bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating eosinophils in asthma, allergic rhinitis, and atopic dermatitis lack morphological signs of degranulation
Authors: Malm‐Erjefält M. , Greiff L. , Ankerst J. , Andersson M. , Wallengren J. , Cardell L.‐O. , Rak S. , Persson C. G. A. , Erjefält J. S.
Published: October 2005

Background
In allergic diseases, eosinophils in affected tissues release granule proteins with cytotoxic, immunoregulatory, and remodelling‐promoting properties. From recent observations, it may be assumed that eosinophils degranulate already in circulating blood. If degranulation occurs in the circulation, this could contribute to widespread systemic effects and provide an important marker of disease.

Objective
To determine the degranulation status of circulating eosinophils in common allergic diseases.

Methods
Using a novel approach of whole blood fixation and leucocyte preparation, the granule morphology of blood eosinophils from healthy subjects, non‐symptomatic patients, symptomatic patients with asthma, asthma and Churg–Strauss syndrome, allergic rhinitis, and atopic dermatitis was evaluated by transmission electron microscopy (TEM) and eosinophil peroxidase (TEM) histochemistry. Plasma and serum levels of eosinophil cationic protein were measured by fluoroenzymeimmunoassay. Selected tissue biopsies were examined by TEM.

Results
Regardless of symptoms, circulating eosinophils from allergic patients showed the same granule morphology as cells from healthy subjects. The majority of eosinophil‐specific granules had preserved intact electron‐density (96%; range: 89–98%), while the remaining granules typically exhibited marginal coarsening or mild lucency of the matrix structure. Abnormalities of the crystalline granule core were rarely detected. Furthermore, granule matrix alterations were not associated with any re‐localization of intracellular EPO or increase in plasma eosinophil cationic protein. By contrast, eosinophils in diseased tissues exhibited cytolysis (granule release through membrane rupture) and piecemeal degranulation (loss of granule matrix and core structures).

Conclusion
In symptomatic eosinophilic diseases, circulating blood eosinophils retain their granule contents until they have reached their target organ.
Long‐term exposure to background air pollution related to respiratory and allergic health in schoolchildren
Authors: Pénard‐Morand C. , Charpin D. , Raherison C. , Kopferschmitt C. , Caillaud D. , Lavaud F. , Annesi‐Maesano I.
Published: October 2005

Background
The impact of air pollution on asthma and allergies still remains a debate.

Objective
Our cross‐sectional study was intended to analyse the associations between long‐term exposure to background air pollution and atopic and respiratory outcomes in a large population‐based sample of schoolchildren.

Methods
Six thousand six hundred and seventy‐two children aged 9–11 years recruited from 108 randomly schools in six French cities underwent a clinical examination including a skin prick test (SPT) to common allergens, exercise‐induced bronchial reactivity (EIB) and skin examination for flexural dermatitis. The prevalence of asthma, allergic rhinitis (AR) and atopic dermatitis was assessed by a standardized health questionnaire completed by the parents. Three‐year‐averaged concentrations of air pollutants (NO2, SO2, PM10 and O3) were calculated at children' schools using measurements of background monitoring stations.

Results
After adjusting for confounders, EIB, lifetime asthma and lifetime AR were found to be positively related to an increase in the exposure to SO2, PM10 and O3. The adjusted odds ratios (aOR) per increase of 5 μg/m3 of SO2 was 1.39 (95% confidence interval (CI)=1.15–1.66) for EIB and 1.19 (1.00–1.41) for lifetime asthma. The aOR for lifetime AR per increase of 10 μg/m3 of PM10 was 1.32 (CI=1.04–1.68). Moreover, SPT positivity was associated with O3 (aOR=1.34; CI=1.24–1.46). Associations with past year symptoms were consistent, even if not always statistically significant. Results persisted in long‐term resident (current address for at least 8 years) children. However, no consistent positive association was found with NO2.

Conclusions
A moderate increase in long‐term exposure to background ambient air pollution was associated with an increased prevalence of respiratory and atopic indicators in children.
Bacterial and fungal agents in house dust and wheeze in children: the PARSIFAL study
Authors: Schram‐Bijkerk D. , Doekes G. , Douwes J. , Boeve M. , Riedler J. , Üblagger E. , von Mutius E. , Benz M. R. , Pershagen G. , van Hage M. , Scheynius A. , Braun‐Fahrländer C. , Waser M. , Brunekreef B. ,   on behalf of the PARSIFAL study group
Published: October 2005

Background
Growing up on a farm and an anthroposophic lifestyle are associated with a lower prevalence of allergic diseases in childhood. This might be related to increased inhalatory exposure to microbial agents.

Objective
To assess the association between microbial agents in house dust and atopic wheeze in farm children, Steiner school children and reference children.

Methods
Levels of bacterial endotoxin, fungal β(1,3)‐glucans and fungal extracellular polysaccharides (EPS) in mattress and living room floor dust were measured in a population of 270 atopic (=Phadiatop‐positive) children with self‐reported wheezing, including 168 current atopic wheezers, and 441 non‐atopic, non‐symptomatic controls. These children were selected from a cross‐sectional study in five European countries.

