bluuebunny/crossref_metadata_2025 huggingface dataset:
Risks factors for highly unstable response to oral anticoagulation: a case‐control study
Authors: Palareti Gualtiero , Legnani Cristina , Guazzaloca Giuliana , Lelia Valdré , Cosmi Benilde , Lunghi Barbara , Marchetti Giovanna , Poli Daniela , Pengo Vittorio ,   the ad hoc study group of the Italian Federation of Anticoagulation Clinics
Published: March 2005

The factors associated with persistent instability of oral anticoagulant treatment (OAT) were investigated in a case‐control study. The most unstable patients from 35 Italian anticoagulation clinics were matched with stable controls, for gender, age and OAT indication. Socio‐demographic data, medical history, dietary and life habits, cytochrome P450 CYP2C9 variants, blood cell count, liver and renal functions were investigated. An ‘Abbreviated Mental Test’ (AMT) and a questionnaire to assess patient compliance to, and comprehension of, OAT indications and mechanisms were administered. An International Normalized Ratio (INR) above 4·5 was more frequently found in cases (n = 77) than controls (n = 80) (12·3% vs. 0·4%; P < 0·0001). The odds ratio for instability was significantly higher for: people who worked versus pensioners, acenocoumarol versus warfarin, and an insufficient score in the AMT and/or in the questionnaire. Cytochrome P450 CYP2C9 variants *1/*3 or *2/*3 or *3/*3 were more frequent among cases than controls (29·9% vs.15·0%; P = 0·042). No differences were observed as regards the other variables. In conclusion, we found that high intra‐individual variability in OAT control was multifactorial, but poor OAT comprehension was prevalent.
Does cryosupernatant plasma improve outcome in thrombotic thrombocytopenic purpura? No answer yet
Authors: Rock Gail , Anderson David , Clark William , Leblond Pierre , Palmer Douglas , Sternbach Marion , Sutton David , Wells George ,   members of the Canadian Apheresis Group and members of the Canadian Association of Apheresis Nurses
Published: March 2005

A randomized prospective trial compared cryosupernatant plasma (CSP) to fresh frozen plasma (FFP) for treatment of thrombotic thrombocytopenic purpura (TTP). A total of 236 patients were required: 28 patients were treated with CSP and 24 with FFP within 30 months. There were no differences in survival at 1 month. By day 9, 17 of 26 patients with CSP and 18 of 24 with FFP had a platelet count >100 × 109/l. At entry, von Willebrand factor (VWF) multimers were normal in all patients (range 1·1–3·95 IU/ml). ADAMTS‐13 levels showed large variations ranging from 10% to 100% activity. At entry, no individual had <5% VWF cleaving protease. By day 9 (end of cycle), 89% (FFP) and 67% (CSP) had levels >50% of the controls. At 6 months some patients showed inhibitors to the enzyme in spite of adequate or normal platelet counts. The data from this study do not show an apparent advantage to the use of CSP in TTP. A large number of patients will be required to determine appropriate replacement therapy. We were not able to find a statistically significant relationship between the low level of protease activity at presentation of TTP and response.
FRETS‐VWF73, a first fluorogenic substrate for ADAMTS13 assay
Authors: Kokame Koichi , Nobe Yuko , Kokubo Yoshihiro , Okayama Akira , Miyata Toshiyuki
Published: March 2005

A plasma metalloprotease, ADAMTS13, cleaves von Willebrand factor (VWF) multimers and downregulates their activity in platelet aggregation. Functional ADAMTS13 deficiency leads to the accumulation of hyperactive large VWF multimers, inducing a life‐threatening disease, thrombotic thrombocytopenic purpura (TTP). Although measuring ADAMTS13 activity is important in TTP diagnosis, existing methods require time and skill. Here, we report a fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity. We developed a synthetic 73‐amino‐acid peptide, FRETS‐VWF73. Cleavage of this substrate between two modified residues relieves the fluorescence quenching in the intact peptide. Incubation of FRETS‐VWF73 with normal human plasma quantitatively increased fluorescence over time, while ADAMTS13‐deficient plasma had no effect. Quantitative analysis could be achieved within a 1‐h period using a 96‐well format in commercial plate readers with common filters. The FRETS‐VWF73 assay will be useful for the characterization of thrombotic microangiopathies like TTP and may clarify the importance of ADAMTS13 activity as a predictive marker for various thrombotic diseases.
Early response to induction is predictive of survival in childhood Philadelphia chromosome positive acute lymphoblastic leukaemia: results of the Medical Research Council ALL 97 trial
Authors: Roy Anindita , Bradburn Mike , Moorman Anthony V. , Burrett Julie , Love Sharon , Kinsey Sally E. , Mitchell Chris , Vora Ajay , Eden Tim , Lilleyman John S. , Hann Ian , Saha Vaskar ,   Medical Research Council Childhood Leukaemia Working Party
Published: March 2005

