin‐10 causes BALB/c mice to induce a T helper 2‐type response despite the expected T helper 1‐type response with a low‐dose infection ofLeishmania major
Authors: Yamakami Kazuo , Akao Shinkichi , Tadakuma Takushi , Nitta Yoshio , Miyazaki Jyun‐Ichi , Yoshizawa Nobuyuki
Published: March 2003

BALB/c mice are susceptible to developing an infection with Leishmania major as a result of a fatal T helper 2 (Th2)‐type response. However, mice infected with a low dose of parasites are reported to be able to overcome the lesions associated with a T helper 1 (Th1)‐type response. To clarify why a difference in the dose of parasites induces a difference in the polarization of the Th phenotype, we first attempted to measure cytokine production. Soon after infection, the mice given high doses of parasites produced elevated levels of both Th1 [interferon‐γ (IFN‐γ)] and Th2 [interleukin (IL)‐4 and IL‐10] cytokines. However, when assessed at 1 and 2 weeks after infection, no significant difference in the balance of Th1 and Th2 cytokines could be detected between mice infected with low or high doses of L. major. These results support the notion that the Th2 cytokine levels at an early phase of infection could be a key factor for the induction of a Th2 response. In order to assess the efficacy of Th2 cytokines, the mice infected with low doses of L. major were co‐administered IL‐4 plasmid and IL‐10 plasmid. Consequently, the mice (which originally exhibited a Th1 response) showed progressive disease and developed a Th2 response. However, administration of these plasmids at 7 days postinfection could not alter the Th polarization. Furthermore, production of IL‐12 from the spleen cells stimulated by L. major was suppressed in the presence of IL‐4 and IL‐10. These results strongly suggest that the susceptibility to L. major in BALB/c mice depends on the persistence of Th2 cytokine levels at an early phase of infection.
T‐cell activation occurs simultaneously in local and peripheral lymphoid tissue following oral administration of a range of doses of immunogenic or tolerogenic antigen although tolerized T cells display a defect in cell division
Authors: Smith Karen M. , Davidson Joanne M. , Garside Paul
Published: March 2003

How the mucosal immune system promotes active immunity against harmful organisms but tolerance to commensal bacteria or dietary antigens is poorly understood. Thus, the antigen‐presenting cell (APC), site of antigen presentation, and effector mechanisms responsible for oral priming and tolerance remain unclear. Characterizing differences between oral priming and tolerance may improve the exploitation of oral tolerance for therapeutic applications and aid the design of oral vaccines. To address these questions we compared the mucosal and systemic activation and localization of antigen‐specific T cells during the induction of oral priming and tolerance. Activation marker expression and cell division by tg T cells was determined in conjunction with their anatomical location. These studies show that after feeding, T cells are activated in both peripheral and local lymphoid tissues within 6 hr, irrespective of the presence of adjuvant. Subsequently, T‐cell accumulation can be detected simultaneously in peripheral and mesenteric lymph nodes and Peyer's patches within 24 hr of feeding, but only after 3 days post feeding in the lamina propria. Primed and tolerized T cells adopted similar phenotypes as assessed by activation marker expression. However, within the mesenteric lymph nodes (MLN) tolerized T cells underwent significantly fewer divisions than primed T cells. Thus, T‐cell activation and expansion occurs throughout the animal after feeding a range of doses of antigen, irrespective of whether priming or tolerance is the eventual outcome. However, the presence of an adjuvant enhances clonal expansion in the MLN while tolerized T cells display defective cell division.
Hydroquinone, a reactive metabolite of benzene, enhances interleukin‐4 production in CD4+ T cells and increases immunoglobulin E levels in antigen‐primed mice
Authors: Lee M. H. , Chung S. W. , Kang B. Y. , Kim K.‐M. , Kim T. S.
Published: March 2003

Exposure to cigarette smoke is known to increase the risk of the development of allergic disease. The mechanism is not well understood. In this study, we determined the effect of hydroquinone (HQ), a major metabolite of benzene present in large quantities in cigarette tar, on interleukin‐4 (IL‐4) production by CD4+ T cells. HQ significantly enhanced IL‐4 production by keyhole limpet haemocyanin (KLH)‐primed CD4+ T cells in a dose‐dependent manner. The enhancing effect of HQ on IL‐4 production was maximal at a concentration of 50 µm. It increased the level of IL‐4 production approximately 10‐fold. HQ enhanced IL‐4 mRNA expression and also IL‐4 gene promoter activity, suggesting that the enhancing effect of HQ on IL‐4 production may occur at the transcriptional level. Furthermore, the injection of KLH‐primed mice with HQ resulted in a significant increase in the levels of IL‐4 and immunoglobulin E. These findings provide evidence that HQ, a major component of cigarette tar, may enhance allergic immune responses by inducing the production of IL‐4 in CD4+ T cells.
Mistletoe lectins enhance immune responses to intranasally co‐administered herpes simplex virus glycoprotein D2
Authors: Lavelle E. C. , Grant G. , Pusztai A. , Pfüller U. , Leavy O. , McNeela E. , Mills K. H. G. , O'Hagan D. T.
Published: March 2003