Results
In the study population as a whole, average levels of mattress dust endotoxin, EPS and glucans were slightly (1.1–1.2‐fold; P<0.10) higher in control children than in atopic wheezers. Atopic wheeze was related to mattress levels of endotoxin, EPS and glucans in farm and farm‐reference children. However, when adjusting for group (farm vs. farm‐reference children), the associations became non‐significant whereas the group effect remained. No associations between atopic wheeze and microbial agents were observed in Steiner and Steiner‐reference children. For current atopic wheeze, the farm effect became non‐significant after adjustment for microbial agent levels.

Conclusion
Not only bacterial endotoxin but also mould components might offer some protection against atopic wheeze in children. However, the protective effect of being raised on a farm was largely unexplained by the mattress microbial agent levels measured in this study.
Nitric oxide synthase polymorphisms and asthma phenotypes in Chinese children
Authors: Leung T. F. , Liu E. K. H. , Tang N. L. S. , Ko F. W. S. , Li C. Y. , Lam C. W. K. , Wong G. W. K.
Published: October 2005

Background
Nitric oxide (NO) is a key factor for balancing T‐helper type 1/T‐helper type 2 immunity. Single nucleotide polymorphisms (SNPs) in nitric oxide synthase (NOS) genes have been associated with atopy and exhaled NO concentrations in Caucasians. We investigated the association between asthma traits and genetic polymorphisms in neuronal NO synthase (NOS1) and endothelial NO synthase (NOS3) in Chinese children.

Methods
Asthmatic children between 5 and 18 years of age and non‐allergic controls were recruited. Plasma total IgE was measured by microparticle immunoassay, whereas allergen‐specific IgEs were measured by fluorescent enzyme immunoassay. Fractional exhaled NO concentration (FeNO) was measured by a chemiluminescence analyser. NOS1 C5266T and NOS3 G894T were genotyped by restriction fragment length polymorphism, and (AAT)n polymorphism in intron 20 of NOS1 was determined by GeneScan analysis.

Results
The mean (SD) ages of 295 asthmatics and 174 controls were 11.1 (3.8) years and 11.6 (4.0) years, respectively (P=0.162). NOS1 C5266T and NOS3 G894T were not associated with asthma, atopy or FeNO. However, significantly more subjects with T/T in NOS1 C5266T had increased plasma total IgE as compared with those with C/T or C/C (P=0.017). This SNP was also associated with sensitization to Dermatophagoides pteronyssinus (P=0.049). Among asthmatic patients, log‐transformed plasma total IgE levels were significantly higher among those homozygous for 5266T of NOS1 [mean (SD): 2.84 (0.44) for T/T, 2.68 (0.42) for C/T, 2.59 (0.69) for C/C; P=0.021]. This study found a significant inter‐ethnic difference in the allele frequencies of AAT repeats, and this polymorphism was associated with high plasma total IgE levels (P=0.044) but not FeNO (P=0.158). NOS3 G894T was not associated with any asthma or atopy phenotype.

Conclusions
NOS1 C5266T and AAT repeats affect plasma IgE concentrations in Chinese children. On the other hand, neither NOS1 nor NOS3 SNP was associated with FeNO or the risk of having asthma.
Double‐blind study of tolerability and antibody production of unmodified and chemically modified allergen vaccines ofPhleum pratense
Authors: Casanovas M. , Sastre J. , Fernández‐Nieto M. , Lluch M. , Carnés J. , Fernández‐Caldas E.
Published: October 2005

BackgroundThe physicochemical modification of allergen extracts provides a chance for administering higher doses of allergen vaccines.

ObjectiveTo evaluate the safety of a chemically modified (depigmented‐glutaraldehyde polymerized) therapeutic vaccine ofPhleum pratenseadministered at doses that are 10 times higher than those used in clinical practice, in comparison with conventional doses of the corresponding non‐modified alum‐adsorbed vaccine.

Materials and methodsThe design of the study was randomized, double‐blind, parallel and included two groups of patients. Twenty‐three patients were treated weekly during nine visits for the build‐up phase, followed by two weekly maintenance doses (a total of 11 injections per patient). Twelve patients received a vaccine containing the standardized unmodified extract, at a maximum concentration of 308.5 mcg of freeze dried material/mL (Group A). Eleven patients received a standardized modified allergen extract (Group B). The maximum dose used was 2400 mcg/mL. Safety was evaluated recording all adverse events. Skin test results and specific antibody levels were evaluated at the beginning and at the end of the study.

ResultsGroup A patients experienced three local immediate (two clinically irrelevant and one with a diameter > 5 cm) and 18 delayed reactions (15 irrelevant and three with a diameter > 10 cm), while Group B experienced six local immediate and 12 delayed reactions (all clinically irrelevant). Nine Group A patients experienced 12 systemic reactions (one immediate of grade 1, one of grade 2; and one delayed of grade 1; four of grade 2 and three of grade 3), while Group B patients experienced one immediate systemic reaction of grades 1, and 1 delayed reaction of grade 1.

