bluuebunny/crossref_metadata_2025 huggingface dataset:
Double‐stranded conformation polymorphism of heterologous RNA transcripts and its use for virus strain differentiation
Authors: Rosner  , Maslenin  , Spiegel
Published: March 2003

Polymorphism of heterologous duplexes of RNA transcripts of PNRSV isolates was demonstrated. Complementary RNA trancripts were copied from PCR products of PNRSV isolates, to which the T7 RNA promoter was added on either the 3′ or 5′‐ends. Heterologous duplexes made from complementary RNA transcripts originating from several virus isolates differed in their electrophoretic mobilities. This polymorphism is hypothesized to be conferred by conformational variations among the transcript duplexes, and was termed by us ‘double‐stranded transcript conformation polymorphism (ds‐TCP)’. This method may serve as an additional tool in virus strain differentiation.
Peach red marbling and peach sooty ringspot, two new virus‐like degenerative diseases of Prunus
Authors: Grasseau  , Macquaire  , Boyé  , Cornaggia  , Desvignes
Published: March 2003

More than 600 Prunus samples were examined by using a nonradioactive digoxigenin‐labelled RNA probe specific for hop stunt viroid (HSVd). Prunus salicina and Prunus armeniaca appeared to be better hosts than Prunus persica. The weak viroid concentration in flowers and young leaves of peach trees growing in the field did not permit its detection in such samples. The diagnosis was more reliable (about 85%) with bark and leaves aged 4 months and more, from regrowths of GF 305 peach seedlings inoculated and kept in the greenhouse. Detection of HSVd in leaves and bark of apricot and Japanese plum plants aged 3 months or more also proved reliable (about 80% and 90%, respectively). HSVd could be transmitted in apricot, peach and plum nucleic acid preparations to GF 305 peach seedlings by repeated stem slashing, and to cherries (Prunus avium and Prunus serrulata) by approach grafting with an infected P. salicina source. The viroid was eliminated from 18% of the clones obtained after thermotherapy.

In the course of this study, 25 selected Prunus accessions suspected to be infected by unusual diseases were analysed by hybridization with a HSVd‐specific probe and by indexing on GF 305 peach seedlings in the greenhouse. Fifteen of these accessions were found to be infected by HSVd, 19 induced reddish marbling, and four induced small blackish spots on the leaves aged about 4 months. Repeated assays showed that these foliar symptoms were not caused by the viroid. Peach red marbling (PRMa) has not been associated with any known virus and seems to be caused by an infectious agent not yet described. That could also be the case with the agent of peach sooty ringspot (PSRS). PRMa and PSRS symptoms were reproduced by grafting and indexing, and their causal agents eliminated by thermotherapy in a significant fraction of the treated plants. They behave like viral agents and can infect the different Prunus species studied.
Interactions between isolates of Puccinia smyrnii, an autoecious, demicyclic rust and accessions of Smyrnium olusatrum
Authors: Agro  , Shattock
Published: March 2003

Plants derived from bulked seed samples of Smyrnium olusatrum collected from small patches of plants in Anglesey, Gwynedd, Shropshire and Suffolk were all susceptible, but expressed quantitative differences in disease severity, when inoculated with either aeciospores or teliospores of Puccinia smyrnii from the same or similar locations under controlled environmental conditions. Latent periods (mean 16 and 11 days for appearance of telia and spermagonia, respectively) were longer in 1996 compared with repeat inoculation in 1997 when the rust was more severe. Summation of mean numbers for each year of both aecia and telia, for rust isolate × host accession combinations, showed significantly different numbers of aecia in both years but not of telia. Incidence and severity of P. smyrnii varied on plants derived from seed from Anglesey, Shropshire and Suffolk when transplanted into a naturally rust‐affected population of S. olusatrum at Gwynedd, but all plants were susceptible. Experimental transfer of spermatia between spermagonia confirmed that P. smyrnii is heterothallic.
Genetic differentiation in the French population of Erysiphe cichoracearum, a causal agent of powdery mildew of cucurbits
Authors: Bardin  , Carlier  , Nicot
Published: March 2003