The mucosal adjuvant properties of the three type 2 ribosome‐inactivating proteins (RIPs) from the European mistletoe, Viscum album L., were investigated. Mistletoe lectins were compared with cholera toxin (CT) as adjuvants when delivered nasotracheally together with herpes simplex virus glycoprotein D2 (gD2). All three mistletoe lectins (MLI, MLII, MLIII) were potent mucosal adjuvants. Co‐administration of MLI, MLII or MLIII with gD2 led to significantly higher levels of gD2‐specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) antibody than when the antigen was delivered alone. The levels of antibodies induced were similar to those generated in mice immunized with gD2 and the potent mucosal adjuvant CT. Administration of ML1 with gD2 enhanced the antigen‐specific splenic T‐cell proliferative response. Interleukin‐5 (IL‐5), but not interferon‐γ (IFN‐γ), was detected in supernatants from splenocytes stimulated in vitro with gD2. This indicates that MLI enhanced type 2 T‐helper cell (Th2) responses to the bystander antigen, gD2. Analysis of the gD2‐ and lectin‐specific IgG subclass titres in mice immunized with gD2 and MLI, MLII or MLIII revealed a high ratio of IgG1 : IgG2a, which is compatible with the selective induction of Th2‐type immune responses.
Generation and characterization of ovine dendritic cells derived from peripheral blood monocytes
Authors: Chan Simon S. M. , Mcconnell Ian , Blacklaws Barbara A.
Published: March 2003

Dendritic cells (DCs) are professional antigen‐presenting cells with a highly immunostimulatory function and the capacity to activate naïve T cells. In recent years the rapid progress in mouse and human DC research can be mainly attributed to the generation of DCs from precursor cells in vitro, although a lack of reagents has hampered DC research in many large animal models. Here we describe the generation and characterization of ovine monocyte‐derived DCs in vitro. In addition to the characteristic morphology and non‐adherence of DCs, peripheral blood mononuclear cell monocytes cultured with ovine granulocyte–macrophage colony‐stimulating factor (GM–CSF) and interleukin‐4 (IL‐4) expressed CD11c and major histocompatibility complex (MHC) class II, but did not express CD14. High levels of endocytosis and an ability to stimulate antigen‐specific proliferation of CD4 T lymphocytes were also demonstrated.
Down‐regulation of the myeloid homeobox protein Hex is essential for normal T‐cell development
Authors: Mack David L. , Leibowitz David S. , Cooper Scott , Ramsey Heather , Broxmeyer Hal E. , Hromas Robert
Published: March 2003

The haematopoietic homeobox gene Hex (also called Prh) is expressed in myeloid cells and B cells but not T cells. To investigate whether Hex levels might play a role in myeloid versus T‐cell development, two types of transgenic mouse lines were constructed, each with ectopic expression of Hex in T cells (CD11a/Hex and Lck/Hex). Both these types of transgenic mouse had the same defects in T‐cell maturation, indicating that proper T‐cell development may be dependent not just on the up‐regulation of lymphoid‐specific transcriptional regulators but also on the co‐ordinated down‐regulation of myeloid‐specific transcriptional regulators such as Hex. In addition, Hex over‐expression significantly increased myeloid progenitor cycling, which may explain its role in retrovirally induced murine leukaemia.
Carbohydrate‐based particles: a new adjuvant for allergen‐specific immunotherapy
Authors: Grönlund Hans , Vrtala Susanne , Wiedermann Ursula , Dekan Gerhard , Kraft Dietrich , Valenta Rudolf , Van Hage‐Hamsten Marianne
Published: March 2003