ConclusionsThe modified extract ofP. pratenseis safe to treat sensitive patients, even at concentrations that are 10 times higher than those regularly administered in clinical practice. The majority of the local reactions were clinically irrelevant. No systemic reactions of grade 2, 3 or 4 were reported using the modified extract.
Exposure of the fetus and infant to hens' egg ovalbumin via the placenta and breast milk in relation to maternal intake of dietary egg
Authors: Vance G. H. S. , Lewis S. A. , Grimshaw K. E. C. , Wood P. J. , Briggs R. A. , Thornton C. A. , Warner J. O.
Published: October 2005

Background
Maternally derived allergens may be transferred to the developing infant during pregnancy and lactation. However, it is not known how manipulation of environmental allergen levels might impact on this early‐life exposure.

Objective
To measure dietary egg allergen (ovalbumin (OVA)) in gestation‐associated environments, in relation to maternal dietary egg intake.

Method
OVA was measured by allergen‐specific ELISA in maternal blood collected throughout pregnancy, infant blood at birth (umbilical cord) and in breast milk at 3 months post‐partum. Samples derived from pregnant women undergoing diagnostic amniocentesis at 16–18 weeks gestation who were not subject to any dietary intervention, and from pregnant women, with personal or partner atopy, randomized to complete dietary egg exclusion or an unmodified healthy diet before 20 weeks gestation as a primary allergy prevention strategy. Maternal dietary egg intake was monitored closely throughout the study period by diary record and serial measurement of OVA‐specific immunoglobulin G concentration.

Results
Circulating OVA was detected throughout pregnancy in 20% of women and correlated with both presence (P<0.001) and concentration (r=0.754, P<0.001) of infant OVA at birth (umbilical cord). At 3 months post‐partum OVA was detected in breast milk samples of 35% women, in higher concentrations than measured in blood. Blood and breast milk OVA were not related to maternal dietary intake or atopic pre‐disposition.

Conclusions
Rigorous dietary egg exclusion does not eliminate trans‐placental and breast milk egg allergen passage. This early‐life exposure could modulate developing immune responses.
Constructing a 7‐day ahead forecast model for grass pollen at north London, United Kingdom
Authors: Smith M. , Emberlin J.
Published: October 2005

Background A number of media outlets now issue medium‐range (∼7 day) weather forecasts on a regular basis. It is therefore logical that aerobiologists should attempt to produce medium‐range forecasts for allergenic pollen that cover the same time period as the weather forecasts.Objective the objective of this study is to construct a medium‐range (7 day) forecast model for grass pollen at north London.Method the forecast models were produced using regression analysis based on grass pollen and meteorological data from 1990 to 1999 and tested on data from 2000 and 2002. The modelling process was improved by dividing the grass pollen season into three periods; the pre‐peak, peak and post‐peak periods of grass pollen release. The forecast consisted of five regression models: two simple linear regression models predicting the start and end date of the peak period, and three multiple regression models forecasting daily average grass pollen counts in the pre‐peak, peak and post‐peak periods.Results overall, the forecast models achieved 62% accuracy in 2000 and 47% in 2002, reflecting the fact that the 2002 grass pollen season was of a higher magnitude than any of the other seasons included in the analysis.Conclusion this study has the potential to make a notable contribution to the field of aerobiology. Winter averages of the North Atlantic Oscillation were used to predict certain characteristics of the grass pollen season, which presents an important advance in aerobiological work. The ability to predict allergenic pollen counts for a period between five and seven days will benefit allergy sufferers. Furthermore, medium‐range forecasts for allergenic pollen will be of assistance to the medical profession, including allergists planning treatment and physicians scheduling clinical trials.
Atopic dermatitis, extrinsic atopic dermatitis and the hygiene hypothesis: results from a cross‐sectional study
Authors: Zutavern A. , Hirsch T. , Leupold W. , Weiland S. , Keil U. , von Mutius E.
Published: October 2005

Background
Atopic Dermatitis (AD), hayfever and asthma are commonly summarized as atopic diseases. The spatial distribution of AD differs from that of asthma and hayfever, suggesting that AD might follow a different risk pattern than these diseases. AD can be differentiated into an allergic extrinsic form (EAD) and a non‐allergic intrinsic form (IAD). Only EAD might follow the distribution and risk pattern that have been ascribed to asthma and hayfever.

Objective
To investigate the distribution and risk factor profile of AD and EAD focusing on environmental factors relating to the hygiene hypothesis.

Methods
Population‐based cross‐sectional study on 12 601 children aged 5–7 and 9–11 years from Dresden (Eastern Germany) and Munich (Western Germany). Information was obtained by International Study of Asthma and Allergic Childhood questionnaires, dermatological examinations and skin prick testing. AD‐diagnosis ever, current AD‐symptoms and visible eczema were investigated with their respective extrinsic forms.

Results
Maternal and paternal history of AD were equally strong determinants of the child's AD status. Factors related to the hygiene hypothesis like day‐care attendance and number of older siblings were not associated with a decreased risk of AD. The proportion of EAD within AD was higher in Eastern than in Western Germany. The determinants of the diseases appeared to be similar for both EAD and IAD.

