bluuebunny/crossref_metadata_2025 huggingface dataset:
Inheritance of resistance to stem canker (Phomopsis helianthi) in sunflower
Authors: Deglène  , Alibert  , Lesigne  , Tourvieille de Labrouhe  , Sarrafi
Published: March 2003

Six male sterile sunflower lines were crossed with seven restorers in a factorial mating design. The 13 inbred lines and their 42 F1 hybrids were planted in a randomized block design with three replicates. Each replicate consisted of two rows, 5 m long (30–35 plants per replicate). Resistance to natural Phomopsis infection, presented as the percentage of plants with no encircling necrosis lesions of the fungus on the main stem, was determined at physiological maturity. Analysis of variance showed that female and male general combining abilities (GCA) and specific combining abilities (SCA) of F1 hybrids were significant. The ratio of additive variance to total variance was 0.662, a high value which indicates prevailing additive effects. The additive variance due to females was more important than that of males, probably because of the existence of maternal effects or more effective genes for resistance in the female lines used in this experiment. The estimates of GCA were significant and positive for LC1004A, KO549A, 50KD8 and LC1064C inbred lines. These lines should be considered in developing hybrids with improved resistance to Phomopsis in sunflower breeding programmes.
Visual disease and PCR assessment of stem base diseases in winter wheat
Authors: Turner  , O'hara  , Rezanoor  , Nuttall  , Smith  , Nicholson
Published: March 2003

Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four‐week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum, F. culmorum, both varieties of Microdochium nivale, both eyespot‐causing species of Tapesia and Rhizoctonia cerealis. PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R‐type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var. majus, accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.
Development of symptoms caused by Erwinia carotovora ssp. atroseptica under field conditions and their effects on the yield of individual potato plants
Authors: Hélias  , Andrivon  , Jouan
Published: March 2003

Infection of seed tubers by pectinolytic Erwinia species can lead to the development of various symptoms during vegetative growth of potato crops, including non‐emergence of plants, chlorosis, wilting, haulm desiccation and typical blackleg. The relationships between types of symptoms and yield are poorly documented, and are investigated by following the development of symptoms in potato plants grown under field conditions from seed tubers artificially inoculated with E. carotovora ssp. atroseptica (Eca), and measuring the yield of each plant. Symptoms were classified into five main types (non‐emergence, wilting/chlorosis, blackleg, haulm desiccation and plant death). Each plant was scored for types of symptom on four successive dates; plants without visible symptoms were scored as healthy. The method of inoculation and inoculum concentration proved major factors for the subsequent development of symptoms. Disease development was more severe after vacuum infiltration of bacteria into seed tubers than after shaking tubers in contaminated sand. Disease usually progressed from chlorosis and/or wilting to partial or total desiccation on a given plant. Yield losses varied according to symptom type, but the relationship between symptoms recorded and yield also depended on scoring dates. The data suggest that the beginning of tuber growth might be the most suitable stage for predicting yield losses from symptom observations. In both cultivars studied (Bintje, highly susceptible, and Désirée, moderately resistant), the yield of symptomless plants growing from inoculated seed tubers was significantly less than that of control plants, indicating that the presence of bacteria on the seed tuber was detrimental, even in the absence of visible symptoms. Differences in symptom expression in the field between cultivars matched the level of visible infection of tubers at harvest, as Bintje tubers showed a higher incidence of rot than Désirée tubers.
Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach
Authors: Mutasa‐Gottgens E.S. , Chwarszczynska D.M. , Halsey K. , Asher M.J.C.
Published: March 2003

Highly specific rabbit polyclonal antibodies for the obligate sugar‐beet root parasite, Polymyxa betae, were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro, and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta, and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar‐beet roots and with the closely related Polymyxa graminis in barley roots, and to cross‐react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross‐reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea. In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar‐beet, barley, cabbage and potato roots.
Predicting effective fungicide doses through observation of leaf emergence
Authors: Paveley N. D. , Lockley D. , Vaughan T. B. , Thomas J. , Schmidt K.
Published: March 2003