The occurrence of systemic anaphylactic side‐effects in the course of allergen‐specific immunotherapy has been strongly reduced by the adsorption of allergens to aluminium hydroxide, the most frequently used adjuvant in humans. Using the major timothy grass pollen allergen, Phl p 5b, in its recombinant form for immunization of mice, we demonstrate that carbohydrate‐based particles (CBP) exhibit several potential advantages over aluminium‐hydroxide as adjuvant for immunotherapy. Similar to alum‐bound rPhl p 5b, CBP‐bound rPhl p 5b induced a stronger antibody and cytokine response than unbound rPhl p 5b after subcutaneous injection in mice. The antibodies induced by CBP‐bound rPhl p 5b, exhibited potentially beneficial activities as they cross‐reacted with group 5 allergens from five other grass species and inhibited the binding of grass pollen allergic patients IgE to Phl p 5b. Alum‐bound rPhl p 5b induced a preferential allergen‐specific Th2‐response characterized by high immunoglobulin G1 (IgG1) antibody levels and elevated interleukin (IL)‐4 and IL‐5 production in cultured splenocytes. By contrast, CBP‐bound rPhl p 5b, but not rPhl p 5b alone or coadministered with CBP, induced a mixed allergen‐specific T helper 1 (Th1)/Th2 immune response characterized by the additional production of allergen‐specific IgG2a/b antibody responses and elevated interferon‐γ production. Conjugation of rPhl p 5b to CBP yielded a stable vaccine formulation with preserved immunogenic features of the allergen and, in contrast to alum, induced no granulomatous tissue reactions. Based on these results, CBP is suggested as a potentially useful adjuvant for specific immunotherapy of IgE‐mediated allergies.
Identification of a new cluster of T‐cell receptor delta recombining elements
Authors: Przybylski Grzegorz K. , Wanzeck Jens , Verschuren Martie C. M. , Van Dongen Jacques J. M. , Serke Stephan , Schmidt Christian A.
Published: March 2003

Within the human T‐cell receptor δ (TCRD) gene we have identified a new cluster of seven δ recombining elements (δRec2.1–2.7), located 2·6–5·2 kilobases downstream of the Vδ2 gene segment. The δRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Dδ3 and Jδ1 segments of the TCRD gene as well as with the ψJα of the TCRA gene. Rearrangements involving the δRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100‐fold lower than that of classical δRec rearrangements. The total frequency of δRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T‐cell development. The decrease of the frequency of the δRec2‐Dδ3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the δRec2‐Jδ1 rearrangements contained the Dδ3 segment in the junctional region, we assume that they are derived from the δRec2‐Dδ3 rearrangements. In contrast, the majority of δRec2‐ψJα rearrangements did not contain the Dδ3 segment, indicating that they are single step rearrangements. The δRec2‐Jδ1 and δRec2‐ψJα rearrangements seem to be T‐lineage specific, but the δRec2‐Dδ3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that δRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Vα‐Jα rearrangements. We hypothesize, that in a similar manner to the classical δRec rearrangements, the δRec2 rearrangements might also contribute to T‐cell differentiation towards the TCR‐αβ lineage.
Communicative action‐based business process and information systems modelling with DEMO
Authors: Van Reijswoud Victor E. , Mulder Hans B. F. , Dietz Jan L. G.
Published: March 2003

Abstract. Abstract. The field of communicative action‐based modelling of business processes and information systems has attracted more and more attention in recent years. Inspired by the seminal work of Winograd and Flores, researchers have proposed several modelling approaches. In this article we discuss communicative action‐based modelling approaches in general and the DEMO (dynamic essential modelling of organizations) approach in particular. Besides establishing the theoretical foundations of this modelling approach, we also apply DEMO to a case study, and we discuss how the resulting models can be used for information systems design and business process optimization.
IT outsourcing: frameworks for conceptualizing practice and perception
Authors: Hancox Martin , Hackney Ray
Published: March 2003

This paper seeks to conceptualize theory and practice of information technology (IT)‐based outsourcing. It uses four conceptual frameworks in this respect – core competencies, transaction cost economics, agency theory and partnership – in an empirical comparison of IT outsourcing in the public and private sectors within the UK. The qualitative research methodology involved interviews with IT managers in both sectors and documentary analysis of outsourcing vendors’ literature. The findings suggest that practices and perceptions of IT outsourcing found in the two sectors support the basic principles of some frameworks more than others, e.g. core competencies, transaction cost economics and agency theory are well developed with partnerships less so. However, using all four frameworks improves understanding of multifaceted strategic practice in fundamentally different types of organization and helps to expose some issues which have not emerged in previous outsourcing studies.
Current directions in IS security research: towards socio‐organizational perspectives
Authors: Dhillon Gurpreet , Backhouse James
Published: March 2003

Abstract. The purpose of this paper is to map the current territory of information systems and security research. It uses the Burrell and Morgan framework as an intellectual map to analyse the socio‐philosophical concerns in various information systems and security approaches. The paper's contributions are in its analysis of trends in information systems and security research, the former in stressing the socio‐organizational perspectives and the latter in criticizing the preponderance of technical solutions. The paper also sets an agenda for a future research emphasis.
Inferences about orthopteroid phylogeny and molecular evolution from small subunit nuclear ribosomal DNA sequences
Authors: Flook P. K. , Rowell C. H. F.
Published: March 2003

