bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired secretion of interleukin‐4 and interleukin‐13 by allergen‐specific T cells correlates with defective nuclear expression of NF‐AT2 and jun B: relevance to immunotherapy
Authors: Faith A. , Richards D. F. , Verhoef A. , Lamb J. R. , Lee T. H. , Hawrylowicz C. M.
Published: September 2003

Background
Allergen immunotherapy (IT) is a successful treatment associated with decreased Th2 cytokine production by allergen‐specific T cells. We have previously demonstrated (Faith et al., J Immunol 1997; 159:53–57) that inhibition of Th2 cytokine production in vitro correlates with impaired tyrosine kinase activity through the TCR. The transcription factor complex, nuclear factor of activated T cells (NF‐AT), which regulates Th2 cytokine production is controlled by the activity of tyrosine kinases.

Objective
To address whether decreased Th2 cytokine production by allergen‐specific CD4+ T cells following IT is correlated with altered translocation and nuclear expression of the NF‐AT family member, NF‐AT2, and the activator protein 1 (AP1) component of NF‐AT, jun B.

Methods
T cell lines specific for insect venom phospholipase A2 (PLA) were derived from patients prior to and during conventional venom IT. Nuclear expressions of NF‐AT and jun B were assessed following stimulation through the TCR. Th1 and Th2 cytokine and IL‐10 production by insect venom‐specific T cells were also determined. Results were compared with a well‐established model system in which anergy was induced in cloned, allergen‐specific Th2 cells.

Results
Impaired translocation and decreased expression of NF‐AT2 and jun B were detected in PLA‐specific T cell lines derived from bee venom‐allergic individuals following 16 weeks treatment compared to pre‐treatment. These results correlated with significantly reduced production of IL‐4 and IL‐13 and significantly increased production of IFN‐γ and IL‐10 by PLA‐specific T cells. Impaired IL‐4 and IL‐13 production also correlated with defective nuclear expression of NF‐AT2/jun B in cloned, anergic allergen‐specific Th2 cells.

Conclusion
These results suggested that optimal production of IL‐4 and IL‐13 by allergen‐specific T cells is dependent on the nuclear expression of NF‐AT2 and jun B. Thus, specific inhibition of NF‐AT2/jun B might be an option in novel and improved forms of allergen IT.
Non‐bronchoscopic sampling and culture of bronchial epithelial cells in children
Authors: Doherty G. M. , Christie S. N. , Skibinski G. , Puddicombe S. M. , Warke T. J. , De Courcey F. , Cross A. L. , Lyons J. D. M. , Ennis M. , Shields M. D. , Heaney L. G.
Published: September 2003

Background
The bronchial epithelium is likely to play a vital role in airway diseases in children, such as asthma and viral‐associated wheeze. In adults, studies with primary bronchial epithelial cells cultured from samples obtained by fibre‐optic bronchoscopy have provided key insights into the role of the epithelial cell. However, it is difficult to justify bronchoscopy in children to obtain epithelial cells for research purposes.

Objective
To examine the possibility of retrieving and culturing viable epithelial cells using a blind non‐bronchoscopic method from children undergoing elective surgery.

Methods
Subjects were children undergoing elective surgery under general anaesthesia. Following intubation, non‐bronchoscopic bronchoalveolar lavage and non‐bronchoscopic bronchial brushing were performed. A sheathed bronchial cytology brush was advanced through the endotracheal tube, wedged and then withdrawn 2–3 cm before gentle sampling was used to collect bronchial epithelial cells. Initial samples were used to characterize the number, type and viability of epithelial cells recovered compared to a control group of adults undergoing standard bronchoscopic sampling. Subsequent samples were used to establish primary bronchial epithelial cell cultures in children both with and without wheezing illness.

Results
A total of 63 children underwent bronchial brushing [38 male; median age 7.1 years (1.0–14.2 years]. Initial samples (n=30) showed recovery of viable epithelial cells comparable to that from a single brush obtained via a bronchoscope in an adult control group (n=11). In 27 (82%) of the subsequent 33 samples obtained non‐bronchoscopically from children, primary bronchial epithelial cell cultures were successfully established. There were no adverse effects attributable to sampling.

Conclusion
We have shown that non‐bronchoscopic bronchial brushing is a safe and effective technique for recovering viable bronchial epithelial cells that consistently yield primary cultures. This method will facilitate examination of the role of the epithelium in paediatric disease.
Assessment of ocular and nasal irritation in asthmatics resulting from fragrance exposure
Authors: Opiekun R. E. , Smeets M. , Sulewski M. , Rogers R. , Prasad N. , Vedula U. , Dalton P.
Published: September 2003

Background
Many asthmatics report worsening of symptoms following exposure to odours and sensory irritants commonly found in household and cosmetic products. Despite this, little evidence exists to confirm the degree to which such subjective reports are correlated with localized, objective changes in the upper or lower airways following a fragranced product exposure.