The relative incidence of Erysiphe cichoracearum and Sphaerotheca fuliginea, both agents of powdery mildew of cucurbits, was determined from 275 samples of mildewed leaves of cucurbits collected in 1994 from five regions of France. E. cichoracearum was identified in 9 to 39% of the mildewed leaf samples from four of the regions but was not detected in samples from the Mediterranean island of Corsica. The genetic structure of the French population of E. cichoracearum was examined using RFLPs of the ribosomal internal transcribed spacers amplified by PCR, random amplified polymorphic DNA (RAPD) markers, pathogenicity and mating‐type tests. Forty‐one isolates, including one from England, were analysed. Cluster analysis from 147 RAPD fragments using 16 primers revealed the existence of three distinct genetic lineages corresponding to three rDNA haplotypes (designated groups A, B and C). Bootstrap, genetic diversity, gametic disequilibrium and private allele analyses supported this differentiation. The genetic differentiation observed in the French population was not related to the geographical origin of the isolates. Group A isolates may be more specialized on melon as, with one exception, they were of race 1 (growth on four of the five melon cultivars tested) in comparison with group B and C isolates, which were of race 0 (growth on IranH only). Thus, the genetic differentiation observed may indicate a host‐specialized subdivision within the French population of E. cichoracearum from cucurbits. Gametic disequilibrium analysis among RAPD loci and biological observations suggest that the sexual stage is of minor importance for epidemics of E. cichoracearum on cucurbits.
Transmission of tomato spotted wilt tospovirus by Thripstabaci populations originating from leek
Authors: Chatzivassiliou  , Nagata  , Katis  , Peters
Published: March 2003

The transmission of tomato spotted wilt tospovirus (TSWV) by Thrips tabaci collected from leek was studied using the petunia local‐lesion leaf‐disc assay. After an acquisition‐access period of 72 h given to newborn larvae up to 8 h old, the efficiency of transmission by adults was determined in three inoculation‐access periods of 48 h. This efficiency varied for six T. tabaci populations from 0.7 to 11.6% in experiments using the Greek TSWV isolate GR‐04. Males were more efficient transmitters than females (19 out of 176 versus five out of 494). Frankliniella occidentalis transmitted the same virus with a higher efficiency (34.8%). The transmission rate differed also among TSWV isolates, as shown in tests with four T. tabaci using two isolates. The virus was more efficiently acquired from infected leaf material of Datura stramonium than from that of Emilia sonchifolia. Plants of the latter species were more susceptible than Nicotiana tabacum in thrips transmission tests.
Inhibition of the development of leaf rust (Puccinia recondita) by treatment of wheat with allopurinol and production of a hypersensitive‐like reaction in a compatible host
Authors: Ádám A. L. , Galal A. A. , Manninger K. , Barna B.
Published: March 2003

The effect of allopurinol [4‐hydroxypyrazolo (3,4‐ d) pyrimidine], a purine analogue inhibitor of xanthine oxidase (XO) enzyme, was studied in the host–pathogen combination of Triticum aestivum–Puccinia recondita f.sp. tritici. Analysis of purines and pyrimidines in the allopurinol‐treated wheat seedlings showed marked accumulation of xanthine, suggesting the inplanta inhibition of XO activity. In the incompatible wheat–rust interaction application of allopurinol as a drench, even at the highest concentration (50 μm), did not change the hypersensitive reaction phenotype; only the number of lesions was slightly reduced. Allopurinol treatment decreased the augmented rate of electrolyte leakage and lipid peroxidation associated with the hypersensitive response (HR), an effect probably related to the inhibition of rust development by allopurinol. By contrast, in the case of the compatible wheat–leaf‐rust combination the reaction type was strongly affected. The formation of uredia and production of uredospores were diminished or completely inhibited depending on the concentration of allopurinol, which was applied either as a drench (3.125–50 μm) or as a foliar spray (100–400 μm) to plants grown in perlite. At the highest allopurinol concentration in the drench, the compatible reaction type changed to a hypersensitive‐like necrotic reaction. Significant increases in electrolyte leakage and lipid peroxidation (characteristic of the HR) were found 4–6 days after infection in susceptible plants treated with allopurinol. Staining of leaf slices from allopurinol‐treated and compatible rust‐infected plants with Evans blue indicated cell death surrounding the pustules, while at this stage no cell death was detected in infected leaves without allopurinol treatment. The above results suggest that XO is not the main source of the generation of active oxygen species in wheat during the HR to leaf rust.
Effects of plant hormones on white clover mosaic potexvirus double‐stranded RNA
Authors: Clarke S. F. , Burritt D. J. , Jameson P. E. , Guy P. L.
Published: March 2003