We determined DNA sequences of SSU rRNA genes in twenty‐nine polyneopteran insect species and aligned these with homologues from eight other insects. In a phylogenetic analysis we recovered the classic divisions of Palaeoptera and Neoptera, with the latter divided into monophyletic Paraneoptera and Polyneoptera. The polyneopterans divided into three lineages: one includes the Grylloblattodea, Dermaptera and Plecoptera, the second contains the Blattodea, and the third (Orthopteroidea sensu Hennig) contains the Embiidina, Phasmida, and Orthoptera, in that order. The monophyly of the Orthoptera is supported by the analyses, as is the separation between taxa from its suborders Caelifera and Ensifera. The Caelifera are not always supported as a monophyletic group; the basal Tridactyloidea are separated from the rest of the Caelifera in some analyses. Inside of Tridactyloidea, the Acridoidea, Pamphagoidea, Pneumoroidea and Trigonopterygoidea are always recovered as a monophyletic group. We also examined the basal orthopteran relationships, with the specific aim of assessing the antiquity of the Ensifera. Character state reconstructions indicated that the ancestral ensiferan sequence is very similar to the ancestral orthopteran sequence. However, likelihood ratio tests rejected the null hypothesis of a molecular clock and we conclude that a change in substitution rate has occurred within the Orthoptera and several of the other polyneopteran orders. Similar observations have been made in holometabolous insects, suggesting that variation in substitution rate is a general feature of insect nuclear rRNA evolution.
Structure and expression of tandemly duplicated xanthine dehydrogenase genes of the silkworm (Bombyx mori)
Authors: Kômoto N. , Yukuhiro K. , Tamura T.
Published: March 2003

Xanthine dehydrogenase (XDH) is a molybdoenzyme which catalyses oxidation of xanthine and hypoxanthine to uric acid. We isolated genomic clones of silkworm (Bombyx mori) XDH genes (BmXDH1 and BmXDH2). The BmXDH2 The BmXDH2 gene is located upstream from the BmXDH1 gene and they show a tandemly duplicated structure. Both BmXDH genes were expressed in the fat body and Malpighian tubules, whereas only the BmXDH1 gene was expressed in the midgut. Phylogenetic analysis indicates that BmXDH gene duplication occurred after the divergence of the silkworm and dipteran species. Intron insertion site comparison shows that some introns were lost during insect XDH gene evolution.
Cloning of an aquaporin‐like cDNA and in situ hybridization in adults of the mosquito Aedes aegypti (Diptera: Culicidae)
Authors: Pietrantonio P. V. , Jagge C. , Keeley L. L. , Ross L. S.
Published: March 2003

A cDNA encoding a putative water channel protein, aquaporin, was cloned from a cDNA library of Aedes aegypti Malpighian tubules. The cDNA encodes a 26.11 kDa protein similar to insect aquaporins from Haematobia irritans exigua (Diptera) and Cicadella viridis (Homoptera), and to mammalian aquaporin 4. Localization of the messenger RNA (mRNA) was performed by in situ hybridization of Malpighian tubules and analysed by fluorescence and confocal microscopy. The mRNA was localized in tracheolar cells associated with the Malpighian tubules. No signal was detected in the Malpighian tubule epithelium. The molecular mechanisms for water movement between tissues and tracheoles are not yet elucidated in insects. Our results suggest a model to explain fluid movements in tracheoles during insect respiration.
The tetraspanin superfamily in insects
Authors: Todres E. , Nardi J. B. , Robertson H. M.
Published: March 2003

We describe four members of the tetraspanin/TM4SF superfamily of proteins that were identified in expressed sequence tag projects on the antennae of Manduca sexta moths and Apis mellifera honey bees. The three moth genes are expressed in the sensillar epithelium of male antennae, and some are expressed in female antennae, haemocytes, wing scale cell primordia and/or embryonic tissues. These proteins are probably involved in diverse cellular processes, much like their vertebrate homologues. A phylogenetic analysis of all known tetraspanins, including thirty‐seven members of the superfamily revealed by the Drosophilamelanogaster genome and twenty in the nematode Caenorhabditiselegans genome, reveals some phylum‐specific gene amplification, in particular a contiguous array of eighteen genes in the D. melanogaster genome.
Sequence, secondary structure and phylogenetic analyses of the ribosomal internal transcribed spacer 2 (ITS2) in the Timarcha leaf beetles (Coleoptera: Chrysomelidae)
Authors: Gómez‐Zurita J. , Juan C. , Petitpierre E.
Published: March 2003