We report on the outcome of children with Philadelphia positive acute lymphoblastic leukaemia (Ph+ ALL) treated on the UK Medical Research Council (MRC) trial for childhood ALL, MRC ALL 97, between January 1997 and June 2002. Forty‐two (2·3%) patients were Ph+. Nineteen (45%) had <25% blasts in bone marrow (BM) within the first 2 weeks of treatment and were defined as a good response group (GRG), the others as a poor response group (PRG). Thirty‐six (86%) achieved first complete remission (CR1) at the end of induction, of which 28 underwent BM transplantation (BMT). The median follow‐up was 42 months (range, 21–84). The 3‐year event‐free survival (EFS; 52%, 95% CI, 36–66%) was a considerable improvement on the previous MRC UKALL XI trial (27%). EFS for the GRG and PRG were 68% (43–84%) and 39% (18–59%), respectively (P = 0·03); presenting white cell count <50 × 109/l (P = 0·02) was predictive for overall survival. Changes in the MRC ALL97 trial within the study period resulted in some Ph+ ALL receiving daunorubicin and either prednisolone or dexamethasone during induction. Though the use of daunorubicin during induction was not a prospective study question, EFS was significantly better for those whose induction included this drug (P = 0·02). Steroid randomization was not stratified for Ph+ ALL patients and was not predictive for EFS. BMT in CR1 appeared to reduce the risk of a subsequent BM relapse. These results show significant improvement on previous MRC trials; future therapeutic strategies should include early intensive therapy and BMT in CR1.
Therapeutic administration of Budesonide ameliorates allergen‐induced airway remodelling
Authors: McMillan S. J. , Xanthou G. , Lloyd C. M.
Published: March 2005

Background
Airway inflammation and remodelling are important pathophysiologic features of chronic asthma. Although current steroid use demonstrates anti‐inflammatory activity, there are limited effects on the structural changes in the lung tissue.

Objective
We have used a mouse model of prolonged allergen challenge that exhibits many of the salient features of airway remodelling in order to investigate the anti‐remodelling effects of Budesonide.

Methods
Treatment was administered therapeutically, with dosing starting after the onset of established eosinophilic airway inflammation and hyper‐reactivity.

Results
Budesonide administration reduced airway hyper‐reactivity and leukocyte infiltration in association with a decrease in production of the Th2 mediators, IL‐4, IL‐13 and eotaxin‐1. A reduction in peribronchiolar collagen deposition and mucus production was observed. Moreover, our data show for the first time that, Budesonide treatment regulated active transforming growth factor (TGF)‐β signalling with a reduction in the expression of pSmad 2 and the concomitant up‐regulation of Smad 7 in lung tissue sections.

Conclusions
Therefore, we have determined that administration of Budesonide modulates the progression of airway remodelling following prolonged allergen challenge via regulation of inflammation and active TGF‐β signalling.
1,3‐β‐glucanases as candidates in latex–pollen–vegetable food cross‐reactivity
Authors: Palomares O. , Villalba M. , Quiralte J. , Polo F. , Rodríguez R.
Published: March 2005

Background
1,3‐β‐glucanases (group 2 of pathogenesis‐related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens.

Objective
The aim of this work was to study the potential implication of 1,3‐β‐glucanases in cross‐reactivities among latex, pollen and vegetable foods.

Methods
The cDNA encoding the N‐terminal domain (NtD) of Ole e 9, a major allergenic 1,3‐β‐glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N‐terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD‐specific polyclonal antiserum were used.

Results
The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3‐β‐glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD‐specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3‐β‐glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell‐pepper, banana and latex.

Conclusion
rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.
The predictive value of specific immunoglobulin E levels in serum for the outcome of oral food challenges
Authors: Celik‐Bilgili S. , Mehl A. , Verstege A. , Staden U. , Nocon M. , Beyer K. , Niggemann B.
Published: March 2005

Background
Specific serum IgE is considered as one of the important diagnostic measures in the diagnostic work‐up of food allergy.

Objective
To evaluate the role of specific serum IgE in predicting the outcome of oral food challenges, and to determine threshold concentrations of specific serum IgE that could render double‐blind, placebo‐controlled food challenges unnecessary.

Methods
In 501 children (median age 13 months), 992 controlled oral challenges were performed with cow's milk (CM), hen's egg (HE), wheat and soy. 440/501 (88%) children suffered from atopic dermatitis. For all children, specific IgE concentrations in serum were determined. Sensitivity, specificity, positive and negative predictive values, receiver operator characteristics‐curves as well as predictive decision points were calculated.

Results
Four hundred and forty‐five out of 992 oral food challenges with allergens were assessed as positive. Sensitivity of specific serum IgE was 97% for HE, 83% for CM, 69% for soy, and 79% for wheat. Specificity was 51% for HE, 53% for CM, 50% for soy, and 38% for wheat. Calculating 90%, 95% and 99% predicted probabilities using logistic regression revealed predictive decision points of 6.3, 12.6, and 59.2 kU/L for HE, respectively. Subdividing our children in those of below or above 1 year of age resulted in a markedly different predicted probability for HE. For CM, only the 90% predicted probability (88.8 kU/L) could be calculated. No decision points could be determined for CM, wheat and soy.