Experimental data were used to test the hypothesis that the effective fungicide dose (ED) – the dose required to achieve a given level of disease suppression – varies in a predictable manner according to the pattern of development of the wheat canopy. Replicated and randomized field plots received a single systemic fungicide spray at either zero (control), 0·25, 0·5, 0·75 or 1·0 dose (the recommended dose), at one of eight timings from April to June. Wheat cultivars and locations for experiments were selected to promote epidemics of septoria tritici spot and yellow rust caused bySeptoria tritici(anamorph ofMycosphaerella graminicola) andPuccinia striiformis,respectively. Logistic or exponential disease progress curves were fitted to disease severity data and used to estimate the date of disease onset (t0) and relative epidemic growth rate (r) on each leaf layer for each treatment. Area under the disease progress curve (AUDPC) values were used to construct fungicide dose by spray timing response surfaces for each of the upper four leaves. A parsimonious function, with an exponential form in the dose–response dimension and a normal distribution in the timing dimension described a high proportion of the variation in AUDPC (R2values ranging from 0·73 to 0·97). Consistent patterns of treatment effect were noted across pathogen species, leaf layers, sites and seasons. Fungicide applications that coincided with full leaf emergence delayedt0on that leaf layer. Treatments applied after full leaf emergence did not delayt0, but reducedr. Progressively earlier or later treatments, or lower doses, had decreasing effects. AUDPC was affected more byt0thanr. AUDPC response surface parameter estimates showed that curvature of the dose–response was not affected by spray timing, but appeared to be a characteristic of the fungicide–pathogen combination. However, the lower asymptote of the dose–response curve, and hence the ED, varied substantially with spray timing. The pattern of change in ED with spray timing was consistent across a range of leaf layers, pathosystems and seasons, and the spray timing at which the ED was minimized varied only within a small range, around the time of leaf emergence. In contrast, variation in untreated disease severity, resulting from variation in initial inoculum and weather, was large. It was concluded that the main value of disease forecasting schemes may be in their capacity to predict the level of untreated disease, to which the economic optimum, or ‘appropriate’, dose relates. Spray timing determines the part of the canopy where disease will be efficiently controlled and hence the green leaf area saved. Timing decisions should relate to observations of emergence of those leaf layers important to yield.
Fungal communities on roots of wheat and barley and effects of seed treatments containing fluquinconazole applied to control take‐all
Authors: Dawson W. A. J. M. , Bateman G. L.
Published: March 2003

Communities of fungi on roots increased in diversity between second and third cereals, and were more diverse on winter wheat than on winter barley in a field experiment conducted over 3 years. Seed treatment with formulations containing fluquinconazole or fluquinconazole plus prochloraz had little effect on these communities, which include potentially beneficial, antagonistic fungi. Seed treatment decreased take‐all and increased yield of wheat, especially in the second wheat in which take‐all was building up, and in the third wheat when take‐all was at its peak. It was less effective in the fourth wheat when take‐all appeared to be in decline in plots that had no seed treatment throughout the experiment, and in barley, in which take‐all was usually less severe. Take‐all increased in untreated fourth wheat crops grown after third wheat crops that had been treated, suggesting that treatment had delayed take‐all build‐up and eventual progress into take‐all decline.
Population structure and possible origin of Amylostereum areolatum in South Africa
Authors: Slippers B. , Wingfield M. J. , Coutinho T. A. , Wingfield B. D.
Published: March 2003

The woodwasp, Sirex noctilio, and its symbiotic fungus, Amylostereum areolatum, cause extensive damage to pine plantations in the Southern Hemisphere. S. noctilio was first reported from South Africa in 1994. In this study, the population diversity of A. areolatum isolates from South Africa, South America, Australasia and Europe was determined by vegetative incompatibility testing. All 108 South African and 26 South American isolates belonged to the same vegetative compatibility group (VCG). This VCG showed a weak incompatibility reaction with the single Tasmanian and single New Zealand isolates tested. This VCG differed from VCGs from Europe. It also differed from isolates associated with the biocontrol nematode, Deladenus siricidicola, which is produced in Australia. It is concluded that the South African and South American populations of A. areolatum share a common origin.
Rapid‐cycle PCR detection of Pyrenophora graminea from barley seed
Authors: Taylor E. J. A. , Stevens E. A. , Bates J. A. , Morreale G. , Lee D. , Kenyon D. M. , Thomas J. E.
Published: March 2003

Sixty RAPD primers were used to screen for a diagnostic marker that could be used to identify Pyrenophora graminea, a fungal seedborne pathogen that causes leaf stripe on barley. Primer pairs were designed to differentiate P. graminea from other Pyrenophora spp. using a sequence‐characterized amplified region (SCAR) approach. A pair of P. graminea‐specific primers (PG2 F/R) was obtained that amplified a single fragment from 37 isolates of P. graminea tested, but not from 29 isolates of other Pyrenophora spp. or 12 saprophytes isolated from barley seed. Rapid PCR detection was achieved using a LightCycler, in which the emission of fluorescence from the binding of SYBR Green I dye to the PCR products is measured. The P. graminea‐specific product resulting from amplification with PG2 F/R can be distinguished from any nonspecific products by post‐PCR melting point analysis. The PCR assay involves 40 amplification cycles of PCR, and the total PCR test including melting point analysis takes 25 min to complete. The rapidity of this test, combined with the closed ‘in‐tube’ detection of PCR products, which reduces the potential for contamination, offers significant advantages compared with conventional laboratory and PCR analyses.
Evaluation of diagnostic and quantitative PCR for the identification and severity assessment of eyespot and sharp eyespot in winter wheat
Authors: Turner A. S. , Nicholson P. , Edwards S. G. , Bateman G. L. , Morgan L. W. , Todd A. D. , Parry D. W. , Marshall J. , Nuttall M.
Published: March 2003