Internal transcribed spacer 2 (ITS2) sequences of the nuclear rDNA in forty‐seven specimens (thirty‐four species) of the leaf beetle genus Timarcha have been studied. Timarcha ITS2 (523 bp on average) share some sequence features with other Chrysomeloidea relatives (Chrysolina, Diabrotica and Bruchus) but have no clear similarity with any other arthropod ITS2 sequences. Interspecific divergences are in the range 0.002–0.166, and 0.124–0.206 in the comparisons between subgenera. No evidence of intragenomic divergent ITS2 sequences has been found. Secondary structures are concordant with the four‐domain model proposed for vertebrates and yeast, but differs from those proposed for dipterans. Phylogenetic analysis of the ITS2 data confirms the results of a previous study based on mitochondrial sequences, as the basality of the Metallotimarcha subgenus and the absence of phylogenetic support for the Timarchostoma subgenus.
Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.
Authors: Favia G. , Lanfrancotti A. , Spanos L. , Sidén‐Kiamos I. , Louis C.
Published: March 2003

The sequence of a 2.3 kb long DNA segment derived from the 5′‐most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako.
The D7 family of salivary proteins in blood sucking diptera
Authors: Valenzuela J. G. , Charlab R. , Gonzalez E. C. , De Miranda‐Santos I. K. F. , Marinotti O. , Francischetti I. M. B. , Ribeiro J. M. C.
Published: March 2003

The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty‐one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.
Association of midgut defensin with a novel serine protease in the blood‐sucking fly Stomoxys calcitrans
Authors: Hamilton J. V. , Munks R. J. L. , Lehane S. M. , Lehane M. J.
Published: March 2003

Using ELISA we provide direct evidence that the midgut defensins of the blood‐sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post‐transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS‐stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.
Expression of D7 and D7‐related proteins in the salivary glands of the human malaria mosquito Anopheles stephensi
Authors: Suwan N. , Wilkinson M. C. , Crampton J. M. , Bates P. A.
Published: March 2003

Full‐length cDNA clones encoding D7 (AnsD7) and D7‐related (AnsD7r1) secreted salivary gland proteins were isolated from Anopheles stephensi. Corresponding proteins were separated by SDS‐PAGE and analysed by N‐terminal sequencing, which also identified a second D7‐related protein (AnsD7r2). AnsD7 encodes a protein of 37 kDa, AnsD7r1 of 18 kDa, and AnsD7r2 of 16 kDa. Polyclonal antibodies against recombinant AnsD7 showed immunological cross‐reactivity with the D7‐related proteins, and alignment demonstrated sequence similarity between the C‐terminal region of AnsD7 and the D7‐related proteins. AnsD7, AnsD7r1 and AnsD7r2 were major female‐specific salivary gland proteins, and Western blotting, immunohistochemistry and immunogold labelling demonstrated expression was predominantly in the secretory cavities of the distal‐lateral and median lobes. Expression and localization of D7 and D7‐related proteins was similar in Plasmodium berghei‐infected and uninfected mosquitoes.
Linkage map organization of expressed sequence tags and sequence tagged sites in the mosquito, Aedes aegypti
Authors: Severson D. W. , Meece J. K. , Lovin D. D. , Saha G. , Morlais I.
Published: March 2003

A composite genetic linkage map for the yellow fever mosquito Aedes aegypti was constructed based on restriction fragment length polymorphism (RFLP), single nucleotide polymorphism (SNP) and single strand conformation polymorphism (SSCP) markers. The map consists of 146 marker loci distributed across 205 cM, and includes several morphological mutant marker loci. Most of the genetic markers are derived from random cDNAs or Ae. aegypti genes of known function. A number of markers are derived from random genomic DNAs, including several cloned RAPD‐PCR fragments, and also several cDNAs from Drosophila melanogaster. Most of the random cDNAs (80.2%) have high BlastX sequence identities to known genes, with the majority of matches to genes from D. melanogaster. Access to sequence data for all markers will facilitate their continued development for use in high‐throughput SNP marker analyses and also provides additional physical anchor points for an anticipated genome sequencing effort.
Population variability in Chironomus (Camptochironomus) species (Diptera, Nematocera) with a Holarctic distribution: evidence of mitochondrial gene flow
Authors: Martin J. , Guryev V. , Blinov A.
Published: March 2003