Conclusion
In general, specific serum IgE levels showed a correlation with the outcome of positive oral food challenges for CM and HE. Meaningful predictive decision points can be calculated for HE, which may help to avoid oral food challenges in some cases. However, data need to be ascertained for each allergen separately. Furthermore, the age of the patient population under investigation must also be taken into account.
Increase in daytime symptoms is a sensitive and specific criterion for predicting corticosteroid‐treated exacerbations in a clinical asthma trial
Authors: Dennis S. M. , Altmann D. R. , Lee T. H.
Published: March 2005

Background
To determine which diary card variables are the most predictive for administration of additional courses of corticosteroids using the TRUST (The Regular Use of Salbutamol Trial) data set.

Methods
Logistic regression models were used to identify the extent to which a change in diary card variable affected the odds ratio (OR) for administering a course of oral or increased inhaled corticosteroids. The complete TRUST diary card data were used with over 200 000 days of diary card observations from 983 mild to moderate asthmatic subjects.

Results
An increase in daytime symptoms of 1–5 U over baseline was associated with an increase in the OR for starting all types of corticosteroids from two‐ to 60‐fold.

Conclusions
These results indicate that an increase in daytime symptoms of two or more over baseline strongly predicts the administration of additional corticosteroids. The results have significant implications for both clinical practice and design of clinical trials in asthma.
Prenatal lipopolysaccharide‐exposure prevents allergic sensitization and airway inflammation, but not airway responsiveness in a murine model of experimental asthma
Authors: Blümer N. , Herz U. , Wegmann M. , Renz H.
Published: March 2005

Background
Epidemiological evidence underlines the impact of prenatal environmental factors on the development of postnatal allergies. In this regard an inverse correlation between lipopolysaccharide (LPS) exposure and development of childhood allergy has been found.

Objective
To assess the impact of prenatal LPS exposure on the development of postnatal respiratory allergies in a mouse model of experimental asthma.

Methods
Female BALB/c mice were exposed to LPS before conception and during pregnancy. Several weeks after birth offspring were sensitized to ovalbumin (OVA) followed by aerosol allergen challenges.

Results
Prenatal, maternal LPS‐exposure enhanced neonatal IFN‐γ, but not IL‐4 and IL‐2 production. OVA sensitization of prenatally LPS‐exposed mice was accompanied by a marked suppression in anti‐OVA IgG1 and IgE as well as unchanged IgG2a antibody responses, paralleled by a significant reduction in IL‐5 and IL‐13 levels following mitogenic stimulation of splenic leucocytes. Assessment of bronchoalveolar lavage fluids following allergen challenges revealed a marked reduction in eosinophils and macrophages in these mice. Surprisingly, development of airway hyper‐responsiveness, a hallmark of bronchial asthma, was not affected.

Conclusion
This study provides first experimental evidence that LPS may already operate in prenatal life in order to modulate the development of allergies in the offspring.
Glutathione protects human airway proteins and epithelial cells from isocyanates
Authors: Wisnewski A. V. , Liu Q. , Liu J. , Redlich C. A.
Published: March 2005

Background
Glutathione (GSH), one of the major anti‐oxidants of the lung, has been linked to the human response to isocyanate exposure. However, the ability of GSH to modulate key chemical reactions, thought to be central to the development of human isocyanate allergy, has not been directly analyzed under biologic exposure conditions.

Objective
To better understand the potential role of GSH in the response to occupational isocyanate exposure, we evaluated its effects on two processes thought to be involved in the development of isocyanate allergy, isocyanate–protein conjugation and epithelial cell toxicity.

Methods
The effects of GSH on (1) isocyanate conjugation with albumin, its major target in the airway fluid and (2) isocyanate‐induced toxicity to human airway epithelial cell lines, A549 and NCI‐H292, were tested using two different in vitro models. For protein conjugation studies, a newly described vapour exposure system was used to model the air/liquid interface at the surface of the epithelial fluid in the airways. Epithelial cell exposures were performed in fluid phase to mimic the in vivo exposure of airway cells covered by epithelial lining fluid.

Results
Reduced GSH prevented hexamethylene diisocyanate (HDI) conjugation to albumin in a dose‐dependent manner, while oxidized GSH (GSSG) conversely increased conjugation rates. GSH levels equivalent to those found in normal human airway fluid (100 μm) provided >90% protection against HDI‐protein conjugation when albumin was exposed to HDI vapour levels 10‐fold above permissible occupational limits. Physiologic levels of GSH, but not GSSG, also reduced HDI toxicity to human airway epithelial cells in a dose‐dependent manner, when present extracellularly, however, drugs that modulate intra‐cellular GSH levels did not significantly alter isocyanate toxicity.

Conclusions
Together with previously reported genetic and toxicity studies, the data suggest that airway GSH plays an important role in protection against HDI exposure and may help prevent the development of allergic sensitization and asthma.
Cytosine–phosphate–guanine motifs fail to promote T‐helper type 1‐polarized responses in human neonatal mononuclear cells
Authors: Prescott S. L. , Irwin S. , Taylor A. , Roper J. , Dunstan J. , Upham J. W. , Burgner D. , Richmond P.
Published: March 2005

Background
The T‐helper type 1 (Th1) trophic properties of bacterial cytosine–phosphate–guanine (CpG) motifs have made them logical adjuvants both for the suppression of Th2‐mediated allergic disease in early life and for promoting vaccine responses in neonates who have relatively immature Th1 function. However, little is known about their effects on immature immune responses in this period.

