bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA–DrrA from Thermotoga maritima
Authors: Foster J. Estelle , Sheng Qin , McClain Jonathan R. , Bures Mark , Nicas Thalia I. , Henry Kenneth , Winkler Malcolm E. , Gilmour Raymond
Published: April 2004

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA–DrrA histidine kinase–response regulator pair fromThermotoga maritimaand allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis–Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5′-β,γ-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.
Genes for Mn(II)-dependent NahC and Fe(II)-dependent NahH located in close proximity in the thermophilic naphthalene and PCB degrader, Bacillus sp. JF8: cloning and characterization
Authors: Miyazawa Daisuke , Mukerjee-Dhar Gouri , Shimura Minoru , Hatta Takashi , Kimbara Kazuhide
Published: April 2004

A 10 kb DNA fragment was isolated using a DNA probe derived from the N-terminal amino acid sequence of the extradiol dioxygenase purified from naphthalene-grownBacillussp. JF8, a thermophilic naphthalene and polychlorinated biphenyl degrader. The cloned DNA fragment had six open reading frames, designatednahHLOMmocBnahCbased on sequence homology, of which the products NahH_JF8 and NahC_JF8 were extradiol dioxygenases. Although NahC_JF8 and NahH_JF8 exhibit low homology to known extradiol dioxygenases, the active-site residues and metal ion ligands are conserved. The presence of Mn(II) in culture medium was found to be essential for production of active recombinant NahC_JF8, while Fe(II) was necessary for active recombinant NahH_JF8. Inductively coupled plasma mass spectrometry analysis of active NahC_JF8 identified the cofactor to be manganese, indicating a Mn(II)-dependent extradiol dioxygenase. NahC_JF8 exhibitedKmvalues of 32±5 μM for 1,2-dihydroxynaphthalene and 510±90 μM for 2,3-dihydroxybiphenyl at 60 °C. In cell-free extracts, NahH_JF8 exhibited a broad substrate range for 2,3-dihydroxybiphenyl, catechol, and 3- and 4-methylcatechol at 25 °C. Stability studies on the Mn(II)-dependent NahC_JF8 indicated that it was thermostable, retaining 50 % activity after incubation at 80 °C for 20 min, and it exhibited resistance to EDTA and H2O2. Northern hybridization studies clarified that both NahC_JF8 and NahH_JF8 were induced by naphthalene; RT-PCR showed thatnahHLOMmocBnahCis expressed as a single transcript.
Phosphorylation and DNA binding of the regulator DcuR of the fumarate-responsive two-component system DcuSR of Escherichia coli
Authors: Janausch Ingo G. , Garcia-Moreno Inma , Lehnen Daniela , Zeuner Yvonne , Unden Gottfried
Published: April 2004

The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of Escherichia coli was analysed in vitro. Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate. In gel retardation assays with target promoters (frdA, dcuB, dctA), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent K
D (app. K
D) of 0·2–0·3 μM DcuR-P, and a low-affinity (app. K
D 0·8–2 μM) complex. The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters. The binding site of DcuR-P at the dcuB promoter was determined by DNase I footprinting. One binding site of 42–52 nt (position −359 to −400/−410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P. Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters. DcuR-D56N was still able to interact with DcuS. DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.
The diversity within an expanded and redefined repertoire of phase-variable genes in Helicobacter pylori
Authors: Salaün Laurence , Linz Bodo , Suerbaum Sebastian , Saunders Nigel J.
Published: April 2004

Phase variation is a common mechanism used by pathogenic bacteria to generate intra-strain diversity that is important in niche adaptation and is strongly associated with virulence determinants. Previous analyses of the complete sequences of the Helicobacter pylori strains 26695 and J99 have identified 36 putative phase-variable genes among the two genomes through their association with homopolymeric tracts and dinucleotide repeats. Here a comparative analysis of the two genomes is reported and an updated and expanded list of 46 candidate phase-variable genes in H. pylori is described. These have been systematically investigated by PCR and sequencing for the presence of the genes, and the presence and variability in length of the repeats in strains 26695 and J99 and in a collection of unrelated H. pylori strains representative of the main global subdivisions recently suggested. This provides supportive evidence for the phase variability of 30 of the 46 candidates. Other differences in this subset of genes were observed (i) in the repeats, which can be present or absent among the strains, or stabilized in different strains and (ii) in the gene-complements of the strains. Differences between genes were not consistently correlated with the geographic population distribution of the strains. This study extends and provides new evidence for variation of this type in H. pylori, and of the high degree of diversity of the repertoire of genes which display phase-variable switching within individual strains.
Abstract Agreement and Clausal Arguments
Authors: Picallo M. Carme
Published: April 2004