Diagnostic and quantitative polymerase chain reaction (PCR) provided clarification of the causes of symptoms and the extent of infection by eyespot (Tapesia spp.) and sharp eyespot (Rhizoctonia cerealis) on winter wheat at early growth stages. Disease assessments made before stem extension, when decisions to apply fungicides are usually made, often did not agree with the pathogen diagnoses using PCR, suggesting that such early visual diagnoses may be unreliable. Visual and PCR diagnoses made on stems in summer generally supported each other, but there were often discrepancies in relating disease severity to amounts of pathogen present when determined by regression analyses of incidence or severity of symptoms on amount of pathogen DNA. Mixed symptoms caused by different pathogens may sometimes have been confounded. Relationships between symptoms and DNA of eyespot pathogens were less clear on some cultivars, often those with least disease. Sharp eyespot symptoms had a stronger relationship to DNA of its pathogen. Significant regressions often accounted for a small percentage of the variance, suggesting either that pathogens not assayed were contributing to symptoms or that lesions were in some cases persisting longer into the season than pathogen DNA. The frequency of pathogen detection before stem extension was a poor predictor of the amounts of pathogen DNA measured later in the season.
Spatio‐temporal dynamics of brown rot (Monilinia fructigena) on apple and pear
Authors: Xu X.‐M. , Robinson J. D. , Berrie A. M. , Harris D. C.
Published: March 2003

Spatio‐temporal development of brown rot (Monilinia fructigena) on apple and pear was monitored in an apple (cv. Cox) orchard and a pear orchard of several cultivars over several years. Disease on individual trees was recorded weekly from July to harvest, individual fruits with brown rot were tagged but not removed and rot‐origin identified. On apple cv. Cox and pear (cvs Conference and Comice), all primary rot arose from infection via wounds caused by insects, birds and growth cracks. Birds were the most important wounding agents on pear in the field. Secondary (fruit‐to‐fruit contact) rot was considerably less than primary rot, especially for pear. Incidence of disease (percentage of fruits with brown rot) increased gradually from late July up to harvest; the final disease incidence varied with seasons and cultivars, ranging from 1 to 11%. For pear, Comice had greater incidence than Conference. Significant aggregation of diseased fruits among trees was detected for assessment dates when the overall incidence of disease was greater than 0·5%. On Cox and Conference, significant correlation of disease incidence between adjacent trees or trees separated by one or more trees (i.e. spatial lag measured as units of distance between adjacent trees) was detected, but there was no clear relationship between the correlation, the distance or time. For Comice, there was consistent and significant positive correlation of brown rot incidence over 3 years. It is speculated that behavioural characteristics of wounding agents may have played an important role in influencing the spatio‐temporal dynamics of brown rot on apple and pear.
Synergism between plant viruses: a mathematical analysis of the epidemiological implications
Authors: Zhang X.‐S. , Holt J. , Colvin J.
Published: March 2003

Many virus diseases of plants are caused by a synergistic interaction between viruses within the host plant. Such synergism can induce symptoms more severe than would be caused by additive effects. In a synergistic interaction, the virus titre of both, one, or neither virus may be enhanced and, as a consequence, the rate of disease spread may be affected. An epidemiological model was developed in which transmission and loss rates were attributed to the different virus infection possibilities. Sharing the same host population implies competition, and this imposes an increased constraint on the survival of both viruses. It was shown that, in order to ensure virus survival in a mixed infection, the basic reproductive number should exceed a critical value which is larger than unity (R0 > Rc > 1). Here R0 is used in the same sense as in the absence of superinfection. Increased virulence (equivalent to disease severity) in dually infected plants decreases the opportunities for both viruses to coexist, while increased virus transmission from dually infected plants increases such opportunities. The net effect of increased virulence and increased virus transmission on virus persistence was neutral if synergism caused the same proportional effect on both. Total host abundance was, however, reduced. The opportunity for virus persistence was increased if the enhancement of transmission exceeded that of virulence. Indeed, by this mechanism a virus which was nonviable alone could invade and persist in a chronic epidemic of another virus. Where the effect on virulence is greater than that on transmission, the viruses are likely to exclude each other, especially when the transmission rates of both viruses have intermediate values. In such cases, the final outcome is determined by both the parameter values and the initial state.
Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture
Authors: Parmessur Y. , Aljanabi S. , Saumtally S. , Dookun‐Saumtally A.
Published: March 2003

Yellow leaf syndrome (YLS) is a recently reported disease of sugarcane, characterized by yellowing of the leaves. Two pathogens: a virus, Sugarcane yellow leaf virus (SCYLV); and a phytoplasma, sugarcane yellows phytoplasma (SCYP), are associated with the disease. The use of tissue culture was investigated as a means to eliminate both SCYLV and SCYP from exotic varieties undergoing quarantine in Mauritius. Of 43 varieties in quarantine, 28 were infected with SCYLV and 19 with SCYP when checked by RT–PCR and nested PCR, respectively. Seventeen varieties were coinfected with both pathogens. Thirty infected varieties were induced to form callus in vitro using leaf rolls as explants. After two subcultures, 19 varieties were successfully regenerated and tested for SCYLV and SCYP. No pathogen could be detected in any regenerated plantlets. All the regenerated plants remained free from both SCYLV and SCYP over a period of 1 year in the glasshouse, confirming that the pathogens had been eliminated by tissue culture.
Phenotypic Convergence and Divergence of Surface Immunoglobulin and CD40 Signals
Authors: Axcrona  , Åkerblad  , Leanderson
Published: March 2003