Phylogenetic analysis of DNA sequences from mitochondrial (mt) genes (Cytochrome b and Cytochrome oxidase I) and one nuclear gene (globin 2b) was used for the investigation of Nearctic and Palearctic populations representing four Chironomus species of the subgenus Camptochironomus, namely C. biwaprimus, C. pallidivittatus, C. tentans sensu stricto and C. dilutus (the last two species constitute Holarctic C. tentans sensu lato). Phenograms constructed on the basis of mt sequences were not congruent with trees based on nuclear genes, or with morphological and cytological data. The mt tree divided the populations by continental region, rather than by the species groupings recognized by the other data sets. The incongruence is explained by mt gene flow resulting from hybridization between the sympatric species on each continent. Calculation of divergence times, based on the sequence data, suggest that C. tentans (s.l.) and C. pallidivittatus have both been in North America for about 2.5 My.
Two novel strains of Wolbachia coexisting in both species of mulberry leafhoppers
Authors: Mitsuhashi W. , Saiki T. , Wei W. , Kawakita H. , Sato M.
Published: March 2003

Wolbachia is an intracellular symbiont that causes reproductive disorders in many insects. Its presence in the leafhoppers Hishimonoides sellatiformis and Hishimonus sellatus, vectors of mulberry dwarf‐Phytoplasma, was confirmed by the PCR analysis of 16S rDNA, ftsZ and wsp. Sequencing of cloned PCR products revealed that two Wolbachia strains coexist in both leafhoppers. The phylogenetic analysis of wsp revealed that these strains belong in novel positions in the B‐group of Wolbachia. These strains were detected by PCR and/or PCR‐RFLP in all of the tested non‐genital organs including salivary glands, as well as in the tested genital organs of Hishimonoides sellatiformis. In addition, Wolbachia‐like organisms were observed by electron microscopy in all PCR‐positive organs. We discuss the possible horizontal transmission of Wolbachia via mulberry trees.
Toxoneuron nigriceps polydnavirus encodes a putative aspartyl protease highly expressed in parasitized host larvae
Authors: Falabella Patrizia , Varricchio Paola , Gigliotti Silvia , Tranfaglia Antonio , Pennacchio Francesco , Malva Carla
Published: March 2003

Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae). This parasitoid is associated with a polydnavirus (TnBV), injected at oviposition along with the egg, and involved in the disruption of host immune reaction and endocrine balance. This paper reports the molecular characterization of TnBV2, one of the most abundant genes in the TnBV genome. TnBV2 expression produces a mature 0.6 kb transcript in fat body, prothoracic glands and haemocytes, as early as 6 h after parasitoid oviposition. Only in haemocytes a specific longer transcript of 2.5 kb is found 24 h after parasitization. The putative translation product of TnBV2 contains a retroviral type aspartyl protease domain. The possible origin and functional role of this TnBV gene are discussed.
Preparation of the apical part of the root canal by the Lightspeed and step‐back techniques
Authors: Portenier  , Lutz  , Barbakow
Published: March 2003

This study measured in vitro the displacement of natural canal centres in 18 human teeth before and after shaping by the step‐back or Lightspeed techniques. Experimental roots (n= 9 per group), embedded in clear plastic, were cross‐sectioned using a 0.1‐mm‐thick band saw at distances 1.25 mm, 3.25 mm and 5.25 mm from the apices. A stereo microscope was used to take 35 mm slides of the cut surfaces of the sectioned roots and canals. The slides of the uninstrumented canals were scanned into a computer and saved. Each sectioned root was then reassembled and the canals shaped by the step‐back or Lightspeed technique. File size 40 and instrument size 50 were selected as the master apical file and master apical rotary for the step‐back and Lightspeed groups, respectively. The 18 prepared canals were photographed, and the 35 mm slides scanned and computer stored as previously. This allowed the positions of the pre‐ and postinstrumented roots to be electronically superimposed for subsequent analyses. Displacements of the root canal centres before and after preparation were assessed in relation to the cross‐sectional diameter of the files or instruments used. In addition, increases in cross‐sectional area of the root canals after preparation were evaluated in relation to the cross‐sectional area of the files or instruments used. Engine‐driven nickel‐titanium Lightspeed instruments caused significantly less (P < 0.001) displacement of the canal centres, so roots in the Lightspeed group remained better centred than those in the step‐back group. The mean cross‐sectional area after preparation in the Lightspeed group was significantly less (P < 0.001) than that recorded in the step‐back group. Clinically, this implies less apical transportation and less dentine destruction with the Lightspeed technique than with the step‐back technique.
Root canal treatment of mandibular second premolar with four root canals: a case report
Authors: Holtzman
Published: March 2003