CP arguments have been argued to be Caseless and ϕ– featureless. Empirical evidence mainly drawn from Spanish suggests that this claim should be reconsidered. In this paper, I claim that clausal arguments and nominalized clauses have a ϕ content and Case specification. These types of arguments are therefore able to relate by agreement with a functional category. The facts examined support minimalist guidelines by claiming that the concrete realization of the ϕ and Case content of a syntactic object is irrelevant for computational mechanisms to take place.
Review of Phrasal Movement and its Kin
Authors: Simpson Andrew
Published: April 2004

Book reviewed in this article:

David Pesetsky, Phrasal Movement and Its Kin
A molecular phylogenetic analysis of the pleasing fungus beetles (Coleoptera: Erotylidae): evolution of colour patterns, gregariousness and mycophagy
Authors: Robertson James A. , McHugh Joseph V. , Whiting Michael F.
Published: April 2004

Abstract. Phylogenetic relationships of Erotylidae (pleasing fungus beetles) were inferred based on DNA sequence data. Relationships of clades within Erotylidae were examined, as was the relationship of the entire family to Languriidae (lizard beetles). 18S and 28S ribosomal DNA were sequenced for sixty‐one taxa representing major erotylid lineages and outgroups. Phylogenetic analyses under varying parameter settings using standard parsimony and likelihood techniques were performed. These data indicate a paraphyletic Erotylidae and Languriidae. Encaustinae (includingCoptengis), Megalodacninae and Erotylinae are supported as monophyletic, whereas Dacninae and Tritominae are paraphyletic. Taxonomic and biological implications are discussed. Gregariousness has arisen at least three times in Erotylidae. The erotylid clade has experienced at least one evolutionary transition from mycophagy (on Aphyllophorales) to phytophagy, three transitions from Aphyllophorales hosts to Euagarics, and one transition from Euagarics hosts to Mucorales (Zygomycetes). There are no recognizable phylogenetic trends in coloration across higher‐level erotylid lineages.
The impact of parental problem drug use on children: what is the problem and what can be done to help?
Authors: Barnard Marina , McKeganey Neil
Published: April 2004

Aim  To review the literature on the impact of parental problem drug use on children, and indicate the efficacy of key evaluated interventions to reduce the impact of parental drug use on children.

Methods  Comprehensive narrative review of English language published research and intervention spanning the last three decades identified through searching library databases and citation.

Findings  Problem drug use can impede parenting and the provision of a nurturing environment. Although small‐scale, localized and resource‐intensive these key evaluated interventions show cautious optimism that problem drug‐using parents can reduce drug use and achieve better family management. Children have rarely been directly the focus of intervention.

Conclusions  Wider application and more rigorous evaluation of interventions in this area are needed. Given the scale of the problem it is important to establish how statutory services can apply the lessons of these more localized interventions.
Hair testing and self‐report of cocaine use by heroin users
Authors: Tassiopoulos Katherine , Bernstein Judith , Heeren Timothy , Levenson Suzette , Hingson Ralph , Bernstein Edward
Published: April 2004

Aim  Failure to disclose cocaine use can have a negative impact on medical care and research validity. This study was performed to identify predictors of cocaine non‐disclosure among individuals who self‐reported heroin use during a medical care encounter.

Design  A prospective comparison of self‐report of cocaine use among heroin users and hair analysis for cocaine.

Setting  Four health‐care clinics at an academic, inner‐city hospital.

Participants  Patients presenting for a health‐care visit who were willing to self‐report use of heroin and were not engaged in any form of drug treatment.

Measurements  (1) Self‐report using standardized instruments: the Drug Addiction Severity Test (DAST), the Addiction Severity Index (ASI) and quantity/frequency questions for heroin and cocaine use. (2) Biochemical evidence: analysis of hair by radioimmunoassay (RIA) for cocaine and opiate levels.

Findings  Among 336 heroin users who tested positive for cocaine in hair, 34.2% did not report their recent cocaine use. The mean cocaine level for discordant individuals was significantly lower than for concordant individuals (109.6 ng/10 mg versus 470.57 ng/10 mg; P < 0.0001). Multivariate predictors of disclosure included opiate and cocaine levels in hair and the ASI drug severity subscore.

Conclusions  Although self‐report has been validated for treatment system patients, almost a third of the out‐of‐treatment heroin users in this medical clinic study failed to disclose concomitant cocaine use. The likelihood of non‐disclosure was greatest for heavy users of heroin and light users of cocaine. Confirmation of self‐report with biochemical analysis in the medical setting may be necessary to improve both clinical care and research validity.
Does moderate alcohol use affect health‐care costs? A propensity analysis of female health‐care workers
Authors: McMillan Garnett P. , Lapham Sandra C.
Published: April 2004

Aims  To determine differences in health‐care costs associated with moderate alcohol consumption among female health‐care workers while controlling for other risk factors that may be correlated with alcohol use.

Design and setting  Non‐randomized, prospective, observational study of health‐care costs by female health‐care workers in a large managed care organization recruited between 1 January 1998 and 1 July 2000.