The double‐stranded RNAs (dsRNA) produced during the replication of white clover mosaic potexvirus (WClMV) in the primary leaves of Phaseolus vulgaris during the 10 days following inoculation were investigated. Replication of a large dsRNA fragment (dsRNA 1) occurred within 24 h of inoculation and probably represents the replicative form of the genomic RNA. A second dsRNA fragment (dsRNA 5) appeared at day 3, and four other dsRNAs were detected from day 5; the putative functions of these are unknown, however their appearance coincided with a rapid increase in virus titre. Hormone treatments that inhibited virus replication altered the production of these dsRNAs. Dihydrozeatin, jasmonic acid and salicylic acid inhibited production of all dsRNAs except dsRNA 1, while 1‐amino‐1‐cyclopropane carboxylic acid reduced the levels of dsRNAs produced but did not alter the pattern of expression.
Modelling the effect of temperature on the development of Polymyxa betae
Authors: Webb C. R. , Gilligan C. A. , Asher M. J. C.
Published: March 2003

Polymyxa betae is the fungal vector of beet necrotic yellow vein virus (BNYVV), which is the causal agent of the sugar beet disease rhizomania. The within‐season dynamics of the fungus are a crucial factor in the occurrence and severity of rhizomania. Late infection of the host by viruliferous fungi enables host resistance to the virus to develop and hence limits crop damage. A previously published mechanistic model for the dynamics of Polymyxa betae is extended in this paper to incorporate the effect of temperature on the germination of resting spores, and on the latent periods between infection and the production of secondary zoospores and new resting spores. It is shown that, for UK temperature conditions, the effect of sowing date on infection is greater than that of year‐to‐year variations in temperature associated with a single representative sowing date. The variation in inoculum build‐up predicted when temperature data from a range of soil types were used in the model agreed with field observations, where higher levels of infection are observed on sandy soils than on black fen peat soils. The difference was most distinct when daily maximum soil temperature values were used to drive the model rather than rolling 24‐hour average values.
Substantial reductions in winter wheat diseases caused by addition of straw but not manure to soil
Authors: Rodgers‐Gray B. S. , Shaw M. W.
Published: March 2003

Over a period of 3 years, five agronomically distinct crops of winter wheat were grown in plots in which straw (1 kg m−2), manure (4 kg m−2) or nothing were incorporated into the soil. Plant establishment and height, but not leaf area per tiller, were lower in straw‐treated plots. Fertilizer regimes differed between years. Soil and leaf nitrogen were recorded; there was no obvious link between N and any disease or soil amendment. The numbers of leaf layers scorable for disease were similar in all treatments. At the end of the season, plants from straw‐treated plots had consistently reduced septoria tritici blotch (caused by Mycosphaerella graminicola), powdery mildew [caused by Erysiphe (Blumeria) graminis], brown rust (caused by Puccinia recondita) and foot rot (caused by Fusarium spp.). Early on, M. graminicola was worse in straw‐treated plots. In manure‐treated plots, P. recondita was reduced but effects on other diseases were inconsistent and slight. A fungicide, chlorothalonil, was applied in one crop; its effects did not interact with those of other treatments. Mycosphaerella graminicola was not suppressed by straw in outdoor pot experiments. Late in the season, straw‐treated plants had significantly higher leaf silica (P < 0·01). In a glasshouse experiment, plants supplied with silicon had less E. graminis infection (P < 0·001) and higher leaf silica, but effects on M. graminicola were inconsistent. A prior inoculation of M. graminicola primed plant defences against a subsequent attack of E. graminis, but only in the presence of adequate Si. It is postulated that straw acts in the field by increasing Si availability.
Variation in sensitivity of Western Australian isolates of Phytophthora cinnamomi to phosphite in vitro
Authors: Wilkinson C. J. , Shearer B. L. , Jackson T. J. , Hardy G. E. St J.
Published: March 2003