Objective
Subjective symptom reports were compared to objective measures in mild asthmatics, moderate asthmatics and non‐asthmatics following exposure to one of two fragranced household aerosol mixtures and a clean air control condition to determine if asthmatics reported greater subjective symptoms of nasal congestion or exhibited objective measures of elevated ocular irritation and nasal congestion following exposure than did healthy controls.

Methods
Measures of nasal mucosal swelling, using acoustic rhinometry, and photographic assessments of ocular hyperemia, using macro‐photography, were taken before exposure, immediately after an initial 5‐min exposure and again following a 30‐min exposure to either of two, fragranced aerosol products and a clean air control. Self‐reports of nasal patency at each time‐point were also obtained.

Results
Although moderate asthmatics tended to report more nasal congestion following fragranced product exposure than did non‐asthmatics, no exposure‐related changes in ocular redness or nasal mucosal swelling were observed among the three groups. Spirometry readings also failed to show evidence of any exposure‐related changes in pulmonary function.

Conclusion
Despite claims that exposure to fragranced products may trigger ocular and respiratory symptoms among asthmatics, we found no evidence that 30 min of exposure to one of two fragranced aerosols elicited objective adverse effects in the ocular or nasal mucosa of mild and moderate asthmatics. While physiological mechanisms of fragrance impact may yet be responsible for some of the adverse reports among asthmatics following fragrance exposure, such reports may also reflect a non‐physiological locus of symptom perception triggered by other sensory cues.
Measurement of basophil‐activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti‐CD203c
Authors: Kahlert H. , Cromwell O. , Fiebig H.
Published: September 2003

Background
The assessment of the basophil‐activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild‐type and natural allergens.

Methods
Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE‐conjugated anti‐CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously.

Results
Grass pollen allergoids revealed a 10–10 000‐fold reduction of basophil‐activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50–10 000‐fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement.

Conclusions
The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.
CD28/CTLA4 double deficient mice demonstrate crucial role for B7 co‐stimulation in the induction of allergic lower airways disease
Authors: Deurloo D. T. , Van Berkel M. A. T. , Van Esch B. C. A. M. , Hofhuis F. , Nijkamp F. P. , Oosterwegel M. A. , Van Oosterhout A. J. M.
Published: September 2003

Background
The existence of a third B7‐1/B7‐2 receptor was postulated in a recent study using a novel mouse strain lacking both CD28 and CTLA4 (CD28/CTLA4−/−).

Objective
In the present study, it was investigated if T cell co‐stimulation via the putative B7‐1/B7‐2 receptor plays a role in the induction of Th2‐mediated asthma manifestations in mice.

Methods
BALB/c wild‐type, CD28/CTLA4−/− and B7‐1/B7‐2−/− mice were sensitized and aerosol challenged with ovalbumin (OVA).

Results
At 24 h after the last aerosol, wild‐type mice showed airway hyper‐responsiveness in vivo and up‐regulated levels of serum OVA‐specific IgE compared with the situation shortly before OVA challenge. In addition, eosinophil numbers and IL‐5 levels in the broncho‐alveolar lavage fluid and Th2 cytokine production by lung cells upon OVA re‐stimulation in vitro were observed. In agreement with an earlier study, we failed to induce any of the asthma manifestations in B7‐1/B7‐2−/− mice. Importantly, also CD28/CTLA4−/− mice showed no asthma manifestations upon OVA sensitization and challenge.

Conclusion
These data clearly demonstrate that T cell co‐stimulation via the putative B7‐1/B7‐2 receptor appears to have no role in the induction of Th2‐mediated asthma manifestations in this murine model and, conversely, that CD28 signalling is crucial.
Immunological analysis of allergenic cross‐reactivity between peanut and tree nuts
Authors: De Leon M. P. , Glaspole I. N. , Drew A. C. , Rolland J. M. , O'Hehir R. E. , Suphioglu C.
Published: September 2003

Background
Peanut and tree nut allergy is characterized by a high frequency of life‐threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross‐reactive allergens remains unknown.

Objective
The aim of this study was to investigate the presence of allergenic cross‐reactivity between peanut and tree nuts.

Methods
Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross‐reactivity between peanut and tree nuts.

Results
Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut‐ and tree nut‐allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre‐incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross‐reactivity. Pre‐incubation of sera with cashew nut extract did not cause any inhibition.

Conclusion
These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross‐reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross‐reactivity.
Assessment of T lymphocyte cytokine production in induced sputum from asthmatics: a flow cytometry study
Authors: Boniface S. , Koscher V. , Mamessier E. , El Biaze M. , Dupuy P. , Lorec A.‐M. , Guillot C. , Badier M. , Bongrand P. , Vervloet D. , Magnan A.
Published: September 2003

Background
Asthma results from a bronchial inflammation in which Th2 lymphocytes play a pivotal role, as shown in invasive bronchial biopsies and broncho‐alveolar lavages. Induced sputum (IS) is a non‐invasive method of recovery of bronchial cells, which can be repeated in the same patients. However, lymphocyte activation has not been studied in IS to date, because of the low number of T cells recovered. Herein we took advantage of flow cytometry, a method suitable for the study of small cell populations, to assess T cell cytokine production in IS.