Participants  Six hundred and eighty‐five female employees, continuously and stably employed by the managed care organization, who received health‐care through the affiliated managed care organization. All women completed a health risk appraisal as part of the company's Employee Wellness Plan; 218 women were categorized as moderate drinkers and 467 as abstainers/light drinkers.

Measurements  Total costs of in‐plan and out‐of‐plan health‐care utilization, by type of service, during the 6 month period after completing the health risk survey were calculated.

Findings  Using 218 one‐to‐one matched pairs of moderate drinkers and abstainers/light drinkers, no significant differences in total, outpatient or inpatient costs were observed during the 6 month observation period. Pharmacy costs were significantly lower for moderate drinkers (–$43, 95% CI = –$88.82 to –$2.41), primarily due to differences in costs from anxiolytic (including barbiturates and benzodiazepines), hypnotic and sedative drug fills.

Conclusions  Findings demonstrate the value of risk factor matching when studying the relationship between alcohol use and health‐care utilization. The discovery of differential pharmacy utilization raises the possibility that alcohol consumption may reduce the use of prescribed central nervous system depressants.
New Mexico's 1998 drive‐up liquor window closure. Study II: economic impact on owners
Authors: Lapham Sandra C. , Skipper Betty J.
Published: April 2004

Aims  This study examined the economic impact of the New Mexico legislative action closing drive‐up liquor windows on the retail establishments that operated them.

Design  A telephone survey was conducted 20 months after the closure seeking information and owners’ opinions about how their outlets had changed since the closure and how this affected their business. In addition, 2 years of aggregated pre‐ and post‐closure total gross receipts revenues were obtained from the New Mexico Taxation and Revenue Department, with convenience stores as a comparison group.

Findings  Interviews were completed for 149 of 220 establishments. Over one‐quarter of former drive‐up liquor windows (28%) had been converted to ‘step‐in’ sales, defined as an outside door where customers can stop and enter the premises while their car is running. Almost two‐thirds (61%) of owners reported decreased annual gross revenues following closure, with a reported average 15% reduction in alcohol sales. This is consistent with findings of decreased gross receipts for operators of non‐urban, but not urban, drive‐up liquor windows compared to convenience store gross receipts. Almost three‐quarters (72%) of those surveyed would re‐open the drive‐up window if the law were rescinded.

Conclusion  Over one‐quarter of the drive‐up owners converted to step‐in alcohol sales that still allow a form of drive‐up liquor sales. Despite this, the forced closure of New Mexico's drive‐up liquor windows negatively impacted total sales and liquor sales revenues of establishments that operated them.
Recent trends in diagnosis and treatment of faecal incontinence
Authors: Tuteja A. K. , Rao S. S. C.
Published: April 2004

The inability to control bowel discharge is not only common but extremely distressing. It has a negative impact on a patient's lifestyle, leads to a loss of self‐esteem, social isolation and a diminished quality of life. Faecal incontinence is often due to multiple pathogenic mechanisms and rarely due to a single factor. Normal continence to stool is maintained by the structural and functional integrity of the anorectal unit. Consequently, disruption of the normal anatomy or physiology of the anorectal unit leads to faecal incontinence. Currently, several diagnostic tests are available that can provide an insight regarding the pathophysiology of faecal incontinence and thereby guide management. The treatment of faecal incontinence includes medical, surgical or behavioural approaches. Today, by using logical approach to management, it is possible to improve symptoms and bowel function in many of these patients.
Evaluation of three different tests for the detection of stool antigens to diagnose Helicobacter pylori infection in patients with upper gastrointestinal bleeding
Authors: Gisbert J. P. , Trapero M. , Calvet X. , Mendoza J. , Quesada M. , Güell M. , Pajares J. M.
Published: April 2004

Aim : To evaluate the accuracy of several methods aimed to detect Helicobacter pylori stool antigens in patients with upper gastrointestinal bleeding.

Methods : Thirty‐four patients with upper gastrointestinal bleeding because of peptic ulcer were included. The first stool sample during hospitalization was collected, and stool antigens were determined with: polyclonal enzyme‐linked immunosorbent assay (Premier‐Platinum‐HpSA); monoclonal enzyme‐linked immunosorbent assay (Amplified‐IDEIA‐HpStAR); and rapid monoclonal immunochromatographic test (ImmunoCard‐STAT HpSA). A patient was considered infected when H. pylori was diagnosed with invasive tests (rapid urease test or histology) or with 13C‐urea breath test. When all tests were negative, a new breath test was repeated after stopping proton pump inhibitors.

Results : All patients were infected and, therefore, only sensitivity of the tests could be calculated: polyclonal enzyme‐linked immunosorbent assay (74%), monoclonal enzyme‐linked immunosorbent assay (94%), and rapid monoclonal immunochromatographic test (60%; concordance between the two observers was high, kappa = 0.9). Neither the presence of maelena nor the delay in obtaining stool samples explained false negatives.