Conclusions
There was no evidence of the hygiene hypothesis holding true for AD or EAD. AD might be a separate entity than respiratory atopic diseases. Little is known about the risk factors of AD and factors different from those of respiratory allergic diseases should be considered in future research.
Performance of High Arctic tundra plants improved during but deteriorated after exposure to a simulated extreme temperature event
Authors: Marchand Fleur L. , Mertens Sofie , Kockelbergh Fred , Beyens Louis , Nijs Ivan
Published: October 2005

Arctic ecosystems are known to be extremely vulnerable to climate change. As the Intergovernmental Panel on Climate Change scenarios project extreme climate events to increase in frequency and severity, we exposed High Arctic tundra plots during 8 days in summer to a temperature rise of approximately 9°C, induced by infrared irradiation, followed by a recovery period. Increased plant growth rates during the heat wave, increased green cover at the end of the heat wave and higher chlorophyll concentrations of all four predominating species (Salix arctica Pall., Arctagrostis latifolia Griseb., Carex bigelowii Torr. ex Schwein and Polygonum viviparum L.) after the recovery period, indicated stimulation of vegetative growth. Improved plant performance during the heat wave was confirmed at plant level by higher leaf photochemical efficiency (Fv/Fm) and at ecosystem level by increased gross canopy photosynthesis. However, in the aftermath of the temperature extreme, the heated plants were more stressed than the unheated plants, probably because they acclimated to warmer conditions and experienced the return to (low) ambient as stressful. We also calculated the impact of the heat wave on the carbon balance of this tundra ecosystem. Below‐ and aboveground respiration were stimulated by the instantaneous warmer soil and canopy, respectively, outweighing the increased gross photosynthesis. As a result, during the heat wave, the heated plots were a smaller sink compared with their unheated counterparts, whereas afterwards the balance was not affected. If other High Arctic tundra ecosystems react similarly, more frequent extreme temperature events in a future climate may shift this biome towards a source. It is uncertain, however, whether these short‐term effects will hold when C exchange rates acclimate to higher average temperatures.
Flow cytometric technique for determination of prostasomal quantity, size and expression of CD10, CD13, CD26 and CD59 in human seminal plasma
Authors: CARLSSON LENA , RONQUIST GUNNAR , ELIASSON RUNE , EGBERG NILS , LARSSON ANDERS
Published: October 2005

Prostasomes are prostate‐derived organelles in seminal plasma exhibiting pluripotent properties to facilitate the fertilization process. Seminal prostasome concentration, size distribution and expression of the prostasomal surface antigens CD10, CD13, CD26 and CD59 were examined by flow cytometry. The study group consisted of 79 men with involuntary infertility. Very strong correlations existed between the prostasome expressions of the different CD markers. Significant correlations between prostasome concentration and CD molecules were weak or lacking. Further, no or weak relationships were observed between the prostasomal CD markers and sperm morphology, seminal fructose, neutral α‐glucosidase activity, zinc and tumour necrosis factor α concentrations. Flow cytometry is a practical way to study prostasomes in seminal fluid without prior separation. This is a new technique for evaluation of the role of prostasomes and their functions in male reproductive physiology.
Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase‐dependent control
Authors: Rasmussen Anders Aamann , Eriksen Maiken , Gilany Kambiz , Udesen Christina , Franch Thomas , Petersen Carsten , Valentin‐Hansen Poul
Published: October 2005

The Escherichia coli ompA mRNA, encoding a highly abundant outer membrane protein, has served as a model for regulated mRNA decay in bacteria. The half‐life of this transcript correlates inversely with the bacterial growth rate and is growth stage‐dependent. The stability of the messenger is determined by the 5′‐untranslated region which possesses cleavage sites for RNase E. Hfq binds to this region, is essential for controlling the stability and has been suggested to directly regulate ompA mRNA decay. Here we report that the 78 nucleotide SraD RNA, which is highly conserved among Enterobacteriaceae, acts in destabilizing the ompA transcript when rapidly grown cells enter the stationary phase of growth. During this growth‐stage the expression of SraD RNA becomes strongly increased. The SraD‐mediated decay of ompA mRNA depends on Hfq and in vitro studies revealed that Hfq facilitates binding of the regulatory RNA to the translational initiation region of the messenger. Deletion of sraD, however, does not significantly affect the stability of the ompA mRNA in slowly growing cells. Our results indicate that distinct regulatory circuits are responsible for growth phase‐ and growth rate‐dependent control of the ompA mRNA stability.
Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae
Authors: He Xin‐Jian , Fassler Jan S.
Published: October 2005

The Saccharomyces cerevisiae Yap1p and Skn7p transcription factors collaborate in the activation of oxidative stress response (OSR) genes. Although Yap1p and Skn7p oxidative stress response elements (YRE, OSRE) have been characterized and identified in some OSR genes, many OSR genes lack such elements. In this study, the complex, oxidative responsive, CCP1 promoter was used as a model to investigate the cis‐acting elements responsible for activation by oxidative stress. In addition to consensus YRE and OSRE sequences, novel Yap1p and Skn7p binding sites were identified in the CCP1 promoter. These new sites were found to mediate Yap1p‐ and Skn7p‐dependent activation of OSR genes including TSA1 and CTT1 previously thought to lack Yap1p and Skn7p binding sites. The novel YREs and OSREs were found to be enriched in the promoter regions of a set of 179 OSR genes. The widespread existence of novel Yap1p and Skn7p binding sites strongly suggest that direct binding of Yap1p and Skn7p is responsible for activation of many more OSR genes than previously believed.
Involvement of the Escherichia coli folate‐binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation
Authors: Ote Tomotake , Hashimoto Masayuki , Ikeuchi Yoshiho , Su’etsugu Masayuki , Suzuki Tsutomu , Katayama Tsutomu , Kato Jun‐ichi
Published: October 2005