Objectives
(1) To assess induced sputum T cell cytokine production by flow cytometry in asthmatic subjects and controls. (2) To compare the T cell cytokine production between symptomatic and non‐symptomatic asthmatics.

Methods
Thirteen asthmatics and 19 controls were included. Sputum was induced by a hypertonic saline. Sputum cells were stimulated and intracellular IL‐13 and IFN‐γ were detected in T cells by flow cytometry.

Results
Stimulation induced an increase of IL‐13 and IFN‐γ production by T cells. This increase was higher in asthmatics. IL‐13‐producing T cells were increased in asthmatics after stimulation. In symptomatic asthma, IFN‐γ‐producing T cells were in higher proportion than in controlled asthma.

Conclusion
IS T cell cytokine production indicates a basic Th2 bias in asthma, accompanied during symptoms by a Th1‐like activation. These results open the field for longitudinal studies of the variation of T cell activation in asthma.
Direct and indirect exposure to pets – risk of sensitization and asthma at 4 years in a birth cohort
Authors: Almqvist C. , Egmar A.‐C. , Hedlin G. , Lundqvist M. , Nordvall S. L. , Pershagen G. , Svartengren M. , Hage‐Hamsten M. van , Wickman M.
Published: September 2003

Introduction
There are conflicting data on the association between early exposure to pets and allergic diseases. Bias related to retrospective information on pet ownership has been addressed as a reason for distorted study results.

Objective
To elucidate how early exposure to cat and dog relates to IgE‐sensitization and asthma in children at 2 and 4 years of age, in a prospective birth‐cohort study.

Methods
Four thousand and eighty‐nine families with children born 1994–1996 in predefined areas of Stockholm answered questionnaires on environmental factors and symptoms of allergic disease at birth, one, two and four years of age. Dust samples collected from the mothers' beds at birth were analysed for Fel d 1 and Can f 1 in a subgroup of the cohort. Blood samples taken at four years from 2614 children were analysed for allergen‐specific IgE to common airborne allergens. Risk associations were calculated with a multiple logistic regression model, with adjustment for potential confounders.

Results
A correlation was seen between allergen levels and reported exposure to cat and dog. Exposure to cat seemed to increase the risk of cat sensitization, OR (odds ratio) 1.44 (95% confidence interval 1.03–2.01), whereas dog exposure did not have any effect on dog sensitization, OR 1.16 (0.79–1.72). Dog ownership was related to a reduced risk of sensitization to other airborne allergens, OR 0.36 (0.15–0.83), and a similar tendency was seen for cat ownership OR 0.63 (0.37–1.07). Early dog ownership seemed to be associated with a lower risk of asthma, OR 0.50 (0.24–1.03), with no corresponding effect after cat ownership, OR 0.88 (0.56–1.38).

Conclusion
Early exposure to cat seems to increase the risk of sensitization to cat but not of asthma at 4 years of age. Dog ownership, on the other hand, appears to be associated with lowered risk of sensitization to airborne allergens and asthma. Both aetiological relationships and selection effects have to be considered in the interpretation of these findings.
Histamine‐releasing factors, a heterogeneous group of different activities
Authors: Budde I. Kleine , Aalberse R. C.
Published: September 2003

Histamine‐releasing factor or HRF is a collective term used for a heterogeneous group of factors with different modes of action. The current review is focussed on IgE‐dependent HRF that require the presence of certain types of IgE (designated IgE+) to induce histamine release. IgE+ might be a structurally different IgE molecule, or, alternatively, autoreactive IgE. A subgroup of IgE‐dependent HRF does not bind to IgE, such as cloned HRF p23. This factor turned out to be a basophil‐priming cytokine. Alternatively IgE‐dependent HRF might be an autoallergen. Several groups demonstrated IgE antibodies to human proteins. However, not all IgE autoallergen‐containing extracts induce histamine release of appropriately sensitized basophils. In culture supernatants of human mononuclear cells an autoallergenic activity (Agmn) is found, but no binding to IgE+ was found yet. Agmn might be an autoallergen, since it is cross‐reactive with a grass pollen allergen in the stripped basophil assay.

IgE‐dependent HRF and IgE+ may play a role in the late allergic reaction (LAR). However, IgE+ responsiveness to Agmn (IgEmn+) was not required for a bronchial LAR. IgEmn+ is associated with chronic allergic disease, since the prevalence of IgEmn+ is high in the serum of severe asthmatics and atopic dermatitis patients. Our hypothesis is that exogenous allergens induce IgE antibodies cross‐reactive with an endogenous protein. During a LAR, these endogenous proteins are released and the subsequent IgE‐mediated reaction prolongs and aggravates the allergic and/or asthmatic symptoms.