Objectives
To compare the effects of CpG on adult and neonatal cellular immune responses to various stimuli.

Methods
The immune responses of mononuclear cells (MC) derived from neonates (n=25) and their mothers (n=25) were compared in vitro. These were stimulated with house dust mite (HDM), CpG B, CpG C, non‐CpG oligodeoxynucleotides (ODN) or diphtheria toxoid (DT) in optimized conditions. In parallel cultures, CpGs were combined with HDM or DT antigens to assess the effect of the various ODN on these antigen‐specific responses. Lymphoproliferation and cytokine responses IL‐13, IFN‐γ, IL‐6, IL‐10, TNF‐α) were measured for all of the cultures described above.

Results
Although neonates showed attenuated lymphoproliferation to CpG, the production of antigen‐presenting cell‐derived cytokines such as IL‐6 and IL‐10 and the up‐regulation of major histocompatibility complex class II (HLA‐DR) were detected at adult levels. T cell‐derived cytokines (IL‐13 and IFN‐γ) were not detectable in response to CpG alone. Most neonates also failed to produce detectable IFN‐γ to HDM or DT (unlike adults), but readily produced IL‐13 to these stimuli. The addition of CpG resulted in an increase in neonatal IFN‐γ production in response to HDM (P=0.011) and a similar but non‐significant trend with DT. However, rather than inhibiting Th2 IL‐13 responses, the addition of CpGs was associated with a significant increase in the IL‐13 responses to HDM (P=0.016) and DT (P=0.03), effects not seen in adults.

Conclusions
This study provides further evidence that neonatal MC responses to CpG are functionally different from adults, and do not show clear Th1 polarization. The CpG associated increase in Th2 responses may reflect a potentiation of the normal neonatal Th2 propensity, or non‐specific activation of neonatal MC.
Polymorphisms in the 5‐lipoxygenase activating protein (ALOX5AP) gene are not associated with asthma in an Australian population
Authors: Kedda M.‐A. , Worsley P. , Shi J. , Phelps S. , Duffy D. , Thompson P. J.
Published: March 2005

Background
The cysteinyl‐leukotrienes (cys‐LTs) are important pro‐inflammatory mediators in asthma, and have been shown to have a role in specific disease subtypes, including asthma severity. Few studies have investigated the role of polymorphisms in the ALOX5AP gene, encoding 5‐lipoxygenase activating protein (FLAP), and asthma. We hypothesized that polymorphisms in this gene are associated with asthma and in particular, with asthma severity, in an Australian population.

Objective
To screen the coding region of the ALOX5AP gene for polymorphisms and to determine the association between previously described polymorphisms and asthma and asthma severity in an Australian population.

Methods
We used PCR‐SSCP and PCR‐RFLP analysis to examine a previously described promoter polyA variable repeat polymorphism and two intronic polymorphisms (IVS2+12C>A, IVS2+105T>C), and to screen all five exons of the gene for new polymorphisms, in a large Australian population of randomly selected, non‐asthmatic controls (n=457), mild asthmatics (n=274), moderate asthmatics (n=231) and severe asthmatics (n=79).

Results
We confirmed the presence of two polymorphisms in intron 2 and found no association between these polymorphisms and asthma or asthma severity, nor between a promoter polymorphism in the ALOX5AP gene and asthma or asthma severity. Gene fragment analysis of the promoter polymorphism revealed novel, conserved repeat numbers in our population, and no new polymorphisms were found in the coding region of the gene.

Conclusion
These findings in a large, well characterized asthma population, reveal that, while FLAP is an important enzyme in cys‐LTs biosynthesis, polymorphisms in the ALOX5AP gene are not likely to be functionally associated with the asthma phenotype.
A polymorphism in the promoter region of the human interleukin‐16 gene is not associated with asthma or atopy in an Australian population
Authors: Akesson L. S. , Duffy D. L. , Phelps S. C. , Thompson P. J. , Kedda M.‐A.
Published: March 2005

Background
IL‐16 is an immunomodulatory cytokine whose expression is increased in the bronchial mucosa, bronchoalveolar lavage fluid and induced sputum of asthmatic patients. It has been suggested that IL‐16 has a regulatory role in the pathophysiology of asthma. A single‐nucleotide polymorphism (T−295C) has been described in the promoter region of the gene and it has been hypothesized that this polymorphism may be associated with altered levels of IL‐16 expression, and account for the increased levels of IL‐16 seen in the asthmatic airway.

Objective
To determine the association between the T−295C promoter polymorphism and asthma, disease severity and atopy in a large Australian Caucasian population.

Methods
We used PCR and restriction fragment length polymorphism analysis to establish the allele frequency of the T−295C promoter polymorphism in a random Australian Caucasian population (n=176) and to characterize the polymorphism in a large Australian Caucasian population of mild (n=273), moderate (n=230) and severe (n=77) asthmatic patients, and non‐asthmatic controls (n=455). Genotype association analyses were performed using χ2 tests.