The Escherichia coli hda gene codes for a DnaA‐related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate‐binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over‐initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP‐DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP‐DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2‐methylthio‐6‐iodeadenosine, indicating that YgfZ participates in the methylthio‐group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.
Dissection of functional domains of the polar localization factor PodJ in Caulobacter crescentus
Authors: Lawler Melanie L. , Larson David E. , Hinz Aaron J. , Klein David , Brun Yves V.
Published: October 2005

The polar organelle development protein, PodJ, is important for proper establishment of polarity in Caulobacter crescentus. podJ null mutants are unable to form holdfast or pili, have reduced swarming motility, and have difficulty ejecting the flagellum during the swarmer to stalked cell transition. In this study, we create a series of truncation mutants to investigate functional domains of PodJ. We show that PodJ has a transmembrane domain between amino acids 600 and 670. We identify a periplasmic region important for pili production and a cytoplasmic region required for holdfast formation and swarming motility, and establish that PleC localization is not required for holdfast formation and motility in soft agar. Analysis of the mutants reveals that the last 54 amino acids of the protein negatively regulate processing of the full‐length form of the protein, PodJL, to a shorter form, PodJS. Finally, we identify a cytoplasmic region of PodJ involved in targeting it to the flagellar pole, and a periplasmic region required for localization of PleC.
Effect of atilmotin on gastrointestinal transit in healthy subjects: a randomized, placebo‐controlled study
Authors: Park M‐i. , Ferber I. , Camilleri M. , Allenby K. , Trillo R. , Burton D. , Zinsmeister A. R.
Published: October 2005

Abstract  We studied effects of i.v. atilmotin (BAX‐ACC‐1638, a novel motilin agonist, circulating t1/2 <10 min) on gastrointestinal transit in humans using a randomized, parallel‐group, dose‐response double‐blind study of i.v. atilmotin, 6, 30, 60 μg or vehicle (placebo) given 2 min after standardized breakfast, lunch and dinner. The breakfast meal contained 99mTc‐eggs and 111In‐milk. Full gastrointestinal transit was measured by scintigraphy. Primary endpoints were % gastric emptying (GE) at 30 min, GE t1/2, colonic filling (CF) at 6 h, and geometric centre of colonic transit at 24 h. Analysis included adjustment for age, gender and body mass index, with Bonferroni correction applied for multiple comparisons. A significant treatment effect of atilmotin was detected for GE (%) at 30 min for solids and liquids (P < 0.01 for both). There were no significant effects on CF or CT and no significant adverse clinical events. Thus, atilmotin accelerates GE of solids and liquids in healthy humans. These data suggest that, at the doses tested, atilmotin should be considered for treatment of stomach motility disorders.
The Impact of Electrical Charge on the Viability and Physiology of Dendritic Cells
Authors: Hilpert F. , Heiser A. , Wieckhorst W. , Arnold N. , Kabelitz D. , Jonat W. , Pfisterer J.
Published: October 2005

The use of electrical charge for electroporation or electrofusion is widely applied to customize dendritic cells (DC) and their immunological properties as anticancer vaccines. The aim of this study was to evaluate the influence of various electrical field strengths on the recovery, viability and physiology of DC. Immature DC were transferred into low‐conductive medium and electrically charged within a range of 0–1500 V/cm. Viability was assessed by Trypan Blue dye exclusion or staining with impermeant nucleic acid stains and fluorescence‐activated cell sorter analysis. Additionally, apoptosis was determined by flow cytometry after staining with Annexin‐V, endocytosis by uptake of fluorescein isothiocyanate‐dextran and metabolic activity by a standardized fluorescent live/dead assay. There was a strong correlation between the electrical field strength and the viability and physiology of DC. Field strengths ≥1000 V/cm significantly impaired viability, metabolism and endocytotic activity. Dual fluorescence with 7‐7‐amino‐actinomycin D and Annexin‐V demonstrated that loss of viability was predominantly due to necrosis rather than apoptosis. Field strengths ≤500 V/cm allowed to maintain good cell viability and recovery of DC and did not cause alterations of metabolism and endocytosis. Therefore, the frequently used amplification of field strengths to improve the efficacy of electroporation and electrofusion requires critical re‐evaluation.
Roles of Group IIA Phospholipase A2 and Complement in Killing of Bacteria by Acute Phase Serum
Authors: Grönroos J. O. , Salonen J. H. , Viander M. , Nevalainen T. J. , Laine V. J. O.
Published: October 2005