In conclusion, HRF is a confusing term since it is used for different activities. It might be better to avoid this terminology on and just describe the activity of the factors. Autoallergenic activity is likely to explain most, if not all, IgE‐dependent activity.
Plasma and hepatic tissue levels of thrombomodulin, tissue factor, NFκB and nitric oxide in responders and nonresponders to IFNα therapy
Authors: George M. , Baluch M. , Van Thiel D. H.
Published: September 2003

Summary. Hepatitis C virus (HCV) infects hepatocytes and utilizes the hepatocyte to replicate. In so doing, many hepatocyte activities are shifted from their native state to one reflecting liver cell stress. Thrombomodulin and tissue factor are endothelial cell proteins that are expressed as a result of tissue injury or stress. Urokinase is a serine protease, which has been implicated in a number of physiologic and pathologic processes related to cellular stress and or injury. Nitric oxide is produced by cells in response to injury and functions both as a vasodilator and as an activator of a large number of cytokine cascades. NFκB is a transcription factor that forms one of the first lines of cellular defense against infection and hepatocellular stress.

The levels of these four factors in plasma, hepatocyte cytosol and hepatocyte nuclear extracts provide a precise panoramic measure of cellular stress. Plasma, hepatocyte cytosol and nuclear extracts of hepatocytes were assayed for these four factors in 17 patients treated with αIFN for chronic hepatitis C. Five of the 17 were responders while 12 were nonresponders. Ten normal controls and 1 normal control liver were assayed also for each parameter.

Nonresponders had 2x the plasma urokinase levels of responders and normals. The cytosol prepared from hepatocytes of nonresponders had a urokinase level 15‐fold that of the controls and responders to IFN therapy. Plasma thrombomodulin levels in nonresponders were sixfold greater than those of responders and controls. The levels of all of the other measures in plasma, cytosol and nuclear extracts of liver tissue varied minimally between responders and nonresponders and the normal controls.

These data demonstrate that: (i) urokinase levels in plasma and more clearly in cytosol are greater in nonresponders than responders, and (ii) plasma thrombomodulin levels in nonresponders are sixfold greater than those of responders and controls. These data suggest that urokinase and thrombomodulin may be unique markers of cellular and endothelial stress present in individuals with chronic hepatitis C. These markers might be useful during the clinical course of chronic hepatitis C, as a means of gauging the tissue response to therapy.
Slow viral dynamics of hepatitis C virus genotype 4
Authors: Halfon P. , Neumann A. U. , Bourlière M. , Rieu A. , Chadapaud S. , Khiri H. , Ouzan D. , Cacoub P.
Published: September 2003

Summary. Patients infected with hepatitis C virus genotype 4 (HCV‐4) respond to interferon alpha (IFN) as poorly as those infected with genotype 1. However, there is no information on the viral dynamics of HCV‐4. Interferon–ribavirin treatment was administered to untreated patients infected with HCV‐4. Viral load was assessed with Versant 3.0. Viral dynamics parameters were estimated based on the bi‐phasic model for HCV during IFN treatment. Viral kinetics of HCV‐4 follow a bi‐phasic decline pattern also. The mean effectiveness of IFN in blocking production of HCV‐4 was a decline of 77.8% during the first day of treatment. The half‐life of free virions was estimated at 3.5 h and that of infected cells from 1.9 to over 70 days. The viral dynamics parameters of HCV‐4 appear similar to those of HCV‐1 and slower than those of HCV‐2. HCV‐4 infected patients should be grouped with those with HCV‐1 when therapeutic schemes are considered in relation to genotype.
Mycophenolate mofetil in the treatment of autoimmune HCV‐associated haematological disorders showing steroid resistance or dependence
Authors: Ierardi E. , Rendina M. , Francavilla R. , Barone M. , Castellaneta A. , Panella C. , Francavilla A. , Cuomo R.
Published: September 2003

Summary. We report two cases of hepatitis C virus (HCV) associated autoimmune haematological disorders successfully treated with an unusual protocol (mycophenolate mofetil: MMF). The first case was a male patient with chronic HCV infection who developed, during interferon (IFN)/ribavirin therapy, severe autoimmune thrombocytopenia unresponsive to steroids. MMF was then administered and, simultaneously, the steroid dose was gradually reduced until withdrawal. Following this strategy, a progressive increase in platelet count and complete negativity of anti‐PLT antibodies were achieved without changes in HCV‐RNA quantitative determination. The second case was a woman with HCV liver cirrhosis with severe anaemia and Coombs test positivity partially responsive to continuous administration of steroid high doses. However, this treatment unmasked a severely painful vertebral osteoporosis. For this reason we introduced MMF and simultaneously steroid therapy was progressively reduced until withdrawal. Haemoglobin reached a normal value and the Coombs test became negative within 60 days. These case reports suggest that MMF may represent an interesting therapeutic approach for autoimmune HCV associated haematological disorders.
A randomized trial to assess the efficacy of interferon‐alpha daily in combination with ribavirin in the treatment of naïve patients with chronic hepatitis C
Authors: Tassopoulos N. C. , Ketikoglou I. , Tsantoulas D. , Raptopoulou M. , Hatzis G. , Vafiadis I. , Sidiropoulos L. , Kanatakis S. , Anagnostopoulos G. , Sypsa V. , Hatzakis A. ,   the Hellenic Viral Hepatitis Research Network
Published: September 2003