The importance of an accurate diagnosis of the morphology of the root canal system is a prerequisite for successful root canal treatment and has been emphasized throughout the literature. Root canal morphology of premolar teeth, either maxillary or mandibular, and the discrepancies between first and second premolars, have been investigated and reported. A case report is presented of the root canal treatment of a mandibular second premolar with four root canals. As far as the reviewed literature revealed, up to three root canals in mandibular premolars have been reported. No previous report of a similar case of four root canals in a mandibular premolar was found.
Apical limit of root canal instrumentation and obturation, part 1. Literature review
Authors: Ricucci
Published: March 2003

One of the major controversies in root canal therapy concerns the apical limit of instrumentation and obturation. The results of longitudinal prognostic studies, basic anatomical knowledge of the apical third of the root canal, and the histological pulp reaction to caries progression demonstrated the presence of a vital apical pulp remnant, even in the presence of a periapical lesion. Finally necrosis and bacteria establish themselves in the periapical lesion. All valid prognosis studies confirm the practice of staying short of the apex with a homogeneous obturation to obtain the highest success rate of 90–94% (when done by or under supervision of specialists; results in the general population had a failure rate greater than 50%). The location of the apical foramen(ina) related to root canal treatment most frequently ends short of the apex, often by several millimetres.
Formaldehyde release from root‐canal sealers: influence of method
Authors: Koch M. J.
Published: March 2003

Aim To examine formaldehyde release from three different root‐canal sealers: one phenol resin, one epoxy resin and one paraformaldehyde containing zinc oxide eugenol cement.

Methodology Formaldehyde was measured from freshly mixed material and from specimens allowed to set for 2 days or 2 weeks. Different mixing ratios and sample sizes were tested. Analysis was performed by reacting formaldehyde with acetylacetone and ammonia to form diacetyldihydrolutidine, which was revealed by spectrophotometry.

Results Analysis revealed that mixing ratio, time after mixing and surface : weight ratios of specimens had different influences on the formaldehyde release from different materials.

Conclusions From the results of this study it is difficult to define a standardized in‐vitro test that can be applied universally for the definitive determination of formaldehyde release from endodontic sealers. Prediction of in‐vivo formaldehyde release from endodontic sealers is difficult.
Connective tissue responses to calcium hydroxide‐based root canal medicaments
Authors: Filho P. Nelson , Silva L. A. Bezerra , Leonardo M. R. , Utrilla L. Sabbag , Figueiredo F.
Published: March 2003

Aim The objective of the present study was to evaluate the tissue inflammatory response induced by calcium hydroxide pastes, with or without paramonochlorophenol and camphor.

Methodology Isogenic BALB/c mice were inoculated into the subcutaneous tissue with either 0.1 mL of a suspension of Calen, Calen with camphorated paramonochlorophenol, Calen with paramonochlorophenol, Calasept paste or phosphate‐buffered saline (control). After 6, 12 and 24 h and 2, 3, 5, 7 and 15 days, three animals in each group were sacrificed and the excised lesions processed for histopathological evaluation of the inflammatory response. Events monitored and graded included the assessment of vascular congestion, oedema, haemorrhage, inflammatory infiltrate, necrosis and tissue repair.

Results The pastes induced an inflammatory response at every observation period, although the intensity, duration and extension of inflammation varied. Calen paste always produced an initial short‐term inflammatory response whilst the other pastes produced extended reactions. All pastes allowed repair to take place by the end of the experimental period, although the speed of this process varied between the materials. Calen presented the best biocompatibility; the phenolic compound caused greater tissue response, which was even more severe in the absence of camphor. Calasept paste was damaging and the repair process slower.

Conclusions All calcium hydroxide formulations caused an inflammatory response. The severity and longevity of the responses varied between pastes as a result of the various antiseptic agents. Although irritating, repair was apparent with all formulations.
Dissolution of root canal sealer cements in volatile solvents
Authors: Whitworth J. M. , Boursin E. M.
Published: March 2003

Whitworth JM, Boursin EM. Dissolution of root canal sealer cements in volatile solvents. International Endodontic Journal, 33, 19–24, 2000.

Aim  There are few published data on the solubility profiles of endodontic sealers in solvents commonly employed in root canal retreatment. This study tested the hypothesis that root canal sealer cements are insoluble in the volatile solvents chloroform and halothane.

Methodology Standardized samples (n=5) of glass ionomer (Ketac Endo), zinc oxide‐eugenol (Tubli‐Seal EWT), calcium hydroxide (Apexit) and epoxy resin (AH Plus) based sealers were immersed in chloroform or halothane for 30 s, 1 min, 5 min and 10 min. Mean loss of weight was plotted against time of exposure, and differences in behaviour assessed by multiple paired t‐tests (P <0.01).