Both anti‐CD40 antibodies and anti‐immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)‐induced B‐cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque‐forming assay and Ig RNA steady state levels. Anti‐CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti‐Ig, anti‐CD40 or a combination of the two mitogens could be restimulated by anti‐CD40 but not by anti‐Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti‐Ig‐ or anti‐CD40‐prestimulated cells with anti‐Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti‐CD40.
Constant Region of a κ III Immunoglobulin Light Chain as a Major AL‐Amyloid Protein
Authors: Engvig  , Olsen  , Gislefoss  , Sletten  , Wahlström  , Westermark
Published: March 2003

AL‐amyloidoses are generally described as a group of disorders in which N‐terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence‐Jones protein and the corresponding AL protein as a κ III immunoglobulin light chain from material of a patient with systemic AL‐amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C‐terminal fragments of the Bence‐Jones protein. Furthermore, both the Bence‐Jones protein and the AL protein were glycosylated, with possibly a glycosylation in the constant part of the light chain.
Increased Antibody Production against Gut‐Colonizing Escherichia coli in the Presence of the Anaerobic Bacterium Peptostreptococcus
Authors: Herías  , Midtvedt  , Hanson  , Wold
Published: March 2003

Germ‐free rats were colonized with E. coli alone, or with E. coli plus Lactobacillus acidophilus and a strain of the obligate anaerobic Gram‐positive species, Peptostreptococcus. The presence of Peptostreptococcus reduced translocation of E. coli, but increased the serum antibody response to E. coli antigen. Whereas the immunoglobulin G (IgG) anti‐E. coli antibodies largely represented cross‐reactive antibodies, those of the immunoglobulin M (IgM) isotype represented true anti‐E. coli antibodies because they could not be absorbed by L. acidophilus or Peptostreptococcus but could with E. coli. We suggest that peptostreptococci prime the gut immune system to other bacterial antigens and that this could be a mechanism behind the reduced translocation of facultative anaerobes in the presence of obligate anaerobes.
Distribution of TNF‐α, TNF‐R55 and TNF‐R75 in the Rheumatoid Synovial Membrane: TNF Receptors are Localized Preferentially in the Lining Layer; TNF‐α is Distributed Mainly in the Vicinity of TNF Receptors in the Deeper Layers
Authors: ALSALAMEH  , WINTER  , AL‐WARD  , WENDLER  , KALDEN  , KINNE
Published: March 2003

The expression of TNF‐α and its receptors in the rheumatoid synovial membrane was investigated using immunohistochemistry and immunocytofluorescence. TNF‐α+ cells (< 10% of all cells) were found in all regions, predominantly in sublining and diffuse infiltrates. The highest percentage of TNF‐R+ cells was found in the lining layer (50–90%), with a slight predominance of TNF‐R55. In the sublining, fewer cells expressed TNF‐R (≈ 50%), mostly TNF‐R75. TNF‐R75+ cells were also detectable in diffuse infiltrates and lymphoid aggregates (10–50%). These contained only individual TNF‐R55+ cells. In diffuse infiltrates, there were slightly more TNF‐R55+ cells than in lymphoid aggregates (in both cases < 10%). In sequential sections, TNF‐α+ cells localized mostly in the vicinity of TNF‐R+ cells. Macrophage‐like cells appeared to be the predominant TNF‐R+ cell type. CD3+ T cells in lymphoid aggregates expressed exclusively TNF‐R75. Subsequently, the expression of membrane‐bound TNF‐α, TNF‐R55 and TNF‐R75 was tested by FACS analysis in isolated RA synoviocytes (n = 7 patients). Only four specimens expressed mTNF‐α, and that on a low percentage of cells (2 ± 2.4%; mean ± SD). In contrast, all specimens expressed higher percentages of TNF‐R55 and TNF‐R75 (21 ± 1% and 14 ± 7.1%, respectively). These results demonstrate that: (1) the percentage of cells expressing soluble/transmembrane TNF‐α is greatly outnumbered by the percentage of cells expressing TNF receptors; and (2) TNF‐α‐expressing cells are localized in regions expressing substantial levels of TNF receptors. Therefore, the known pro‐inflammatory and pro‐arthritic effects of TNF‐α are probably mediated by local interactions between the receptors and their soluble and transmembrane ligands.
Cytokine mRNA Expression in Lymphoid Organs Associated with the Expression of IgA Response in the Rat
Authors: PETITPREZ  , KHALIFE  , CETRE  , FONTAINE  , LAFITTE  , CAPRON  , GRZYCH
Published: March 2003

The T‐helper dependency of the IgA antibody response has been investigated in rats injected intravenously with Schistosoma mansoni eggs. This method, allowing the trapping of parasite eggs in the lung tissue, led to a strong anti‐egg IgA antibody response in the bronchoalveolar lavage but not in the serum. To characterize the cytokine pattern associated with the IgA response, kinetic analysis of the cytokine mRNA expression in the lungs, periaortic nodes (PN) and spleen was undertaken. Under such conditions, significant levels of mRNA encoding IL‐5 and IL‐10 were recorded in spleen during the early period following egg injection, as well as a more prolonged expression of TGF‐β and IL‐6 mRNAs. However, neither IFN‐γ nor IL‐4 mRNA could be detected in these samples. Finally, in lungs and in PN, RT–PCR analysis revealed delayed production of cytokine mRNA. Taken together our data suggest that the rat mucosal IgA antibody response is predominantly linked to a Th2 response.
Interleukin‐4‐Mediated Inhibition of Nitric Oxide Production in Interferon‐γ‐Treated and Virus‐Infected Macrophages
Authors: PALUDAN  , ELLERMANN‐ERIKSEN  , LOVMAND  , MOGENSEN
Published: March 2003