Summary. A randomized trial was conducted to assess the efficacy of interferon‐alpha (IFN) daily in combination with ribavirin in 301 naïve patients with chronic hepatitis C (CHC). Patients were randomized to receive ribavirin 1.2 g daily (QD) for 48 weeks with either IFN 5 MU (thrice weekly) TIW for 8 weeks followed by IFN 3 MU TIW for 40 weeks (IFN TIW, n = 154) or IFN 5 MU QD for 8 weeks followed by IFN 3 MU QD for 16 weeks followed by IFN 3 MU TIW for 24 weeks (IFN QD, n = 147). Treatment discontinuation rates, because of adverse events, were similar in the two arms (14.9% in IFN TIW and 14.3% in IFN QD, P = 0.87). The proportion of patients with sustained virological response (SVR) was 27.9% for patients treated TIW and 38.8% for those treated QD (P = 0.046). According to logistic regression analysis, patients in the IFN QD arm had 1.7 times higher probability of achieving SVR, than those receiving IFN TIW (P = 0.038). Low baseline viral load (P = 0.017) and genotype non‐1 (P = 0.036) were associated with higher SVR rates. Combination of IFN/ribavirin for 48 weeks is more effective when IFN is administered daily for the first 24 weeks in naïve patients with CHC.
Intracerebral haemorrhage and hepatitis C treatment
Authors: Ferencz S. , Batey R.
Published: September 2003

Summary. We report 3 episodes of intracerebral haemorrhage in a population of 1460 hepatitis C infected patients. We suggest the possibility of a link between HCV and its treatment and the occurrence of this serious complication.
Comparison of 125I‐interferon‐α binding to peripheral blood cells from African–Americans and Caucasians with hepatitis C
Authors: Kimball P. , Verbeke S. , Shiffman M.
Published: September 2003

Summary. Interferon‐α (IFN‐α) is the major treatment for chronic hepatitis C virus (HCV) infection. Drug resistance is problematic, particularly among African–Americans who typically show poorer clinical outcomes than Caucasians. The reasons for ethnic variation in IFN‐α sensitivity are not clear. We speculated that African–American insensitivity to IFN‐α may be mediated by reduced density of the IFN‐α receptor (IFN‐αR) or reduced internalization of the IFN‐α/IFN‐αR complex. This speculation was evaluated by comparing binding, uptake and release of 125iodine‐labelled IFN‐α (125I‐IFN‐α) to peripheral blood cells from African–Americans and Caucasians with HCV infection and ethnically matched healthy volunteers. Under various in vitro conditions, binding of 125IFN‐α to surface receptors was equivalent (P = ns) between African–Americans and Caucasians with HCV infection as well as healthy volunteers (P = ns). Similarly, internalization and release of the 125I‐IFN‐α/IFN‐αR complex was equivalent (P = ns) between African–Americans and Caucasians with HCV infection and healthy volunteers (P = ns). In addition, ethnicity did not influence (P = ns) IFN‐α suppression of phytohaemagluttinen induced proliferation. However, IFN‐α therapy of the same patients showed that African–Americans had lower response rates than Caucasians (14%vs 54%, P < 0.0001). In summary, IFN‐α resistance among African–Americans is not mediated by intrinsic differences in IFN‐α receptor density or internalization.
Hepatitis B virus: prevalence of precore/core promoter mutants in different clinical categories of Indian patients
Authors: Gandhe S. S. , Chadha M. S. , Walimbe A. M. , Arankalle V. A.
Published: September 2003

Summary. To determine the association of precore (Pre‐C)/basal core promoter (BCP) mutants with clinical outcome of hepatitis B in Western India, 192 hepatitis B virus (HBV) infected individuals were investigated. HBV‐DNA PCR positivity among asymptomatic hepatitis B surface antigen (HBsAg) positive carriers (61/100) was lower (P < 0.0001) than chronic hepatitis B (CHB), acute (P = 0.0001), and fulminant hepatitis B patients (P = 0.047). Pre‐C status was based on restriction fragment length polymorphism (RFLP, n = 153) and sequencing (n = 118). Prevalence of Pre‐C mutants was higher among carriers (23/61) than CHB (10/62, P = 0.0071) or acute (3/22; P = 0.037) patients. Children from carrier and CHB categories showed significantly higher circulation of Pre‐C‐wild than mutant HBV. Clinical manifestations were independent of BCP mutations (1762/64‐T/A). Hepatitis B e antigen (HBeAg) negative CHB patients [62.5% (15/24)] were circulating wild HBV. Higher HBV‐DNA levels were associated with chronic hepatitis and HBeAg positivity, whilst Pre‐C mutant positives had lower levels. BCP mutations did not affect HBV‐DNA levels. Multivariate regression analysis identified HBeAg (OR = 4.3) and Pre‐C mutants (OR = 3.1) to be associated with chronic hepatitis and carriers respectively. In a separate sub‐set analysis (n = 59), HBV‐DNA level was identified as the only variable. In conclusion, chronic or fulminant hepatitis B was not associated with Pre‐C or BCP mutants and switching over to Pre‐C mutant was beneficial for the infected individual in maintaining disease free status for extended periods.
Hepatitis C virus infection: prevalence, predictor variables and prevention opportunities among drug users in Italy
Authors: Quaglio G. L. , Lugoboni F. , Pajusco B. , Sarti M. , Talamini G. ,   GICS, Mezzelani P. , Des Jarlais D. C.
Published: September 2003

Summary. The study assessed rates and predictor variables of hepatitis C virus (HCV) infection among drug users receiving pharmacological treatment for opiates addiction.