Conclusions : Neither the polyclonal enzyme‐linked immunosorbent assay stool antigen test nor the rapid immunochromatographic stool antigen test can be recommended to diagnose H. pylori infection in patients with upper gastrointestinal bleeding. However, the monoclonal enzyme‐linked immunosorbent assay stool antigen test is highly sensitive for detecting the infection in patients with this complication, although more studies are necessary to evaluate the specificity of the method.
Detection of circulating soluble CD28 in patients with systemic lupus erythematosus, primary Sjögren's syndrome and systemic sclerosis
Authors: HEBBAR M , JEANNIN P , MAGISTRELLI G , HATRON P-Y , HACHULLA E , DEVULDER B , BONNEFOY J-Y , DELNESTE Y
Published: April 2004

The aim of this study was to evaluate the presence and the role of the serum soluble costimulatory molecule CD28 in patients with systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), and systemic sclerosis (SSc). Soluble CD28 concentration was determined by ELISA in 45 patients with SLE, 45 patients with primary SS, 30 patients with SSc, and 45 healthy subjects. We also evaluated CD28 mRNA expression by semiquantitative RT-PCR, and the biological activity of recombinant soluble CD28 on T lymphocyte activity. Concentrations of soluble CD28 were significantly higher in patients with SLE, primary SS and SSc than in healthy subjects. Soluble CD28 concentrations were higher in patients with systemic primary SS than in patients with glandular-limited primary SS. PCR analysis suggested that soluble CD28 resulted from the shedding of the membrane form. In vitro assay revealed that soluble CD28 inhibits the anti-CD3 mAb induced T cell proliferation. Soluble CD28, which modulates the proliferation of T lymphocytes, could be associated with disease severity in patients with autoimmune disease, especially primary SS. These results suggest that soluble CD28 could play an important role in the regulation of autoimmune diseases.
Neutrophils migrate across intestinal epithelium usingβ2 integrin (CD11b/CD18)-independent mechanisms
Authors: BLAKE K M , CARRIGAN S O , ISSEKUTZ A C , STADNYK A W
Published: April 2004

Recruitment of polymorphonuclear leucocytes (PMN) across the intestinal epithelium is dependent on specific adhesion molecules and chemoattractants diffusing from the intestinal lumen. The present understanding is that in response to fMLP, PMN migration across a T84 colon carcinoma monolayer is dependent on the β2 integrin, Mac-1 (CD11b/CD18). To further understand PMN transepithelial migration, we sought to determine whether migration to C5a, IL-8 and LTB4 was similarly Mac-1-, or even CD18-dependent. T84 epithelial cell monolayers growing on Transwell filters were used in combination with radiolabelled peripheral blood PMN. The number of migrated PMN was established by the amount of radioactivity recovered from the well after the migration period. Monoclonal antibodies were used to block integrin function. Whereas essentially all migration to fMLP across T84 monolayers was prevented by anti-CD18 antibody, significant migration to C5a, IL-8 or LTB4 persisted despite anti-CD18 antibody, indicating PMN are capable of β2 integrin-independent transepithelial migration. An antibody to CD11b but not CD11a blocked migration to an extent similar as with anti-CD18. CD18-independent PMN migration to C5a occurred only in the basolateral-to-apical direction across epithelial cells. Co-stimulation of PMN with C5a and fMLP or IL-8 plus LTB4 and fMLP still resulted in CD18-independent migration. Thus CD18 use during PMN migration across this model epithelium is a function of the chemoattractant inducing migration. The finding of CD18-independent migration mechanisms needs to be considered when developing antiadhesion molecule strategies to reduce or reverse intestinal inflammation.
The nuclear pore complex protein Tpr is a common autoantigen in sera that demonstrate nuclear envelope staining by indirect immunofluorescence
Authors: OU Y , ENARSON P , RATTNER J B , BARR S G , FRITZLER M J
Published: April 2004

We studied the autoantigen targets of 75 human sera that had antibodies to the nuclear envelope (NE) as identified by indirect immunofluorescence (IIF) on HEp-2 cells. Several different IIF staining patterns could be identified when antibodies to different components of the nuclear membrane (NM) and nuclear pore complexes (NuPC) were identified: a smooth membrane pattern characteristic of antibodies to nuclear lamins, a punctate pattern typical of antibodies to the nuclear pore complex and more complex patterns that included antibodies to nuclear and cytoplasmic organelles. Western immunoblotting of isolated nuclear and NE proteins and immunoprecipitation of radiolabelled recombinant proteins prepared by using the full-length cDNAs of the Translocated promoter region (Tpr), gp210 and p62 were used to identify specific autoantibody targets. Fifty-two of the 75 (70%) sera bound to Tpr, 25 (33%) bound to lamins A, B or C, 15 (20%) reacted with gp210 and none reacted with p62. Sixteen (21%) did not react with any of the NE components tested in our assays. The clinical features of 37 patients with anti-NE showed that there were 34 females and three males with an age range of 16–88 years (mean 59 years). The most frequent clinical diagnosis (9/37 = 24%) was autoimmune liver disease (ALD; two with primary biliary cirrhosis), followed by seven (19%) with systemic lupus erythematosus (SLE), four (11%) with a motor and/or sensory neuropathy, three (8%) with anti-phospholipid syndrome (APS), two with systemic sclerosis (SSc), two with Sjögren's syndrome (SjS), and others with a variety of diagnoses. This report indicates that Tpr, a component of the NuPC, is a common target of human autoantibodies that react with the NE.
Differential expression of Toll-like receptor (TLR)-2 and TLR-4 on monocytes in human sepsis
Authors: ARMSTRONG L , MEDFORD A R L , HUNTER K J , UPPINGTON K M , MILLAR A B
Published: April 2004