Upon interferon‐γ (IFN‐γ) stimulation, murine macrophages (MΦ) produce nitric oxide (NO) through expression of inducible nitric oxide synthase (iNOS). Interleukin (IL)‐4 treatment, even delayed 12 h relative to IFN‐γ, antagonized this induction, whereas infection with herpes simplex virus type 2 (HSV‐2) or treatment with tumour necrosis factor‐α exerted a synergistic effect, which partly compensated for the antagonistic effect of IL‐4. Neither IL‐4 nor HSV‐2 affected the IFN‐γ‐activated Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway or altered the levels of IFN‐γ‐induced interferon regulatory factor (IRF)‐1 expression, which is STAT1‐dependent and known to play a central role in IFN‐γ‐mediated gene induction. The effect of IL‐4 was completely dependent on de novo protein synthesis, indicating that a direct activation of latent inhibitors is not sufficient to explain the inhibitory effect of IL‐4. Furthermore, IL‐4 substantially augmented the IFN‐γ‐induced expression of IRF‐2, which is known to compete with IRF‐1 for the DNA recognition site, ISRE (interferon‐stimulated response element). Our findings could indicate that IL‐4 suppresses IFN‐γ‐stimulated iNOS transcription by elevating the level of IRF‐2 which, through competition, prevents IRF‐1 from binding to ISRE in the iNOS promoter. The virus‐induced effects on iNOS and NO levels in IFN‐γ‐stimulated MΦ do not seem to involve the Jak/STAT pathway or a differential expression of IRF‐1 and IRF‐2.
Epitope Mapping of Aleutian Mink Disease Parvovirus Virion Protein VP1 and 2
Authors: COSTELLO  , STEENFOS  , JENSEN  , CHRISTENSEN  , GOTTSCHALCK  , HOLM  , AASTED
Published: March 2003

Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL‐c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV‐infected mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid‐phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive mink sera, indicating the presence of one dominant linear epitope.
Administration of Interleukin‐12 Prevents Mite Der p 1 Allergen‐IgE Antibody Production and Airway Eosinophil Infiltration in an Animal Model of Airway Inflammation
Authors: LEE  , FU  , YE  , CHIANG
Published: March 2003

The aim of the present study was to examine the in vivo effect of interleukin (IL)‐12 on a murine model of asthma induced by Dermatophagoides pteronyssinus‐derived Der p 1 allergen. C57BL/6 mice immunized with Der p 1 allergen adsorbed to alum/pertussis toxin developed a T‐helper type 2 (Th2)‐dominant immune response characterized by the presence of IgE antibody, airway eosinophil infiltration and increased production of Th2 cytokine. Intraperitoneal injection of IL‐12 (1 or 0.1 μg per day) for 5 days (day − 1 to + 3) simultaneously with each immunization, inhibited the production of IgE and IgG1 antigen‐specific antibodies, whereas production of IgG2a was strongly enhanced. In addition, mice receiving both doses of IL‐12 showed a strong inhibition of IL‐5 but up‐regulation of IFN‐γ production by spleen cells stimulated with antigen. Administration of IL‐12 also prevented antigen‐induced eosinophil infiltration into the bronchoalveolar area in a dose‐dependent manner and the primary inflammatory mediator serotonin in bronchoalveolar lavage (BAL) fluids was also reduced significantly. Taken together, the data indicate that IL‐12 has a potent immunomodulatory effect on house‐dust‐mite‐induced allergic disorders and may be used as an efficient agent for immunotherapy.
Human Mast Cells Produce and Differentially Express Both Soluble and Membrane‐Bound Stem Cell Factor
Authors: Welker  , Grabbe  , Gibbs  , Zuberbier  , Henz
Published: March 2003

Stem cell factor (SCF), characterized as mast cell growth factor, is known to be produced by fibroblasts, keratinocytes and endothelial cells. Two different splice variants encode for either a soluble (SCF‐1) or a membrane‐bound (SCF‐2) form. In order to explore whether mast cells themselves can produce SCF, we examined cultured cord blood (CBMC) and peripheral‐blood‐derived mast cells (PBMC), mast/basophil cell lines (HMC‐1 and KU‐812), and skin mast cells for SCF expression. On immunocytochemistry, cytoplasmatic SCF‐reactivity was observed in HMC‐1 cells, with additional cell membrane staining in KU‐812, skin and cultured mast cells. Low amounts of SCF could be detected by ELISA in lysates of isolated and unstimulated mast cells and in supernatants of skin cells stimulated with anti‐IgE or Ca‐ionophore A23187. SCF mRNA was detected in all cells, although marked quantitative differences were observed among the various cell types. SCF‐2 mRNA expression was low in HMC‐1 cells while it was marked in skin mast cells, KU‐812 cells, CBMC and PBMC. A time‐dependent, increasing induction of both SCF forms was seen in CBMC and PBMC during culture. After stimulation with A23187 and phorbol myristate acetate, an up‐regulation of SCF mRNA was noted in HMC‐1 and KU‐812 cells, without changes in the relationship of the two splice variants. The differential expression of SCF‐specific mRNA splice variants in immature and mature human mast cells and the secretion of this molecule by these cells may play a role in autocrine stimulation, maintenance of survival and the differentiation of tissue mast cells.
The Intravenous Model of Murine Tuberculosis is Less Pathogenic Than the Aerogenic Model Owing to a More Rapid Induction of Systemic Immunity
Authors: CARDONA  , COOPER  , LUQUÍN  , ARIZA  , FILIPO  , ORME  , AUSINA
Published: March 2003