There was a large cohort study in 16 public centres for drug users in north‐eastern Italy, with data collected by standardized face‐to‐face interviews between February 2001 and August 2001.

Of 1095 participants, 74.2% were HCV seropositive. Anti‐HCV status was independently associated with duration of drug use of over 10 years, injecting as a route of drug administration, and hepatitis B virus (HBV) and human immunodeficiency virus (HIV) seropositivity. Further statistical analysis was conducted by dividing the subjects on the basis of the duration of heroin use: more or <10 years. In the multivariate analyses, route of drug administration and HBV status were associated with HCV seropositivity among both groups. Less education was associated with HCV among the shorter term drug users. HIV status and having a sexual partner with a history of drug use were associated with HCV seropositivity among the longer term drug users.

Half of the short‐term heroin users were still HCV seronegative when starting treatment, suggesting opportunities for reducing new HCV infections. Remarkable was the relationship between vaccination for hepatitis B and HCV serostatus. Being HBV seropositive was strongly associated with being HCV seropositive. But heroin users who had been vaccinated for HBV were not significantly more likely to be HCV seropositive than heroin users who were HBV seronegative. HBV vaccination does not provide biological protection against HCV; however, vaccinating heroin users against HBV may help to create a stronger pro‐health attitude among heroin users, leading to a reduction in HCV risk behaviour.
Apoptosis in hepatitis C
Authors: Kountouras J. , Zavos C. , Chatzopoulos D.
Published: September 2003

Summary. The apoptotic process appears to be a host defence mechanism against viral infections and tumourigenesis. However, many viral genomes encode proteins, which repress apoptosis so as to escape from immune attack by the host. Therefore, virus–host interactions may determine viral persistence, extent and severity of liver inflammation and possibly viral hepatocarcinogenesis. Apoptosis of liver cells may play a significant role in the pathogenesis of hepatitis C. Pathomorphologic features of increased apoptosis include shrinkage and fragmentation of nuclei/cytoplasm in piecemeal necrosis areas, acidophilic bodies, and focal cell dropout in the liver lobule. The hepatitis C virus (HCV) core protein exhibits both proapoptotic or antiapoptotic actions. Modulation of apoptosis may involve binding of HCV core protein to the intracellular signal transducing portion of death receptors and displacement of signalling molecules. Apoptosis may occur in the absence of significant transaminase elevation, thereby explaining the lack of correlation between biochemical activity and liver cell histological injury. Monitoring caspase activation might provide a reliable tool to estimate the efficacy of HCV therapy, and might open challenging therapeutic strategies in HCV infection. The antiviral effect of interferon may be mediated through induction of apoptosis. Lastly, administration of the antiapoptotic ursodeoxycholic acid in HCV infection is compatible with the notion that apoptosis may represent a mechanism for viral shedding rather than for viral elimination, thereby raising the concept that inhibition of apoptosis could ameliorate hepatitis C.
Improved detection of hepatocyte proliferation using antibody to the pre‐replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection
Authors: Freeman A. , Hamid S. , Morris L. , Vowler S. , Rushbrook S. , Wight D. G. D. , Coleman N. , Alexander G. J. M.
Published: September 2003

Summary. To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby predisposing HCV carriers to cirrhosis and hepatocellular carcinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre‐replication complex. Antibody to Ki‐67, a conventional marker of cell division, was also studied. Eighty‐seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from normal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV‐infected patients and the proportion of hepatocytes expressing Mcm‐2 (3–40%) always exceeded that expressing Ki‐67 (1–14%) and correlated positively with increasing stage of fibrosis (P = 0.0001) and viral replication (P = 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm‐2 and inflammatory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm‐2 and age, gender or past alcohol consumption, but there was a weak association with current consumption of alcohol (P = 0.0067). The proportion of Ki‐67 hepatocytes did not correlate with any clinical, laboratory or histological parameter. Mcm‐2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm‐2 is a more sensitive marker of hepatocyte proliferation than Ki‐67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy.
Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis
Authors: Klaus Sebastian M. J. , Huang Fong‐Chin , Eibl Christian , Koop Hans‐Ulrich , Golds Timothy J.
Published: September 2003

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA‐6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis‐deficient plastid mutants, ΔpetA, Δycf3 and ΔrpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild‐type as source tissue for transformation, re‐introducing deleted plastid sequences and using the aphA‐6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild‐type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.
Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress
Authors: Maathuis Frans J. M. , Filatov Victor , Herzyk Pawel , C. Krijger Gerard , B. Axelsen Kristian , Chen Sixue , Green Brian J. , Li Yi , Madagan Kathryn L. , Sánchez‐Fernández Rocío , Forde Brian G. , Palmgren Michael G. , Rea Philip A. , Williams Lorraine E. , Sanders Dale , Amtmann Anna
Published: September 2003