Results  Clear differences were shown in the solubility profiles of major classes of root canal sealer cements in two common volatile solvents. In comparison with other classes of material, Ketac Endo was the least soluble in chloroform and halothane (P <0.01), with less than 1% weight loss after 10 min exposure to either solvent. Apexit had low solubility with 11.6% and 14.19% weight loss after 10 min exposure to chloroform and halothane, respectively. The difference between solvents was not significant (P >0.01). Tubli‐Seal EWT was significantly less soluble in halothane than chloroform (5.19% and 62.5% weight loss after 10 min exposure, respectively (P <0.01)). Its solubility in halothane was not significantly different from that of Apexit. AH Plus was significantly more soluble than all other materials in both chloroform and halothane (96% and 68% weight loss after 10 min exposure, respectively (P <0.01)).

Conclusions  There are significant differences in the solubility profiles of major classes of root canal sealer in common organic solvents. Efforts should continue to find a more universally effective solvent for use in root canal retreatment.
Necrosis of the gingiva caused by calcium hydroxide: a case report
Authors: De Bruyne M. A. A. , De Moor R. J. G. , Raes F. M.
Published: March 2003

De Bruyne MAA, De Moor RJG, Raes FM. Necrosis of the gingiva caused by calcium hydroxide: a case report. International Endodontic Journal, 33, 67–71, 2000.

Case report The present case demonstrates the possible detrimental effect of an overextension of a calcium hydroxide intracanal dressing into the periradicular and soft tissue after iatrogenic buccal root perforation of a maxillary central incisor.

At first this perforation was not recognized by the dentist, which resulted in the introduction of a large amount of non‐setting calcium hydroxide paste under the gingival tissues through a dehiscence on the buccal side of the root.

This report describes the consequences and management of the necrosis of the buccal gingiva and mucosa, and the subsequent treatment and follow‐up of the root perforation.
Root canal treatment and general health: a review of the literature
Authors: Murray C. A. , Saunders W. P.
Published: March 2003

Murray CA, Saunders WP. Root canal treatment and general health: a review of the literature. International Endodontic Journal,33, 1–18, 2000.

Review The focal infection theory was prominent in the medical literature during the early 1900s and curtailed the progress of endodontics. This theory proposed that microorganisms, or their toxins, arising from a focus of circumscribed infection within a tissue could disseminate systemically, resulting in the initiation or exacerbation of systemic illness or the damage of a distant tissue site. For example, during the focal infection era rheumatoid arthritis (RA) was identified as having a close relationship with dental health. The theory was eventually discredited because there was only anecdotal evidence to support its claims and few scientifically controlled studies.

There has been a renewed interest in the influence that foci of infection within the oral tissues may have on general health. Some current research

suggests a possible relationship between dental health and cardiovascular disease and published case reports have cited dental sources as causes for several systemic illnesses. Improved laboratory procedures employing sophisticated molecular biological techniques and enhanced culturing techniques have allowed researchers to confirm that bacteria recovered from the peripheral blood during root canal treatment originated in the root canal. It has been suggested that the bacteraemia, or the associated bacterial endotoxins, subsequent to root canal treatment, may cause potential systemic complications. Further research is required, however, using current sampling and laboratory methods from scientifically controlled population groups to determine if a significant relationship between general health and periradicular infection exists.
Alpha‐ and beta‐forms of gutta‐percha in products for root canal filling
Authors: Combe E. C. , Cohen B. D. , Cummings K.
Published: March 2003

Aim

This study was undertaken to evaluate which materials were in the α‐form of gutta‐percha as claimed, and which were in the more conventional β‐form, and to explore the effect of heating on the materials.

Methodology

Samples of gutta‐percha without chemical additives, and dental gutta‐percha formulations which included (i) two products previously studied; (ii) 12 newer products; and (iii) one newer product that had been stored at high temperature, were analysed by simultaneous differential thermal analysis and thermogravimetry.

Results

It was found that only four of the newer materials contained the α‐form; all the rest comprised β‐gutta‐percha. No weight loss was found for any material under the conditions of the present experiments. A typical heating cycle up to 130°C caused changes in material behaviour – that is, on reheating fewer endothermic peaks were present. Storage of gutta‐percha samples for 10 years under ambient temperature and storage in a heater at 80°C appeared to have no effect on properties.

Conclusions

It was concluded that heating dental gutta‐percha to 130°C causes physical changes; this was not seen with chemically pure gutta‐percha. The presence of additives in the dental samples altered material behaviour.