The detection of mRNA in the murine model of tuberculosis for key cytokines involved in protective immunity in the lung tissues revealed a much faster emergence of the interferon (IFN)‐γ response in the intravenous route than in the aerosol route of inoculation. This slower response in the lungs was associated with a stronger inflammatory response, resulting in large granulomatous structures and eventual tissue damage.
Immunological and Ultrastructural Disruptions of T Lymphocytes Following Exposure to the Glycopeptidolipid Isolated from the Mycobacterium avium Complex
Authors: POURSHAFIE  , SONNENFELD  , BARROW
Published: March 2003

In this study, we examined the effects of the serovar‐specific glycopeptidolipid (GPL) on the ultrastructure of purified T lymphocytes and the interleukin secretion by spleen and purified T lymphocytes. Electron microscopy indicated extensive disruption of the cytoplasmic compartment of T lymphocytes, which could result in altered function of immune cells. Despite the cellular damage as viewed by the electron microscopy, the expression of T‐cell surface markers, Thy 1.2 and Lyt‐2, were not affected. The data indicate that GPL is capable of inducing in‐vitro interleukin (IL)‐6 and IL‐2 production by whole spleen or purified spleen T lymphocytes. The level of production of IL‐6 and IL‐2 following the exposure of the mycobacteria‐infected cells to GPL was approximately the same as the uninfected control. A similar finding was also obtained with the total lipid extraction from the mycobacterium. The results suggest that the ability of the total lipid extraction, in inducing cytokine production, may be attributed to its GPL content.
Low Responsiveness to Immunization with Immunoglobulin E/Antigen and Immunoglobulin G/Antigen Complexes in H‐2Ab Mice
Authors: Gustavsson  , Chomez  , Heyman
Published: March 2003

Immunoglobulin (Ig)E and IgG antibodies specific for 2,4,6‐trinitrophenyl (TNP) are able to enhance the carrier‐specific antibody response to TNP‐conjugated soluble proteins such as bovine serum albumin (BSA). We have recently reported that mice carrying the MHC class II Ab molecule are low responders to immunization with IgE/antigen complexes and now show that H‐2Ab mice are also low responders to IgG/antigen complexes. In addition, we found that spleen cells from naive low‐ and high‐responder mice captured IgE/antigen complexes exclusively on B cells, and that the binding was completely inhibited by monoclonal antibodies (MoAbs) against the low‐affinity receptor for IgE (FcεRII or CD23). The IgG/antigen complexes were targeted both to B cells and macrophages. The binding of IgG/antigen to B cells primarily seemed to be dependent on the low‐affinity receptor for IgG (FcγRII or CD32), although some influence of complement receptor 2 (CR2 or CD21) was seen. Capture of IgG/antigen complexes on macrophages was partially blocked by MoAbs against FcγRII/III. There was no difference in expression of FcεRII, FcγRII/III, CR1, CR2, and CR3 between low‐ and high‐responder strains, thus excluding low levels of these FcRs and CRs as a reason for low responsiveness in H‐2Ab mice.
Disruption of Nocturnal Melatonin Rhythm and Immunological Involvement in Ischaemic Stroke Patients
Authors: Fiorina  , Lattuada  , Silvestrini  , Ponari  , Dall'Aglio
Published: March 2003

The aim of this study was to evaluate nocturnal and diurnal urinary melatonin excretion in a group of ischaemic stroke patients, and to verify if a possible impairment of this excretion could be linked with some changes of immunological and neuro‐psychic status. We assessed 13 ischaemic stroke patients and five healthy controls for nocturnal urinary melatonin excretion, which was found to be impaired in stroke patients (patients, 0.05 ± 0.01 ng/ml; controls, 30 ± 3.0 ng/ml; P < 0.001). No differences were found between the groups with regard to diurnal excretion. Patients with impaired nocturnal melatonin excretion presented: a decreased cell‐mediated immunity (assessed by a skin‐test); a prevalence of anergic status (69% of patients were anergic compared with 0% of controls; P < 0.05); a slight (but not significant) enhancement of plasma cortisol levels; and some changes in lymphocyte subsets (an overall decrease in CD3 lymphocyte number). In almost all the ischaemic stroke patients there was an impairment of cortisol/melatonin ratio (a marker of depression) and an altered sleep rhythm with mood disorders. Our data suggest that ischaemic stroke patients had an impairment of nocturnal urinary melatonin excretion, which was associated with an impaired cell‐mediated immunity and some changes of lymphocyte subsets. In addition, reduced melatonin excretion might be associated with neurological and psychical symptoms.
Levels of Interleukin‐15‐Expressing Blood Mononuclear Cells Are Elevated in Multiple Sclerosis
Authors: Pashenkov  , Mustafa  , Kivisäkk  , Link
Published: March 2003