Plant nutrition critically depends on the activity of membrane transporters that translocate minerals from the soil into the plant and are responsible for their intra‐ and intercellular distribution. Most plant membrane transporters are encoded by multigene families whose members often exhibit overlapping expression patterns and a high degree of sequence homology. Furthermore, many inorganic nutrients are transported by more than one transporter family. These considerations, coupled with a large number of so‐far non‐annotated putative transporter genes, hamper our progress in understanding how the activity of specific transporters is integrated into a response to fluctuating conditions. We designed an oligonucleotide microarray representing 1096 Arabidopsis transporter genes and analysed the root transporter transcriptome over a 96‐h period with respect to 80 mm NaCl, K+ starvation and Ca2+ starvation. Our data show that cation stress led to changes in transcript level of many genes across most transporter gene families. Analysis of transcriptionally modulated genes across all functional groups of transporters revealed families such as V‐type ATPases and aquaporins that responded to all treatments, and families – which included putative non‐selective cation channels for the NaCl treatment and metal transporters for Ca2+ starvation conditions – that responded to specific ionic environments. Several gene families including primary pumps, antiporters and aquaporins were analysed in detail with respect to the mRNA levels of different isoforms during ion stress. Cluster analysis allowed identification of distinct expression profiles, and several novel putative regulatory motifs were discovered within sets of co‐expressed genes.
Misregulation of tetrapyrrole biosynthesis in transgenic tobacco seedlings expressing mammalian biliverdin reductase
Authors: Franklin Keara A. , Linley Philip J. , Montgomery Beronda L. , Lagarias J. Clark , Thomas Brian , Jackson Stephen D. , Terry Matthew J.
Published: September 2003

Previous studies have established that the expression of mammalian biliverdin IXα reductase (BVR) in transgenic tobacco (Nicotiana tabacum cv. Maryland Mammoth) resulted in the loss of photoregulatory activity of all phytochromes together with a pronounced chlorophyll deficiency. This study was undertaken to assess the contribution of BVR‐mediated alteration of tetrapyrrole metabolism to the observed phenotypes of BVR transgenic plants. BVR expression in dark‐grown plants led to the reduced accumulation of protochlorophyllide and transcripts for the two committed enzymes for 5‐aminolevulinic acid (ALA) synthesis despite the marked increased capacity for synthesis of ALA. Together with the observation that Mg‐porphyrin accumulation in dark‐grown seedlings treated with an iron chelator was unaffected by BVR expression, these results indicate that BVR diverts tetrapyrrole metabolism toward heme synthesis while also reducing heme levels to de‐repress ALA synthesis. By contrast with dark‐grown seedlings, light‐grown BVR plants showed a marked inhibition of ALA synthesis compared with wild‐type plants – a result that was correlated with the disappearance of the CHL I subunit of Mg‐chelatase and an increase in heme oxygenase protein levels. As transcript levels of all tetrapyrrole biosynthetic genes tested were not strongly affected by BVR expression, these results implicate misregulated tetrapyrrole metabolism to be a major mechanism for BVR‐dependent inhibition of chlorophyll biosynthesis in light‐grown plants.
The biosynthesis of the branched‐chain sugar d‐apiose in plants: functional cloning and characterization of a UDP‐d‐apiose/UDP‐d‐xylose synthase from Arabidopsis
Authors: Mølhøj Michael , Verma Rajeev , Reiter Wolf‐Dieter
Published: September 2003

d‐Apiose is a plant‐specific branched‐chain monosaccharide found in rhamnogalacturonan II (RG‐II), apiogalacturonan, and several apioglycosides. Within RG‐II, d‐apiose serves as the binding site for borate, which leads to the formation of cross‐links within the wall. Biochemical studies in duckweed and parsley have established that uridine 5′‐diphospho‐d‐apiose (UDP‐d‐apiose) is formed from UDP‐d‐glucuronate by decarboxylation and re‐arrangement of the carbon skeleton, leading to ring contraction and branch formation. The enzyme catalyzing this reaction also forms UDP‐d‐xylose by decarboxylation of UDP‐d‐glucuronate, and has therefore been named UDP‐d‐apiose/UDP‐d‐xylose synthase. Using a bioinformatics approach, we identified a candidate gene (AXS1) for this enzyme in Arabidopsis and functionally expressed its cDNA in Escherichia coli. The recombinant enzyme catalyzed the conversion of UDP‐d‐glucuronate to a mixture of UDP‐d‐apiose and UDP‐d‐xylose with a turnover number of 0.3 min‐1. AXS1 required NAD+ for enzymatic activity, and was strongly inhibited by UDP‐d‐galacturonate. It was highly expressed in all plant organs consistent with a function in synthesizing an essential cell wall precursor. Database searches indicated the presence of closely related sequences in a variety of crop plants. The cloning of the AXS1 gene will help to investigate the biosynthesis of RG‐II, and permit insights into the mechanism by which d‐apiose and other branched monosaccharides are formed.
Deletion of the tobacco plastid psbA gene triggers an upregulation of the thylakoid‐associated NAD(P)H dehydrogenase complex and the plastid terminal oxidase (PTOX)
Authors: Baena‐González Elena , Allahverdiyeva Yagut , Svab Zora , Maliga Pal , Josse Eve‐Marie , Kuntz Marcel , Mäenpää Pirkko , Aro Eva‐Mari
Published: September 2003