Toll-like receptors (TLRs) are a recently described family of immune receptors involved in the recognition of pathogen-associated molecular patterns (PAMPs). The central role of TLR-2 and TLR-4 in microbial responses suggests they may be implicated in the pathogenesis of human sepsis. We hypothesized that the incidence and outcome of sepsis would be influenced by the expression of TLR-2 and TLR-4 on monocytes. We have examined the expression of TLR-2 and TLR-4 mRNA and protein and their response to pro- and anti-inflammatory agents on monocytes from subjects in the intensive therapy unit (ITU) with and without Gram-negative, Gram-positive or polymicrobial sepsis. We compared these data to ITU and healthy control subjects. TLR-2 mRNA was significantly up-regulated on monocytes from subjects with both Gram-positive and Gram-negative sepsis. Similarly, we detected increased levels of TLR-2 protein on the surface of monocytes from sepsis subjects relative to ITU controls. TLR-4 mRNA was increased in Gram-positive subjects; however, there was no corresponding increase in TLR-4 protein. Although TLR-4 mRNA expression in healthy control monocytes could be modulated in vitro by culture with lipopolysaccharide or interleukin-10, this was not observed in monocytes obtained from sepsis and ITU control subjects, suggesting that septic and ITU control milieus may alter the immunoregulation of TLR-4 mRNA expression on monocytes. TLR-2 mRNA was not modulated in culture by any stimulus in any group. We suggest that expression and regulatory response of monocyte TLR-2, and to a lesser extent TLR-4 may be abnormal in human sepsis.
The influence of partial or total thymectomy during open heart surgery in infants on the immune function later in life
Authors: EYSTEINSDOTTIR J H , FREYSDOTTIR J , HARALDSSON A , STEFANSDOTTIR J , SKAFTADOTTIR I , HELGASON H , OGMUNDSDOTTIR H M
Published: April 2004

Infants undergoing open heart surgery often have all or part of their thymus removed. The activity of the immune system has not been investigated thoroughly in these children, and only shortly after the operation. Therefore, it was decided to investigate the activity of the immune system in more detail in children several years after their operation. Peripheral blood samples from 19 children who had undergone open heart surgery during their first months of life was collected (study group) and from 19 age- and gender-matched children (control group). The activity of the immune system was evaluated by measuring the number of different cell types in peripheral blood, the phenotype of lymphocytes and the response of T cells following in vitro stimulation by mitogen, tetanus toxoid and measles antigen. The study group had significantly lower counts of total lymphocytes, which was reflected in a lower number of T cells but not B cells. Furthermore, the study group had significantly lower proportion of T cells (CD3+) and helper T cells (CD4+), but not cytotoxic T cells (CD8+). The level of neutrophils in peripheral blood was significantly higher in the study group. This may indicate enhanced innate immunity when the acquired immunity is defective. The results indicate a shift to extrathymic T cell maturation, which is less efficient for CD4+ helper cells than for CD8+ cytotoxic cells.
Development of an anti-Salmonella typhi ViELISA: assessment of immunocompetence in healthy donors
Authors: FERRY B L , MISBAH S A , STEPHENS P , SHERRELL Z , LYTHGOE H , BATEMAN E , BANNER C , JONES J , GROOME N , CHAPEL H M
Published: April 2004