Seventy‐one Australian isolates of Phytophthora cinnamomi (68 from Western Australia) were tested for sensitivity to phosphite on Ribeiro's modified medium. Isolates formed a continuum in their response to phosphite, but could be divided into sensitive (9% of isolates), intermediate (82%) and tolerant (9%) groups. Sensitivity varied between isolates, with EC50 values ranging from 4 to 148 µg phosphite mL−1. Phytophthora cinnamomi A1 mating‐type isolates were at the upper end of the range of tolerance shown by the A2 mating‐type isolates.
Control of the anthracnose pathogen of banana (Colletotrichum musae) using antioxidants alone and in combination with thiabendazole or imazalil
Authors: Khan S. H. , Aked J. , Magan N.
Published: March 2003

Two isolates of Colletotrichum musae (CM100 and CM103) which differed significantly in their sensitivity to the fungicides thiabendazole (TBZ) and imazalil were grown on malt extract agar amended with the following antioxidants: ascorbic acid, benzoic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene, dimethyl sulphoxide, propionic acid, propyl gallate (PG), propyl paraben (PP) and thiourea. The compounds showing the greatest antifungal activity were BHA, benzoic acid, PG and PP. At concentrations of 15 mm or less, these chemicals inhibited conidial germination (at 72 h) and mycelial growth (by day 14) of isolate CM100 and gave high levels of inhibition of isolate CM103. Combinations of low concentrations of BHA (0·5 mm) and imazalil (1·8 µm) completely prevented mycelial growth and germination of the two isolates tested in vitro and showed some synergism when combined at even lower concentrations of BHA. BHA, PP and benzoic acid, in combination with each other and with imazalil and TBZ, applied to banana stalk tissues and crowns previously inoculated with C. musae, were less effective at controlling the fungus in vivo compared with inhibiting mycelial growth and conidial germination in vitro. BHA at 5 mm inhibited fungal growth at 25°C by 35–41%. When BHA (5 mm) was combined with imazalil (0·45 mm), better control (63–80% inhibition) of simulated crown rot was achieved than when imazalil (1·78 mm) or TBZ (2·46 mm) were applied on their own (53–58% and 43–54% inhibition, respectively). The results suggest that BHA, which is a food‐grade chemical, might have the potential to enhance the activity of fungicides currently used to control C. musae on bananas, allowing lower concentrations of fungicide to be used.
Effect of the antioxidant ascorbic acid on sclerotial differentiation in Rhizoctonia solani
Authors: Georgiou C. D. , Petropoulou K. P.
Published: March 2003

Rhizoctonia solani produces ascorbic acid at levels dependent on its developmental stage and its ability to differentiate and form sclerotia. It was found that oxidized exceeded reduced ascorbic acid by a maximum of fourfold in the undifferentiated mycelia, and 2·5‐fold in the differentiated mycelia and sclerotia. Ascorbic acid profiles were very distinctive between differentiating and nondifferentiating R. solani isolates. In the nondifferentiating isolate the ratio of reduced : oxidized ascorbic acid was constant throughout growth, while in the differentiating isolate it increased 1·7‐fold and decreased 1·3‐fold in the middle and at the end of sclerotial differentiation, respectively, compared with the undifferentiated stage. The differentiation‐associated marker of oxidative stress – lipid peroxidation – was 2·8‐fold higher in colonies of the differentiating isolate before they differentiated than in same‐day colonies of the same age of the nondifferentiating isolate. In the differentiating isolate, exogenous ascorbic acid up to 8 mg mL−1 caused a dose‐dependent inhibition of up to 100% of fully developed sclerotia and a delay of 3·5 days in formation of sclerotial initials, apparently acting as an antioxidant and lowering lipid peroxidation to levels similar to those in the nondifferentiating isolate. The data from this study support the hypothesis that oxidative stress induces differentiation in sclerotium‐producing plant pathogenic fungi.
Variability associated with the official USDA sampling plan used to inspect export wheat shipments for Tilletia controversa spores
Authors: Whitaker T. B. , Wu J. , Peterson G. L. , Giesbrecht F. G. , Johansson A. S.
Published: March 2003