The complement system is regarded as an important component of the innate defence system against invading bacteria. However, synergistic actions between the complement and the other components of innate immunity are incompletely known. Human group IIA phospholipase A2 (hGIIA PLA2) is an effective antibacterial enzyme in serum of patients with severe bacterial infections. Our aim was to investigate the significance of complement and hGIIA PLA2 in acute phase serum. Serum samples were collected from patients with acute bacterial infections and from healthy control subjects. We prepared hGIIA PLA2‐depleted serum by immunoadsorption and inhibited the activity of complement by a specific inhibitor, compstatin. The bactericidal effects of treated and untreated serum were compared by incubating Staphylococcus aureus and Listeria monocytogenes in the presence of serum. Acute phase serum effectively killed S. aureus and L. monocytogenes, and depletion of hGIIA PLA2 significantly reduced the antibacterial effect. Complement had a weak bactericidal effect against L. monocytogenes. We conclude that hGIIA PLA2 is the major antibacterial factor in human acute phase serum against the gram‐positive bacteria S. aureus and L. monocytogenes, exceeding complement in efficiency.
Adjuvant Effect of Synthetic Oligodeoxyribonucleotides (CpG‐ODN) From the Paracoccidioides brasiliensis gp43 gene on the Th2–Th1 Immunomodulation of Experimental Paracoccidioidomycosis
Authors: Amaral C. C. , Garcia I. P. , Fernandes G. F. , Almeida S. R. , Camargo Z. P. , Souza M. C.
Published: October 2005

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis. Immunostimulatory effects of P. brasiliensis DNA and CpG‐oligodeoxyribonucleotides (CpG‐ODN) have shown a Th2–Th1 immunomodulation of the isogenic murine model of susceptibility, which develops a progressive and disseminating disease. In this study, we investigated the optimum time interval and doses of CpG‐ODN which are able to induce Th2–Th1 immunomodulation. The optimum concentrations for the induction of a decrease in antibody production were 0.5 and 1 µg. Mice immunized twice with CpG‐ODN and gp43 (5 and 7 days before the challenge) showed a 60% higher chance of survival compared with the control group (nonimmunized), and an increase in Th1 isotype (IgG2a) was also observed. In vitro assays of naive and preimmunized mice showed discrete cellular proliferation when stimulated by suitable concentrations of CpG‐ODN. Type 1 cytokines interleukin‐12 (IL‐12) and interferon‐γ were increased in cell culture supernatants, but no significant difference was found in Th2 IL‐4 cytokines in stimulated or nonstimulated cell cultures. Concerning the Th2–Th1 kinetics in experimental PCM models by adjuvant effect of CpG‐ODN, there are still many questions to be answered and clarified. However, the gathering of data obtained in this investigation has led us to suggest that the modulation of Th2–Th1 in experimental PCM depends on time and CpG‐ODN concentration.
Interleukin‐2‐Activated Natural Killer Cells May Have a Direct Role in the Control of Leishmania (Leishmania) amazonensis Promastigote and Macrophage Infection
Authors: Aranha F. C. S. , Ribeiro U. , Basse P. , Corbett C. E. P. , Laurenti M. D.
Published: October 2005

To study the role of Natural Killer (NK) cells in Leishmania infection, peritoneal macrophages from BALB/c mice were infected with Leishmania (Leishmania) amazonensis promastigotes and incubated with interleukin‐2 (IL‐2)‐activated NK (A‐NK) cells at different ratios of A‐NK cells to infected macrophages (5:1, 1:1, 0.2:1). The A‐NK cells were added either together with the parasites (0‐h group) or 24 h later (24‐h group). Morphological studies of the cultures revealed predominance of parasitic debris within macrophages that were in close contact with A‐NK cells and the decrease in parasite recovery was directly proportional to the A‐NK cell concentration used. Interferon‐γ (IFN‐γ) and IL‐12 were detected in the supernatant at levels proportional to the A‐NK cell concentration used. No significant difference was observed between the groups with respect to NO levels in the culture supernatant. When A‐NK cells were added directly to the L. (L.) amazonensis promastigote cultures, the parasite recovery decreased proportional to the number of A‐NK cells added. In vivo studies demonstrated smaller lesion sizes in animals inoculated with both parasites and A‐NK cells compared with parasites alone. Histopathology of the skin lesions from animals receiving A‐NK cells together with the parasites showed moderate parasitism and a nodular inflammatory infiltrate formed by mononuclear cells and a few vacuolized macrophages. In contrast, animals inoculated only with the parasites showed a highly parasitized dermis with infiltration of intensely vacuolized macrophages. These results demonstrate the role of A‐NK cells in parasite lysis and in resistance of macrophages to L. (L.) amazonensis in the early phase of infection.
Fungal contamination of bedding
Authors: Woodcock A. A. , Steel N. , Moore C. B. , Howard S. J. , Custovic A. , Denning D. W.
Published: October 2005

Background:  It is currently believed that most fungal exposure occurs external to the home.

Aims:  To enumerate the fungal flora of used synthetic and feather pillows and the dust vacuumed from them, in the UK.

Methods:  10 pillows aged between 1.5 and >20 years in regular use were collected and quantitatively cultured for fungi. Swatches were taken from nine sections of the pillow and dust was also collected by vacuum from five pillows. Pillow vacuuming was carried out prior to pillow culture. All were cultured at room temperature, 30 and 37°C for 7 days in broth before plating, and a subset were also cultured for 24 h in broth and then plated. Fungi were identified by standard morphological methods.

Results:  The commonest three species isolated were Aspergillus fumigatus (n = 10), Aureobasidium pullulans (n = 6) and Rhodotorula mucilaginosa (n = 6). Another 47 species were isolated from pillows and vacuum dust. The number of species isolated per pillow varied from 4 to 16, with a higher number from synthetic pillows. Compared with the nonallergenic A. pullulans, more A. fumigatus was found in synthetic than feather pillows.