We have developed a solid-phase enzyme-linked immunosorbent assay (ELISA) to study the vaccination responses to Vi capsular polysaccharide of Salmonella typhi (S. typhi Vi) vaccine. Purified S. typhi Vi polysaccharide was biotinylated and bound to streptavidin coated microtitre plates. Reproducibility was determined across a range of IgG antibody levels: mean interassay coefficients of variation (CVs) were <11·9% for non-vaccinated sera with low levels and <11·1% for sera with very high levels of anti-S. typhi Vi IgG. Specificity was assessed by inhibition studies using salmonella antigen. We have developed the ELISA based on normal adult serum responses to test immunization with S. typhi Vi vaccine. We also report here anti-S. typhi Vi IgG levels in a group of healthy preschool children. In non-vaccinated adult sera (n = 104), the median value of anti-S. typhi Vi IgG, expressed in S. typhi Vi arbitrary units (AU/ml), was 5·3 AU/ml and in non-vaccinated sera from children (n = 44) the median value was 1·4 AU/ml. The data from immunization of healthy volunteers (n = 23) show that geometric mean levels of anti-S. typhi Vi IgG were significantly higher (P < 0·0001) for post-vaccination subjects (39·2 AU/ml) compared to paired prevaccination (3·9 AU/ml) values. A total of 21/23 vaccine recipients had <8 AU/ml S. typhi Vi IgG in their sera prior to vaccination and of these 20/21 (95%) exhibited threefold increases and 14/21 (67%) fourfold increases in their S. typhi Vi IgG following vaccination. Based on the data in this study, we propose a threefold increase in anti-S. typhi Vi IgG post-vaccination to be considered a positive vaccination response. The ability to demonstrate clearly an antibody rise in response to immunization with S. typhi Vi capsular polysaccharide vaccine suggests that this is likely to be a useful vaccine for the assessment of B cell function in patients with suspected immune deficiency.
Phenotypic characterization of regulatory T cells in the human decidua
Authors: HEIKKINEN J , MÖTTÖNEN M , ALANEN A , LASSILA O
Published: April 2004

Pregnancy is a unique situation for the maternal immune system. We have studied and identified a CD4+CD25+ regulatory T (Treg) cell population isolated from the human decidua. This mucosal surface in the uterus is in direct contact with semiallogenic fetal cells. We observed that about 14% of the decidual CD4+ T cells have the CD4+CD25+ phenotype. The decidual CD4+CD25+ T cells expressed high frequency of intracellular CTLA-4 (CTLA-4i). The majority of CD4+CD25+CTLA-4i+ cells were also positive for GITR and OX40, typical markers for human Treg cells. The frequency of CD4+CD25+ T cells in the peripheral blood from pregnant women was found to be increased during the first and second trimester of gestation when compared to nonpregnant controls. Being an important molecule for Treg cells, the role of CTLA-4 in the regulation of indoleamine 2,3-dioxygenase (IDO) expression was also examined. The stimulation with CTLA-4Ig did not increase IDO mRNA expression in CD14+ cells from pregnant women, while IFN-γ was observed to up-regulate IDO expression. The presence of Treg cells in the human decidua suggests that these cells are important in protecting the fetus from alloreactive immune responses at the maternal–fetal interface.
A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin
Authors: CASTELLETTI D , FRACASSO G , RIGHETTI S , TRIDENTE G , SCHNELL R , ENGERT A , COLOMBATTI M
Published: April 2004

Hodgkin's lymphoma patients treated with an anti-CD25 Ricin toxin A-chain (RTA)-based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti-RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail, considering antibody titre, IgG and IgM content, average binding efficacy and ability to inhibit in vitro the cytotoxicity of a RTA-based Immunotoxin. No correlations were found between these parameters and the clinical features of the patients or the total amount of Immunotoxin administered. However, using a peptide scan approach we have identified a continuous epitope recognized by all patients studied, located within the stretch L161-I175 of the RTA primary sequence, close to a previously identified T-cell epitope. The ability of anti-L161-I175 antibodies to recognize folded RTA and to affect the biological activity of RTA by inhibiting RTA-IT cytotoxicity in vitro revealed that they may exert an important role in IT neutralization in vivo. Discovery of RTA immunodominant epitopes which are the target of anti-RTA immune response may lead to the development of immunomodulating strategies and to more successful treatment schedules.
Induction of iNOS expression and antimicrobial activity by interferon (IFN)-β is distinct from IFN-γ in Burkholderia pseudomallei-infected mouse macrophages
Authors: UTAISINCHAROEN P , ANUNTAGOOL N , ARJCHAROEN S , LIMPOSUWAN K , CHAISURIYA P , SIRISINHA S
Published: April 2004

Burkholderia pseudomallei is a causative agent of melioidosis. This Gram-negative bacterium is able to survive and multiple inside both phagocytic and nonphagocytic cells. We previously reported that exogenous interferons (both type I and type II) enhanced antimicrobial activity of the macrophages infected with B. pseudomallei by up-regulating inducible nitric oxide synthase (iNOS). This enzyme thus plays an essential role in controlling intracellular growth of bacteria. In the present study we extended our investigation, analysing the mechanism(s) by which the two types of interferons (IFNs) regulate antimicrobial activity in the B. pseudomallei-infected macrophages. Mouse macrophage cell line (RAW 264·7) that was exposed simultaneously to B. pseudomallei and type I IFN (IFN-β) expressed high levels of iNOS, leading to enhanced intracellular killing of the bacteria. However, neither enhanced iNOS expression nor intracellular bacterial killing was observed when the macrophages were preactivated with IFN-β prior to being infected with B. pseudomallei. On the contrary, the timing of exposure was not critical for the type II IFN (IFN-γ) because when the cells were either prestimulated or co-stimulated with IFN-γ, both iNOS expression and intracellular killing capacity were enhanced. The differences by which these two IFNs regulate antimicrobial activity may be related to the fact that IFN-γ was able to induce more sustained interferon regulatory factor-1 (IRF-1) expression compared with the cells activated with IFN-β.
Mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system
Authors: CEDZYNSKI M , SZEMRAJ J , SWIERZKO A S T , BAK-ROMANISZYN L , BANASIK M , ZEMAN K , KILPATRICK D C
Published: April 2004