Results
The polymorphic C allele was present in 19% of the asthmatic population and 21% of the non‐asthmatic population. There was no association between the polymorphism and asthma (P=0.153) nor with asthma severity (P=0.417) or atopy (P=0.157) in this population.

Conclusion
Although it has been hypothesized that the T−295C promoter polymorphism may be associated with increased IL‐16 gene expression, it is not associated with asthma, disease severity or atopy in this Australian population.
Percutaneous application of peptidoglycan from Staphylococcus aureus induces an increase in mast cell numbers in the dermis of mice
Authors: Matsui K. , Nishikawa A.
Published: March 2005

Background
Atopic dermatitis (AD) is a chronic inflammatory skin disease with immunopathologic features that vary depending on the duration of the lesion. The dermis of lesional skin of AD patients shows an increased number of inflammatory cells such as mast cells, eosinophils and mononuclear cells and superficial Staphylococcus aureus colonization.

Objective
The purpose of this study was to determine the effects of peptidoglycan (PEG) from S. aureus on mast cell induction in murine skin.

Methods
PEG was applied to barrier‐disrupted abdominal skin of mice every 5 days and the number of mast cells in the abdominal skin was counted 20 days after the first application. The cytokine response was investigated by RT‐PCR and immunohistologic analysis.

Results
The number of mast cells in the skin of mice treated with PEG was increased significantly compared with that of mice given phosphate‐buffered saline. In addition, application of PEG to the abdominal skin increased the expression of mRNA for transforming growth factor‐β1 (TGF‐β1), which supports mast cell migration, but not that for IL‐3 or stem cell factor, which support both mast cell proliferation and mast cell migration. Immunohistologic analysis demonstrated that levels of TGF‐β1 transcripts corresponded with those of protein synthesis in the epidermis. TGF‐β1 was found to be highly expressed in keratinocytes of the basal epidermis of PEG‐treated skin. Furthermore, intraperitoneal injection of anti‐TGF‐β1 antibodies neutralized the induction of mast cells into the skin.

Conclusion
These results suggest that PEG may have the ability to induce an increase in mast cell numbers in the skin through TGF‐β1 production by epidermal keratinocytes. Skin inflammation might therefore be linked to colonization with S. aureus in AD patients.
Why generic and disease‐specific quality‐of‐life instruments should be used together for the evaluation of patients with persistent allergic rhinitis
Authors: Leong K. P. , L. Yeak S. C. , M. Saurajen A. S. , H. Mok P. K. , Earnest A. , Siow J. K. , C. Chee N. W. , Yeo S. B. , Khoo M. L. , Y. Lee J. C. , Seshadri R. , Chan S. P. , Tang C. Y. , Chng H. H.
Published: March 2005

Background
The importance of assessing health‐related quality of life (HRQL) of patients with allergic rhinitis (AR) has been well established, but the specific roles of rhinitis‐specific or general health instruments have not been delineated.

Objective
We analysed the psychometric properties of a disease‐specific instrument, the Rhinoconjunctivitis Quality‐of‐Life Questionnaire (RQLQ) and the general health instrument, the Medical Outcome Short‐Form 36 (SF‐36) as they are employed in combination in patients with persistent AR in clinical practice.

Method

We analysed the data collected from a prospective study of 43 newly diagnosed patients with persistent AR and 44 controls. We interviewed the patients four times, at baseline, weeks 4, 8 and 10.

Results
The RQLQ and SF‐36 have good discriminative property, internal consistency, and test–retest reliability. The RQLQ is superior to the SF‐36 as an evaluative instrument because more of its domains respond to change, the magnitude of change was greater, and the response was faster. The SF‐36 is more susceptible to floor and ceiling effects. Both instruments are unsuitable for mildly symptomatic patients based on Rasch model analysis. Each questionnaire assesses a distinct and significant portion of the total HRQL of persistent AR.

Conclusion
The SF‐36 and RQLQ are good for discriminating rhinitis patients from controls, but the former is poor for detecting changes in QOL. Both are inappropriate for mildly symptomatic patients. Each instrument measures non‐overlapping halves of the measurable HRQL. For an assessment of the HRQL in persistent AR that is complete and responsive both instruments should be employed together.
Transforming growth factor‐β1 increases CXCR4 expression, stromal‐derived factor‐1α‐stimulated signalling and human immunodeficiency virus‐1 entry in human monocyte‐derived macrophages
Authors: Chen Shuzhen , Tuttle Daniel L. , Oshier Joseph T. , Knot Harm J. , Streit Wolfgang J. , Goodenow Maureen M. , Harrison Jeffrey K.
Published: March 2005

Stromal‐derived factor‐1 (SDF‐1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus‐1 (HIV‐1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV‐1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV‐1, blood and tissue levels of transforming growth factor‐β1 (TGF‐β1) are increased. The purpose of this study was to evaluate the effects of TGF‐β1 on CXCR4 expression and function in primary human monocyte‐derived macrophages (MDMs) and rat microglia. TGF‐β1 up‐regulated CXCR4 and enhanced SDF‐1α‐stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual‐tropic CXCR4‐using HIV‐1 (D‐X4). In contrast, TGF‐β1 failed to increase CCR5 expression or infection by a CCR5‐using virus in MDMs. Our data demonstrate that TGF‐β1 enhances macrophage responsiveness to SDF‐1α stimulation and susceptibility to HIV‐1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual‐tropic X4 viral variants at later stages of HIV‐1 infection.
Heat shock up‐regulates expression of Toll‐like receptor‐2 and Toll‐like receptor‐4 in human monocytes via p38 kinase signal pathway
Authors: Zhou Jun , An Huazhang , Xu Hongmei , Liu Shuxun , Cao Xuetao
Published: March 2005