We have constructed a tobacco psbA
gene deletion mutant that is devoid of photosystem II (PSII) complex. Analysis of thylakoid membranes revealed comparable amounts, on a chlorophyll basis, of photosystem I (PSI), the cytochrome b6f complex and the PSII light‐harvesting complex (LHCII) antenna proteins in wild‐type (WT) and ΔpsbA leaves. Lack of PSII in the mutant, however, resulted in over 10‐fold higher relative amounts of the thylakoid‐associated plastid terminal oxidase (PTOX) and the NAD(P)H dehydrogenase (NDH) complex. Increased amounts of Ndh polypeptides were accompanied with a more than fourfold enhancement of NDH activity in the mutant thylakoids, as revealed by in‐gel NADH dehydrogenase measurements. NADH also had a specific stimulating effect on P700+ re‐reduction in the ΔpsbA thylakoids. Altogether, our results suggest that enhancement of electron flow via the NDH complex and possibly other alternative electron transport routes partly compensates for the loss of PSII function in the ΔpsbA mutant. As mRNA levels were comparable in WT and ΔpsbA plants, upregulation of the alternative electron transport pathways (NDH complex and PTOX) occurs apparently by translational or post‐translational mechanisms.
Architecture and transcriptional activity of the initiator element of the TATA‐less RPL21 gene
Authors: Achard Patrick , Lagrange Thierry , El‐Zanaty Abdel‐Fattah , Mache Régis
Published: September 2003

The nuclear RPL21 gene coding for the plastid ribosomal protein L21 is a TATA‐less gene that is overexpressed in a leaf‐dependent manner by the specific usage of a strong initiator called P1. We have previously shown that the RPL21 core promoter spanning from −23 to +104 relative to P1 start site activates transcription in the same manner as does the full promoter. Here, we present results of experiments aimed at deciphering the RPL21 core promoter architecture. Results of transient expression using various 5′ deletions of the core promoter fused to a chloramphenicol acetyl transferase (CAT) reporter gene show that 34 bp encompassing the P1 initiation site (from −23 to +11) are required for full transcription activation. Gel‐shift analysis shows that five DNA/protein complexes (C1–C5) are formed on this 34‐bp fragment with protein extracts from green tissues. C1 is the major complex present during seed germination. The other complexes are present in young leaf tissues suggesting a role in transcription activation. Linker scanning mutagenesis experiments show that the five complexes form two independent groups: I (C1–C3) and II (C4 and C5), with a common binding site located on P1. Using transgenic plants, we show that three nucleotides encompassing the P1 start site and three trinucleotides necessary for group I binding are determinant for RPL21 activation. These results identify an unusually compact core structure, which is centred on P1 initiation site and is responsible for transcription activation. A model of the architecture of this region is presented.
The HECT ubiquitin‐protein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development
Authors: Downes Brian P. , Stupar Robert M. , Gingerich Derek J. , Vierstra Richard D.
Published: September 2003

Attachment of one or more ubiquitins (Ubs) to various intracellular proteins has a number of roles in plants including the selective removal of regulatory proteins by the 26S proteasome. The final step in this modification is performed by ubiquitin‐protein ligases (E3s) that promote Ub transfer to appropriate targets. One important family of E3s is defined by the presence of a HECT domain, an active site first found at the C‐terminus of the human E3 (E6‐AP). Using a consensus HECT domain as the query, we identified a family of seven HECT‐containing ubiquitin‐protein ligases (UPL1–UPL7) in Arabidopsis thaliana that can be grouped into four subfamilies. The UPL3 and UPL4 subfamily encodes approximately 200‐kDa proteins with four Armadillo repeats similar to those in the nuclear pore protein importin‐α, suggesting that these E3s identify their targets through binding to nuclear localization sequences. Although T‐DNA disruptions of the UPL3 locus do not affect overall growth and development of Arabidopsis, the mutants show aberrant trichome morphology. Instead of developing three branches, many upl3 trichomes contain five or more branches. The upl3 trichomes also often undergo an additional round of endoreplication resulting in enlarged nuclei with ploidy levels of up to 64C. upl3 plants are hypersensitive to gibberellic acid‐3 (GA3), consistent with the role of gibberellins in trichome development. The phenotype of upl3 mutants is similar to that of kaktus, a previously described set of trichome mutants with supernumerary branches. Genetic analyses confirmed that upl3 mutants and kaktus‐2 are allelic with kaktus‐2 plants harboring a splice‐site mutation within the UPL3‐transcribed region. Collectively, the data indicate that the ubiquitination of one or more activator proteins by UPL3 is necessary to repress excess branching and endoreplication of Arabidopsis trichomes.