Interleukin‐15 (IL‐15) is a novel IL‐2‐like cytokine expressed by cells of the monocyte/macrophage and epithelial lineages. Cytokines might be involved in the pathogenesis of multiple sclerosis (MS). Using immunocytochemistry, we analysed spontaneous expression of IL‐15 by peripheral blood (PB) and cerebrospinal fluid (CSF) mononuclear cells (MNC) from patients with MS, other neurological diseases (OND) and healthy controls. IL‐15‐ positive peripheral blood mononuclear cells (PBMNC) were elevated in patients with MS compared to healthy controls (P < 0.05). The elevation of IL‐15‐ positive PBMNC was restricted to patients with chronic progressive MS and not observed in patients studied during the relapsing‐remitting phase of MS. The numbers of IL‐15‐ expressing PBMNC correlated with the duration and disability of MS (r = 0.45, P < 0.001, and r = 0.39, P < 0.01, respectively). IL‐15 was undetectable in CSF MNC, and ELISA showed low CSF levels of IL‐15 in occasional patients with MS and OND. IL‐15 is a potent growth factor for γδ T cells, but there was no correlation between IL‐15 expression by PBMNC and percentage of γδ T cells in blood from the MS patients. Together, these data demonstrate that IL‐15 expression by PBMNC is upregulated in the chronic stage of MS.
Cryptococcus neoformans Neutralizes Macrophage and Astrocyte Derived Nitric Oxide without Interfering with Inducible Nitric Oxide Synthase Induction or Catalytic Activity — Possible Involvement of Nitric Oxide Consumption
Authors: Trajkovic  , Stepanovic  , Samardzic  , Jankovic  , Badovinac  , Mostarica Stojkovic
Published: March 2003

The effect of Cryptococcus neoformans on the accumulation of nitrite, an indicator of nitric oxide (NO) synthesis, was investigated in cytokine (interferon‐γ [IFN‐γ] and interleukin [IL]‐1)‐stimulated cultures of rat peritoneal macrophages and C6 astrocytoma cells. Cytokine‐induced nitrite generation in cultures of both cell types was inhibited in a dose‐dependent manner by live C. neoformans, but not by heat‐killed cryptococcal cells or conditioned medium from yeast cultures. C. neoformans‐mediated reduction of nitrite formation coincided with impairment of NO‐dependent macrophage tumoricidal activity. Cytokine‐triggered induction of inducible NO synthase (iNOS) was unaffected in C6 cells, and only marginally reduced in macrophages. When cells were pretreated with cytokines for 24 h to induce iNOS, and any further induction was prevented by inhibition of protein synthesis, C. neoformans was still able to reduce nitrite accumulation in cultures of both cell types. Finally, live C. neoformans, but not heat‐killed yeast cells or yeast culture supernatant, significantly reduced nitrite production in a culture solution of NO‐releasing compound S‐nitrosoglutathione (GSNO). Thus, it appears that cryptococcal reduction of nitrite formation in macrophage and C6 cultures was caused by the consumption of NO by some yeast molecule, rather than by the inhibition of cellular NO synthesis.
Increased N‐Linked Glycosylation Leading to Oversialylation of Monomeric Immunoglobulin A1 from Patients with Sjögren's Syndrome
Authors: Basset  , Durand  , Jamin  , Clément  , Pennec  , Youinou  , Dueymes  , Roitt
Published: March 2003

Increased serum immunoglobulin A (IgA) level is a common finding in primary Sjögren's syndrome (pSS). IgA might not be properly eliminated because of an abnormal glycosylation. We reported previously that IgA1 from patients with pSS was oversialylated. We extend this finding by showing that monomeric IgA1 contains more sialic acid (SA) in patients than in controls, as determined by enzyme‐linked immunosorbent assay (ELISA) and Western blot with Sambucus nigra agglutinin (SNA), a lectin specific for SA. To localize this excess of SA on the N‐ and/or O‐linked oligosaccharides, we analysed them separately, using N‐ and O‐linked oligosaccharide profiling kits based on fluorophore‐assisted carbohydrate electophoresis. N‐linked, but not O‐linked, oligosaccharides of patients' IgA1 were oversialylated, and this seemed to be linked to an excess of SA on the same number of polysaccharides as normal IgA1. To localize the abnormality to the Fab and/or Fc fragments, monomeric IgA1 was digested with protease, separated and transferred to nitrocellulose, where SA was identified by SNA. Both Fab and Fc fragments appeared to be oversialylated. Oversialylation of N‐linked oligosaccharides of IgA1 from patients with pSS might prevent the recognition of IgA by receptors that are responsible for their clearance, resulting in an excess of serum IgA and related immune complexes.
Functional and Phenotypic Characteristics of Dendritic Cells Generated in Human Plasma Supplemented Medium
Authors: Pietschmann  , Stöckl  , Draxler  , Majdic  , Knapp
Published: March 2003