Heat stress can alert innate immunity by inducing stress proteins such as heat‐shock proteins (HSPs). However, it remains unclear whether heat stress affects the activation of antigen‐presenting cell (APC) in response to pathogen‐associated molecule patterns (PAMPs) by directly regulating pathogen recognition receptors (PRRs). As an important kind of PRRs, Toll‐like receptors (TLRs) play critical roles in the activation of immune system. In this study, we demonstrated that heat shock up‐regulated the expression of HSP70 as well as TLR2 and TLR4 in monocytes. The induction of TLRs was prior to that of HSP70, which suggesting the up‐regulation of TLR2 and TLR4 might be independent of the induction of HSP70. Heat shock activated p38 kinase, extracellular signal‐related kinase (ERK) and nuclear factor‐kappa B (NF‐κB) signal pathways in monocytes. Pretreatment with specific inhibitor of p38 kinase, but not those of ERK and NF‐κB, inhibited heat shock‐induced up‐regulation of TLR2 and TLR4. This indicates that p38 pathway takes part in heat shock‐induced up‐regulation of TLR2 and TLR4. Heat shock also increased lipoteichoic acid‐ or lipopolysaccharide‐induced interleukin‐6 production by monocytes. These results suggest that the p38 kinase‐mediated up‐regulation of TLR2 and TLR4 might be involved in the enhanced response to PAMP in human monocytes induced by heat shock.
Complement receptors regulate lipopolysaccharide‐induced T‐cell stimulation
Authors: Kaya Ziya , Tretter Theresa , Schlichting Jens , Leuschner Florian , Afanasyeva Marina , Katus Hugo A. , Rose Noel R.
Published: March 2005

Complement receptors type 1 and 2 (CR1 (CD35)/CR2 (CD21)) are known to enhance the adaptive immune response. In mice, CR1/CR2 are expressed on B cells, follicular dendritic cells, and activated granulocytes. Recently, we showed that a subset of CD44high and CD62Llow T cells also expresses CR1 and CR2. We now report that CR1/CR2 are detectable on both CD4+ and CD8+ subsets of T cells. Lipopolysaccharide (LPS) from Gram‐negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other antigen‐presenting cells. We further demonstrate that LPS induced marked up‐regulation of CD25 and CD69 on T cells from CR1/CR2 sufficient (Cr+/+), but significantly lower up‐regulation on T cells from CR1/CR2 deficient (Cr–/–) mice. These findings point to a novel mechanism by which CR1/CR2 modulates the activation of T cells by LPS.
T‐cell homeostasis in mice exposed to airborne xenobiotics
Authors: Drela Nadzieja , Bień Justyna , Kozłowska Ewa
Published: March 2005

Many effects of environmental toxic agents contribute to the deregulation of immune system homeostasis. Here we demonstrate that the effect of airborne suspended matter (ASM) on the generation of mouse T cells is reversible. This reversal can be achieved by an active process that returns the T cells to homeostasis and does not result from the simple effect of ASM deprivation. An accelerated development of thymocytes and increased influx of T‐cell progenitors to the thymus in mice exposed to environmental xenobiotics has been postulated. This hypothesis has been confirmed by parallel increases in the percentages of single‐positive and triple‐negative thymocytes. Enhanced expression of thymocyte surface markers related to positive selection has also been observed. The pathway of T‐cell progenitor development is favoured in the bone marrow of mice exposed to ASM.
B‐cell reconstitution by transplantation of B220+ CD117+ B‐lymphoid progenitors into irradiated mice
Authors: Kawaguchi Susumu
Published: March 2005

Bone marrow cells of 4‐week‐old CBA/J mice were searched for progenitors that can reconstitute CBA‐type B lineage (B220+) cells after transplantation into irradiated (BALB/c × CBA/J) F1 mice. In recipients of B lineage‐committed B220+ CD117+ cells, which were CD19+ CD127+ CD25– IgM–, numbers of CBA‐type B220+ cells had increased greatly by day 8 after transplantation. This increase stopped by day 10 but cell numbers remained at this level for at least 8 weeks. B‐cell production in the bone marrow of B220+ CD117+ cell recipients occurred even 8 weeks after transplantation. Probably, the transplanted B‐lymphoid progenitors are capable of self‐renewing up to 8 weeks after transplantation. B lineage‐ uncommitted CD117high CD71– cells, which were B220– and included haematopoietic stem cells, could also reconstitute B lineage bone marrow cells after transplantation. Sequential development of CD117+, CD25+ and IgM+ cells in CBA‐type B220+ bone marrow cells occurred 2–4 days faster in recipients of B220+ CD117+ progenitors than in recipients of CD117high CD71– progenitors, suggesting that the development of the former progenitors from the latter may take 2–4 days.
Transforming growth factor‐β1 enhances the interferon‐γ‐dependent, interleukin‐12‐independent pathway of T helper 1 cell differentiation
Authors: Smeltz Ronald B. , Chen June , Shevach Ethan M.
Published: March 2005