Conclusions:  We have examined pillows for fungal contamination, and show that the typical used pillow contains a substantial load of many species of fungi, particularly A. fumigatus. Given the time spent sleeping, and the proximity of the pillow to the airway, synthetic and feather pillows could be the primary source of fungi and fungal products. This has important implications for patients with respiratory disease, and especially asthma and sinusitis.
Molecular genetics of the early development of hindbrain serotonergic neurons
Authors: Cordes SP
Published: October 2005

The serotonergic (5HT) system plays a key role in modulating behaviors, such as appetite and anxiety and has been implicated in many human disorders of mood and mind. Recent studies have begun to identify the signaling molecules and transcriptional cascades governing 5HT neuron development in the hindbrain. Already at early stages, local differences in requirements of 5HT neuron development have become apparent. These studies point toward cryptic heterogeneity amongst 5HT neurons and suggest that 5HT neuron determination and differentiation may be more flexible and less absolute biologic processes than might have been expected. Ultimately, the intrinsic heterogeneity and environmental sensitivity of 5HT neruons may help explain the variability observed in some human behavioral disorders, such as autism spectrum disorder, and the less predictable behavioral consequences of fetal alcohol syndrome.
An optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas
Authors: Wuyts W , Radersma R , Storm K , Vits L
Published: October 2005

Hereditary multiple osteochondromas (MO) is an autosomal dominant bone disorder characterized by the presence of bony outgrowths (osteochondromas or exostoses) on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes, which encode glycosyltransferases implicated in heparan sulfate biosynthesis. Standard mutation analysis performed by sequencing analysis of all coding exons of the EXT1 and EXT2 genes reveals a mutation in approximately 80% of the MO patients. We have now optimized and validated a denaturing high‐performance liquid chromatography (DHPLC)‐based protocol for screening of all EXT1‐ and EXT2‐coding exons in a set of 49 MO patients with an EXT1 or EXT2 mutation. Under the optimized DHPLC conditions, all mutations were detected. These include 20 previously described mutations and 29 new mutations – 20 new EXT1 and nine new EXT2 mutations. The protocol described here, therefore, provides a sensitive and cost‐sparing alternative for direct sequencing analysis of the MO‐causing genes.
Influence of Bacillus Calmette‐Guèrin vaccination at birth and 2 months old age on the peripheral blood T‐cell subpopulations [gamma/delta (γδ) and alpha‐beta (αβ) T cell]
Authors: Taştan Y. , Arvas A. , Demir G. , Alikaşifoğlu M. , Gür E. , Kıray E.
Published: October 2005

The neonatal immune system is immature and may be affected by Bacillus Calmette‐Guèrin (BCG) vaccine. We investigated the influence of BCG given at two different ages on the peripheral blood (PB) T‐cell subpopulations. Forty full term healthy newborns were randomly chosen. Twenty of them were vaccinated with BCG at birth (group I) and the remaining at the age of 2 months (group II). The cell analysis were carried out before (pre‐BCGI and pre‐BCGII), and 2 months after (post‐BCGI and post‐BCGII) the vaccination. The analysis of the gamma/delta and alpha/beta T‐cell receptor (TCR) antigens was done by two‐colour flowcytometer. The purified protein derivative (PPD) response was investigated 2 months after vaccination. The results showed that although T‐cell (TCR+ cell) counts showed no difference in PB before and after vaccination in both study groups, the total lymphocyte and non‐T cell (TCR− cell) populations increased significantly whereas αβT‐cell population significantly decreased after vaccination. On the contrary, γδT‐cell counts in PB increased significantly 2 months after vaccination in group I but not in group II. Total lymphocyte and non‐T cell counts in vaccinated infants at 2 months of age (post‐BCGI) were significantly higher than in unvaccinated infants of the same age whereas αβT‐cell count in vaccinated infants was significantly low. However, total T‐cell and γδT‐cell counts showed no difference. PPD positivity was similar in both study groups (61% in group I, 66% in group II). Neither αβT‐ nor γδT‐cell counts were different in PPD positive and PPD negative infants. Our study shows that BCG causes marked quantitative changes in the PB T‐cell subpopulations in young infants.
Comparison of the allergenicity of Actinidia deliciosa (kiwi fruit) and Actinidia chinensis (gold kiwi)
Authors: Lucas Jane S. A. , Lewis Stella A. , Trewin Jennifer B. , Grimshaw Kate E. C. , Warner John O. , Hourihane Jonathan O'B.
Published: October 2005

Actinidia chinensis (gold kiwi) is a newly available fruit which has been shown to have in vitro immunoglobulin E (IgE) cross‐reactivity with green kiwi. This is the first study to investigate clinical reactivity of gold kiwi. Five patients clinically allergic to green kiwi were investigated by skin test and double‐blind placebo controlled food challenge (DBPCFC) with gold kiwi fruit. IgE‐binding patterns of individual sera from the five challenged patients and a pool of sera from a further nine patients with kiwi allergy were compared in the two fruits by Western blotting. Cross reactivity of proteins in the two fruits was assessed by inhibition of immunoblots and by IgE enzyme‐linked immunosorbent assay (ELISA) inhibition. Four of the five patients had a positive DBPCFC to gold kiwi. Western blotting showed marked differences in the allergen patterns of green and gold kiwi. However, inhibition of the immunoblots and ELISA assay reveals extensive inhibition of IgE binding to proteins in each fruit by the alternative species. Gold kiwi fruit is allergenic and patients allergic to green kiwi are at risk of reacting to the gold kiwi fruit. Despite having different protein profiles and IgE‐binding patterns, the two species have proteins that extensively cross‐inhibit the binding to IgE.
Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato
Authors: Mora‐Herrera Martha Elena , López‐Delgado Humberto , Castillo‐Morales Alberto , Foyer Christine H.
Published: October 2005