Blood samples were collected over a 4-year period from 335 children (aged 1–16 years) suffering from recurrent respiratory infections and 78 controls. The patients were subdivided into four groups: I, children with no immune system defects detected (n = 101); II, children with allergies (n = 94); III, children with humoral response defects (n = 93); and IV, children with disturbances of cellular immunity (n = 66). Nineteen patients had both humoral and cellular abnormalities. All patients and controls were investigated to determine the exon 1 and promoter region variants of the mbl-2 gene. MBL serum concentrations were also determined in samples from 291 patients and 75 controls. The proportion of O (B, D or C) alleles was significantly higher in the patient group compared to controls, and this association was strongest for subgroup III. The promoter LX variant frequency was also commoner in the patients as a whole, and significantly so in subgroups II and IV. Genotypes markedly influenced MBL concentrations in all groups, and correlated with ability to activate the lectin pathway of complement activation. The strongest and most significant inverse correlations between serum MBL and respiratory disease were found in patient group III and in 17 patients with multiple humoral and/or cellular abnormalities. Among nine patients with unexpectedly low LP activity in view of their MBL concentrations, one person was found to be MASP-2 deficient. Our results indicate that mannan-binding lectin insufficiency, with or without a coexisting immune defect, is associated with the occurrence of recurrent respiratory infections in childhood, and this relationship is particularly strong and statistically significant in children with concomitant impairments of humoral immunity.
Corneal stromal cells selectively inhibit production of anti-inflammatory cytokines by activated T cells
Authors: HOLÁŇ V , VÍTOVÁ A , PINDJÁKOVÁ J , KRULOVÁ M , ZAJÍCOVÁ A , FILIPEC M
Published: April 2004

The eye has been described as an immunologically privileged site where immunity is purely expressed. It has been demonstrated that administration of antigen into the eye induces only a weak immune response. However, the anterior part of the eye represents an important protective barrier against pathogens and other harmful invaders from the outer environment. Therefore, effective immune mechanisms, which operate locally, need to be present there. Because the cornea has been shown to be a potent producer of various cytokines and other molecules with immunomodulatory properties, we investigated a possible regulatory role for the individual corneal cell types on cytokine production by activated T cells. Mouse spleen cells were stimulated with the T cell mitogen concanavalin A in the presence of either corneal explants or cells of corneal epithelial or endothelial cell lines and the production of T helper 1 (Th1) or Th2 cytokines was determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription–polymerase chain reaction (RT-PCR). We found that the cornea possesses the ability to inhibit, in a dose-dependent manner, production of the inhibitory and anti-inflammatory cytokines interleukin (IL)-4 and IL-10 by activated T cells. The production of cytokines associated with protective immunity [IL-2, IL-1β, interferon (IFN)-γ] was not inhibited under the same conditions. Corneal explants deprived of epithelial and endothelial cells retained the ability to suppress production of anti-inflammatory cytokines. This suppression was mediated by a factor produced by corneal stromal cells and occurred at the level of cytokine gene expression. We suggest that by this mechanism the cornea can potentiate a local expression of protective immune reactions in the anterior segment of the eye.
Susceptibility of T cell receptor-αchain knock-out mice to ultraviolet B light and fluorouracil: a novel model for drug-induced cutaneous lupus erythematosus
Authors: YOSHIMASU T , NISHIDE T , SEO N , HIROI A , OHTANI T , UEDE K , FURUKAWA F
Published: April 2004

The anticancer agent 5-fluorouracil (FU) frequently induces cutaneous lupus erythematosus (LE) lesions on sun exposed sites. Based on this observation, we have tried to establish a cutaneous LE model of C57BL/6 J (B6) mice, B6 T cell receptor (TCR)-α–/– mice and B6 TCR-δ–/– mice treated with FU and/or ultraviolet B light (UVBL) in order to clarify the role of T cells and the cytokine profile of cutaneous lupus lesions. Cutaneous LE-like skin lesions could be induced in TCR-α–/– mice with low FU (0·2 mg) plus UVBL, and in B6 mice treated with a high dose of FU (2·0 mg) plus UVBL. In contrast, low FU plus UVBL induced such skin lesions in TCR-δ–/– mice at a very low incidence. Specifically, the skin lesions of TCR-α–/– mice with low FU plus UVBL appeared more rapidly and were more severe than lesions in B6 mice. The former had the common characteristic features of human chronic cutaneous LE such as typical histology, positive IgG at the dermoepidermal junction, low antinuclear antibody and low mortality. Furthermore, a Th1 response was induced in the development of drug-induced cutaneous LE. FU and UVBL-induced cutaneous LE-like eruption is an excellent model for better understanding the pathomechanisms of skin lesion development in LE.
Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice
Authors: ATRASHEUSKAYA A V , BUKIN E K , FREDEKING T M , IGNATYEV G M
Published: April 2004