One successful approach to generate dendritic cells (DC) is to cultivate peripheral blood monocytes in fetal calf serum (FCS)‐containing medium in the presence of granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin (IL)‐4. Because the use of xenogenic proteins has to be strictly avoided for clinical applications, alternative protocols use human plasma instead of FCS. The aim of our study was to characterize DC generated in the presence of human plasma; moreover, we describe a novel protocol to generate DC directly from peripheral blood mononuclear cells (PBMC). DC generated from purified monocytes in the presence of 1% human plasma (HP‐DC) and GM‐CSF and IL‐4 both in the allogenic mixed leukocyte reaction (MLR) and in the tetanus presentation assay were potent stimulators of T‐cell proliferation. DC generated from PBMC were equally effective stimulators in the allogenic MLR as those generated from purified monocytes. When the immunophenotype of DC generated from FCS containing medium (FCS‐DC) was compared to that of HP‐DC, the surface expression of CD1a and CD80 was significantly lower in HP‐DC. In contrast, the expression of CD83 and CD86 was significantly higher in HP‐DC than in FCS‐DC. The capacity of receptor mediated endocytosis and macropinocytosis was found to be significantly lower in HP‐DC when compared to FCS‐DC. The differences in the immunophenotype, macropinocytosis and endocytosis between the HP‐DC and the FCS‐DC were observed independently of the generation of the cells from PBMC or purified monocytes. Our data indicate that HP‐DC are potent stimulators of T‐cell proliferation and exhibit a characteristic phenotype of intermediate maturity. Moreover, DC can be directly generated from PBMC preparations.
CD4+ T Cells of Schistosomiasis Naturally Resistant Individuals Living in an Endemic Area Produce Interferon‐γ and Tumour Necrosis Factor‐α in Response to The Recombinant 14kdaSchistosoma mansoni Fatty Acid‐Binding Protein
Authors: Brito  , Caldas  , Coura Filho  , Correa‐Oliveira  , Oliveira
Published: March 2003

Cellular immune responses to recombinant (r) Sm14 were examined in chronic, treated patients and uninfected individuals living in an endemic area for schistosomiasis. The lymphocyte proliferative responses and cytokine profile to this antigen were evaluated. Peripheral blood mononuclear cells (PBMC) of all groups studied proliferated to rSm14. However, the highest proliferation index to rSm14 was detected in uninfected endemic normal (EN) individuals who are naturally resistant to schistosomiasis. Regarding the cytokines produced, the levels of interleukin (IL)‐5 and IL‐10, known as Th2 cytokines, were not statistically different among all groups studied. In contrast, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α were produced in significantly higher amounts by PBMC of EN individuals following rSm14 stimulation. Additionally, we have determined by flow cytometry that CD4+ T cells from these individuals are the main lymphocyte subpopulation producing IFN‐γ and TNF‐α. Moreover, we have used rIL‐10 or rIFN‐γ, or monoclonal antibodies (MoAb) against these two cytokines to determine their role on cellular reactivity to rSm14. Exogenous IL‐10 suppressed T‐cell proliferation and neutralization of endogenous IL‐10 restored lymphocyte activation and enhanced IFN‐γ and TNF‐α production in chronically infected patients. In contrast, the addition of anti‐IFN‐γ totally abrogated the PBMC proliferation within the EN group. This study demonstrated that IL‐10 is an important cytokine down‐regulating T‐cell responses in chronic schistosomiasis, whereas lymphocyte proliferation in the uninfected resistant group is dependent on IFN‐γ. Taken together these results suggest that Th1 type of immune response induced in EN individuals to a specific schistosome antigen might be associated with resistance to infection and also highlighted the importance of Sm14 as a potential vaccine candidate.
Comparative Immunization Study Using RNA and DNA Constructs Encoding a Part of the Plasmodium falciparum Antigen Pf332
Authors: Andersson C. , Vasconcelos N.‐M. , Sievertzon M. , Haddad D. , Liljeqvist S. , Berglund P. , Liljeström P. , Ahlborg N. , Ståhl S. , Berzins K.
Published: March 2003

Development of nucleic acid‐based vaccines against parasitic diseases shows great promise, although certain concerns about safety aspects of conventional DNA vaccines have been raised. This study presents a comparison of antibody responses induced in mice by DNA and RNA‐based immunization with vectors encoding a part of the P. falciparum antigen Pf332. Two types of plasmids were used, one conventional DNA plasmid containing a cytomegalovirus promoter and one suicidal DNA plasmid encoding the Semliki Forest virus (SFV) replicase. RNA, encoding the SFV replicase and the relevant antigen, was delivered either as naked RNA or packaged in SFV suicide particles. In general, the antibody responses induced by the DNA plasmids were low and peaking after three injections, the conventional plasmid giving the highest responses. Also the RNA delivered in SFV particles consistently induced antibody responses, although comparatively low. Analyses of the ratio of immunoglobulin (Ig)G1/IgG2a subclasses in the responses indicated that all plasmids resulted in a bias for a Th2‐type of response, while the SFV‐particles elicited a Th1 type of response. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with the corresponding protein, with unchanged patterns of IgG subclasses.