The effects of salicylic acid (SA) and hydrogen peroxide (H2O2) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4‐week‐old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H2O2 were estimated at the following harvest points: stage (a) 4‐week‐old in vitro plants treated with either 0.1 mM SA or 5 mM H2O2; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4‐h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H2O2 accumulation only in Atlantic at stage (a); (4) H2O2 enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H2O2 treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H2O2 accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H2O2 accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA‐induced tolerance in Alpha was not accompanied by H2O2 accumulation. H2O2 was able to induce freezing tolerance only in Atlantic, even though H2O2 accumulated in both cultivars following this treatment.
Immunolocalization of the saposin‐like insert of plant aspartic proteinases exhibiting saposin C activity. Expression in young flower tissues and in barley seeds
Authors: Brodelius Maria , Hiraiwa Masao , Marttila Salla , Al Karadaghi Salam , Picaud Sarah , Brodelius Peter E.
Published: October 2005

The plant‐specific insert (PSI) of cypro11 gene‐encoding cyprosin, an aspartic proteinase from Cynara cardunculus, has been cloned by polymerase chain reaction (PCR) into a bacterial expression vector. A rearranged form of this PSI in which the N‐ and C‐terminal sequences were permutated to make it more similar to the structural arrangement observed in saposins was also cloned and expressed in the same system. The biological activities of the two purified recombinant proteins were compared to those of human saposins B and C. The proteins showed similar activity to saposin C, i.e. capacity to activate human glucosylceramidase. At a concentration of 5 µM, wild‐type PSI, saposin C, and rearranged PSI activated human glucosylceramidase two‐, three‐, and five‐fold, respectively. The Km for 4‐methylumbelliferyl β‐glucopyranoside was around 7 µM in the presence of any of the three activators (5 µM). The neurotropic activity using NS20Y cells and lipid‐binding properties of the plant recombinant proteins were tested. The two plant proteins showed lipid‐binding properties similar to those of saposins but did not have any effect on neurite outgrowth. Immunolocalization of PSI showed its expression in protective tissues in flower meristem – protodermis, in C. cardunculus and embryonic root cap and coleorhiza in mature barley grains – as well as husk, pericarp, and the aleurone layer. Possible biological functions suggested for the plant homologue to saposins besides the general activation of enzymes involved in lipid metabolism would be involvement in plant defence.
Retrospective Study on Vulvar Conformation in Relation to Endometrial Cytology and Fertility in Thoroughbred Mares
Authors: Hemberg E. , Lundeheim N. , Einarsson S.
Published: October 2005

Changes in the conformation of the vulva predispose the mare to uterine infection. Vulvoplasty for closure of the upper vulvar lips improves fertility. Not all mares are resutured after parturition, but are resutured after mating. No clinical data have hitherto been published on the reproductive outcome in mares that are resutured or not after parturition. The objectives of the present study were to investigate relationships between vulvar status (not Caslick‐operated, group A; previously Caslick‐operated and/or not resutured after the last parturition, groups B and C respectively) and endometrial cytology, conception rate and live foal rate for different age groups and categories of Thoroughbred mares. A careful examination of the vulvar area, and endometrial sampling for bacteriology and cytology was performed. The mares were naturally mated once during oestrus (never foal heat) by one of two stallions. Mares belonging to vulvar status group A had the highest conception rate and live foal rate, whereas the lowest conception rate and live foal rate occurred in mares with vulvar status group C. A multifactorial analysis revealed that the vulvar status at arrival had a significant effect on pregnancy rate and live foal rate, but not on fetal loss. Stallion, year and age group had no significant effect on any of the three variables analysed. In conclusion, optimizing managerial factors such as resuturing Caslick‐operated mares immediately after parturition to minimize growth of micro‐organisms with a subsequent endometritis will contribute to their conception and that they carry a pregnancy to term.
Interdigital Involvement in a Case of Primary Cutaneous Canine Histoplasmosis in Japan
Authors: Nishifuji K. , Ueda Y. , Sano A. , Kadoya M. , Kamei K. , Sekiguchi M. , Nishimura K. , Iwasaki T.
Published: October 2005

A 5‐year‐old male Siberian husky bred outdoor in Tokyo had a swollen paw with interdigital granulomatous lesions in the left hindlimb. The dog had no apparent pulmonary or gastrointestinal involvement. Histopathological analysis of the skin lesions demonstrated yeast‐like organisms predominantly within macrophages. Sequence analysis of fungal ribosome RNA gene isolated from a paraffin sample revealed a 100% homology with the teleomorph of Histoplasma capsulatum. The present case may support the concept of primary cutaneous canine histoplasmosis as an endemic phenotype recognized in Japan.