Transforming growth factor (TGF)‐β, a pleiotropic cytokine that has multiple effects on immune responses, has been shown to inhibit interleukin (IL)‐4/GATA‐3 expression as well as T helper 2 (Th2) differentiation. Consistent with these reports, we found that priming T cells from DO11.10 transgenic mice with antigen in the presence of TGF‐β inhibited GATA‐3 expression and the development of IL‐4‐producing T cells. Unexpectedly, the inhibition of Th2 development was accompanied by a substantial increase in the number of interferon‐γ (IFN‐γ)‐producing cells. T cells primed with TGF‐β secreted IFN‐γ in response to both T‐cell receptor ligation and IL‐12/IL‐18 stimulation, and expressed high levels of T‐bet and low levels of GATA‐3. The TGF‐β‐mediated enhancement of T helper 1 (Th1) priming was independent of IL‐12 and signal transducer and activator of transcription (STAT)‐4, but required endogenous IFN‐γ. TGF‐β‐mediated enhancement of the IFN‐γ‐dependent, IL‐12‐independent pathway of Th1 priming was mediated primarily by the inhibition of IL‐4 produced by memory/activated T cells in the unfractionated CD4+ responder population. Nevertheless, TGF‐β did not inhibit this pathway of Th1 differentiation when purified naive CD4+ T cells were used as responders. These data have important implications for strategies being considered for the use of TGF‐β‐producing T cells for the treatment of autoimmune disorders.
Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line
Authors: Echevarria‐Lima Juliana , Kyle‐Cezar Fernanda , P. Leite Daniela F. , Capella Luiz , Capella Márcia A. M. , Rumjanek Vivian M.
Published: March 2005

Multidrug resistance proteins [MRPs and P‐glycoprotein (Pgp)] are members of the family of ATP‐binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti‐cancer agents in tumour cells. These proteins act as ATP‐dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy‐2′‐7′‐dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration‐dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double‐positive cells and CD8 single‐positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation.
Bodyguards and assassins: Bcl‐2 family proteins and apoptosis control in chronic lymphocytic leukaemia
Authors: Packham Graham , Stevenson Freda K.
Published: March 2005

Chronic lymphocytic leukaemia (CLL) is the most common B‐cell malignancy in the Western world and exists as subtypes with very different clinical courses. CLL is generally described as a disease of failed apoptosis. Apoptosis resistance may stem from a combination of microenvironmental survival signals as well as from intrinsic alterations in the apoptotic machinery within the CLL cell. The molecular mechanism involved in controlling apoptosis in CLL is complex and is influenced by many factors, including Bcl‐2 family proteins, which are critical regulators of cell death. Here we review the significance of apoptosis dysregulation in CLL, focusing on the role of Bcl‐2 and related Bcl‐2 family proteins, such as Bax and Mcl‐1. The differential properties of the newly described subsets of CLL are also highlighted.
Analysis of the activation profile of dendritic cells derived from the bone marrow of interleukin‐12/interleukin‐23‐deficient mice
Authors: Bastos Karina R. B. , De Deus Vieira de Moraes Luciana , Zago Cláudia A. , Marinho Cláudio R. F. , Russo Momtchilo , Alvarez José M. M. , D'Império Lima Maria R.
Published: March 2005

We have previously shown that macrophages from interleukin (IL)‐12p40 gene knockout (IL‐12/IL‐23–/–) mice have a bias towards the M2 activation profile, spontaneously secreting large quantities of transforming growth factor‐β1 (TGF‐β1) and producing low levels of nitric oxide (NO) in response to lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ). To verify whether the activation profile of dendritic cells (DCs) is also influenced by the absence of IL‐12/IL‐23, bone marrow‐derived DCs from IL‐12/IL‐23–/– and C57BL/6 mice were evaluated. At first we noticed that ≈ 50% of the C57BL/6 DCs were dead after LPS‐induced maturation, whereas the mortality of IL‐12/IL‐23–/– DCs was < 10%, a protective effect that diminished when recombinant IL‐12 (rIL‐12) was added during maturation. Similarly to macrophages, mature IL‐12/IL‐23–/– DCs (mDCs) produced higher levels of TGF‐β1 and lower levels of NO than C57BL/6 mDCs. NO release was IFN‐γ‐dependent, as evidenced by the poor response of IFN‐γ–/– and IL‐12/IL‐23–/–IFN‐γ–/– mDCs. Nevertheless, IFN‐γ deficiency was not the sole reason for the weak NO response observed in the absence of IL‐12/IL‐23. The high level of TGF‐β1 secretion by IL‐12/IL‐23–/– mDCs could explain why exogenous IFN‐γ partially restored the NO production of IFN‐γ–/– mDCs, while IL‐12/IL‐23–/– IFN‐γ–/– mDCs remained unresponsive. We also showed that CD4+ T‐cell proliferation was inhibited by C57BL/6 mDCs, but not by IL‐12/IL‐23–/– mDCs. IFN‐γ and NO appear to mediate this antiproliferative effect because this effect was not observed in the presence of mDCs from IFN‐γ–/– or IL‐12/IL‐23–/– IFN‐γ–/– mice and it was attenuated by aminoguanidine. We conclude that the presence of IL‐12/IL‐23 during LPS‐induced maturation influences the activation profile of DCs by a mechanism that is, only in part, IFN‐γ dependent.
Various members of the Toll‐like receptor family contribute to the innate immune response of human epidermal keratinocytes
Authors: Köllisch Gabriele , Kalali Behnam Naderi , Voelcker Verena , Wallich Reinhard , Behrendt Heidrun , Ring Johannes , Bauer Stefan , Jakob Thilo , Mempel Martin , Ollert Markus
Published: March 2005