The resistance to mousepox is correlated with the production of type I cytokines: interleukin (IL)-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. We intend to describe the modulation of generalized ectromelia virus (EV) infection with exogenous administration of mrIFN-γ and mrTNF-α separately and in combination using susceptible BALB/c mice. The treatment schemes presented resulted in the localization of the generalized EV infection and its development into non-fatal sloughing of the infected limb. This was accompanied by low virus titres in the treated mice due to control of systemic virus replication and virus clearance. The balance of type I versus type II cytokines was dominated by a type I response in the treated groups. The group treated with the combination of IFN-γ and TNF-α exhibited the best survival with Th1-dominant (IFN-γ and IL-12) cytokine profiles, whereas the TNF-α-treated group of mice was less successful in clearance of virus and demonstrated the lowest survival rate. The successful cytokine treatment schemes in this orthopoxvirus model system may have important implications in the treatment of viral diseases in humans and, in particular, of variola virus infection.
Phytosanitary status of potato in the Bekaa valley in Lebanon
Authors: Choueiri E. , El‐Zammar S. , Jreijiri F. , Mnayer D. , Massaad R. , Saad A. T. , Hanna L. , Varveri C.
Published: April 2004

The incidence, severity and distribution of four virus diseases of potato were determined in the main production areas of Lebanon during the 2001 and 2002 growing seasons. Observations were also recorded on diseases incited by fungi and bacteria, as well as physiological disorders. The surveys covered 40 randomly selected potato fields from three different areas of the Bekaa valley, the main potato production region of Lebanon. 715 samples were individually collected and tested by DAS‐ELISA for Potato virus A (PVA), Potato virus X (PVX), Potato virus Y (PVY) and Potato leafroll virus (PLRV). 51% of the total tested samples were infected by one or more viruses. PVY was the most prevalent virus, detected in 98.8% of the total number of infected samples, followed by PVA and PVX (both at 10.6%) and PLRV (9.3%). Mixed infections were about 17%. Potato tuber necrotic ring spot disease, caused by PVYNTN, was determined by immunocapture reverse transcription polymerase chain reaction. The fungi Thanatephorus cucumeris, Verticillium dahliae, Fusarium sp., Sclerotinia sclerotiorum and the bacterium Erwinia carotovora were the main pathogens found to be associated with the diseased plants. Quarantine pests such as Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum were not encountered. Growth cracks, hollow heart and internal rust spot were the predominant physiological disorders observed.
Trust, planning and benefits in a global interorganizational system
Authors: Ibbott Christopher J. , O'Keefe Robert M.
Published: April 2004

Abstract.  Trust has been shown by many researchers to be an important factor in the success of interorganizational systems (IOS). In this paper, we describe an example of an IOS developed as part of a strong inter‐firm relationship. In 1999, Vodafone of the UK embarked on a transformation to globalize its relationship with Ericsson of Sweden, moving from a set of in‐country relationships to a global relationship mediated through an IOS. As of 2002, the IOS is heavily used, and continues to evolve as part of the overall relationship. Vodafone and Ericsson pursued a journey‐oriented approach to globalization and development, freed from any formal project plan, and identified asymmetric benefits. Using a model previously developed by Gallivan & Depledge (2003), we explore the roles and dynamics of trust, planning and benefits. We posit that a journey or improvisational approach to IOS development works where a relationship is strong and levels of inter‐firm trust are high. We suggest that the nature of the inter‐firm relationship is more important than the approach to IOS development.
Initial cos cleavage of bacteriophage λ concatemers requires proheads and gpFI in vivo
Authors: Sippy Jean , Feiss Michael
Published: April 2004

The development of bacteriophage λ and double‐stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, λ phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of λ, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When λ proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of λ, and although its capsid proteins share ∼60% residue identity with λ’s, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.
The push–pull mechanism of bacteriophage Ø29 DNA injection
Authors: González‐Huici Víctor , Salas Margarita , Hermoso José M.
Published: April 2004

The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid. In contrast to the well‐established DNA packaging mechanism of Bacillus subtilis phage Ø29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell. We have studied this process measuring DNA binding of p6, a viral genome organization protein. The linear DNA penetrates with a right‐left polarity, in a two‐step process. In the first step ∼65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid. Thus, synthesis of viral proteins from the right early operon is allowed. This step is controlled, probably by bacterial protein(s) that slow down DNA entry. In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell. Both steps are energy‐dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.