Toll‐like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)‐κB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1–10 expression profile. First, reverse transcription–polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1–10 mRNA in real‐time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF‐κB‐activated chemokine interleukin‐8 (IL‐8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL‐8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL‐8 secretion, whereas no IL‐8 was induced by LPS, R‐848, loxoribine and cytosine guanine dinucleotide‐containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF‐κB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.
Reduced susceptibility to dextran sulphate sodium‐induced colitis in the interleukin‐2 heterozygous (IL‐2+/–) mouse
Authors: Sund Malin , Xu Li Li , Rahman Arman , Qian Bi‐Feng , Hammarström Marie‐Louise , Danielsson Åke
Published: March 2005

Mice homozygous for an inactivation of the interleukin‐2 (IL‐2) gene develop a T‐cell dependent colitis. Heterozygous (IL‐2+/–) mice are clinically healthy but have been shown to express reduced levels of IL‐2 in the colon. Splenocytes from the IL‐2+/– mice had a poorer proliferative response to polyclonal T‐cell activation and these mice have reduced numbers of intestinal regulatory T cells (CD4+ CD25+ cells) when compared to wild type mice. When exposed to dextran sulphate sodium (DSS) IL‐2+/– mice showed a markedly reduced susceptibility to DSS‐induced colitis. While DSS treatment caused a marked increase in both CD4+ and CD8+ colonic T cells expressing increased levels of IL‐2, IL‐4, and IL‐10 in wild type mice none of these changes were seen in IL‐2+/– mice. On the contrary, cytokine expression in intestinal T cells of IL‐2+/– mice was actually reduced after DSS treatment. These results suggest that reduced levels of IL‐2 leads to attenuated activation and function of intestinal T cells in IL‐2+/– mice and a failure to react adequately to DSS exposure.
Identification and characterization of a functional, alternatively spliced Toll‐like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens
Authors: Philbin Victoria J. , Iqbal Muhammad , Boyd Yvonne , Goodchild Marianne J. , Beal Richard K. , Bumstead Nat , Young John , Smith Adrian L.
Published: March 2005

Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll‐like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8‐like gene with a 6‐kilobase insertion containing chicken repeat 1 (CR1) retroviral‐like insertion elements. The chicken TLR7 gene encodes a 1047‐amino‐acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine‐rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune‐related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin‐1β (IL‐1β) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up‐regulated both chicken IFN‐α and chicken IFN‐β mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up‐regulation of chicken IL‐1β, and chicken IL‐8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.
Tracking the dynamics of T‐cell activation in response to Salmonella infection
Authors: Ravindran Rajesh , McSorley Stephen J.
Published: March 2005

Despite the current availability of Salmonella vaccines, typhoid fever remains a significant public health problem in developing countries. A greater understanding of T‐cell activation and the development of immunological memory during Salmonella infection should lead to the development of more effective prophylactic intervention. Here, we review recent literature on the initiation, expansion and memory development of T‐cell responses using the mouse model of typhoid. We pay particular attention to strategies for tracking T‐cell responses in vivo and ex vivo, and suggest models to integrate some these studies.
Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population1
Authors: SINGH KIRAN , SINGH S. K. , SAH R. , SINGH INDU , RAMAN RAJIVA
Published: March 2005

A mutation (C677T) in the gene, MTHFR, is known to increase susceptibility to various multifactorial disorders. In order to assess this single nucleotide polymorphism (SNP) as risk factor for idiopathic male infertility, a case–control study was done on an Indian population. DNA from 151 cases of non‐obstruction, idiopathic oligo‐/azoospermia and 200 fertile males (controls) was polymerase chain reaction amplified using site‐specific primers, and analysed for the mutation following HinfI‐digestion. Our results show a significantly increased frequency of CT heterozygotes among infertile patients (p value <0.04). More importantly, while there were no T homozygotes in the control population, six of 151 infertile cases were T homozygous. Considering that T allele occurs in very low frequency in the control population, 677T is clearly a risk factor for infertility in the Indian population. We contend that the same could also be true for African and Southeast Asian populations where the frequency of 677T is very low. The lack of similar association in western populations could be because of the overall dietary enrichment of folates, which could nullify or minimize the effect of this polymorphism.