The variability associated with estimating the true concentration of teliospores of dwarf bunt (Tilletia controversa) per 50 g of wheat (TC concentration) in an export wheat shipment was studied by measuring the TC concentration in 16 test samples (50 g) taken from each of 137 export shipments. The variability among the 16 TC test sample results, as measured by the standard deviation, was found to increase with TC concentration. The functional relationship was approximately linear in a full‐log plot and regression analysis was used to determine the coefficients of the regression equation. Using statistical theory, the regression equation was modified to predict the standard deviation among test sample sizes other than the 50 g size used in this study. The standard deviation and coefficient of variation associated with using a 50 g test sample to estimate the true TC concentration of a wheat shipment with 2000 spores per 50 g were estimated to be 1062·8 and 53·1%, respectively. Increasing test sample size to 1600 g reduced the standard deviation and coefficient of variation to 187·9 and 9·4%, respectively.
Factors affecting diseases of winter wheat in England and Wales, 1989–98
Authors: Hardwick N. V. , Jones D. R. , Slough J. E.
Published: March 2003

The above article was published without a number of the author's corrections included. The publisher offers their unreserved apology for this and the sections affected are reprinted below in their entirety.Samples from 360 to 450 randomly selected winter wheat crops in England and Wales were collected annually during the milky ripe development stages (GS 73–75) from 1989 to 1998. The number of samples from each region was proportional to the area of winter wheat grown. The percentage area affected by disease was assessed on the top two leaves and the ear, and the incidence and severity of stem base diseases were also recorded. An estimate of the percentage area of the crop affected by Barley yellow dwarf virus (BYDV) and take‐all (Gaeumannomyces graminis) was made in the field. Septoria leaf blotch (Septoria tritici, teleomorph Mycosphaerella graminicola) was the major foliar disease recorded, with an average maximum severity of 7·8% of the area of leaf 2 affected in 1998. Eyespot (Tapesia spp.) was the major stem base disease, with the highest incidence of stems falling into the damaging moderate plus severe categories (18·9%) in 1998. Levels of powdery mildew (Blumeria graminis) showed a decline from 0·4% of the area of leaf 2 in 1989 to 0·1% in 1998. This fall was associated with a reduction in the proportion of disease‐susceptible cultivars grown. There were significant regional differences in levels of septoria leaf blotch, brown rust (Puccinia recondita), eyespot, sharp eyespot (Rhizoctonia cerealis) and BYDV. The percentage of crops treated with a fungicide rose from 96% in 1989 to 98% in 1998 and the mean number of spray applications per crop rose during this period from 2·1 to 2·5. A higher proportion of crops was treated with fungicides between the end of tillering and fifth node detectable (GS 29–35) than around flag leaf emergence (GS 36–48) or ear emergence (GS 49–71). Prior to 1994, the majority of late fungicide sprays was applied at, or after, ear emergence, but from 1994, the majority was applied around flag leaf emergence. The value and socioeconomic implications of the results are discussed.
Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco
Authors: Fatmi M. , Schaad N. W.
Published: March 2003

The survival and half‐life of Clavibacter michiganensis ssp. michiganensis (C. michiganensis), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Aït Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Aït Melloul and Melk Zhar. The half‐life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half‐life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half‐life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Aït Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half‐life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.
Xylem sap flow of Norway spruce after inoculation with the blue‐stain fungus Ceratocystis polonica
Authors: Kirisits T. , Offenthaler I.
Published: March 2003

In a field experiment, the sap flow of Norway spruce (Picea abies) was measured in response to inoculation with Ceratocystis polonica, a virulent fungal associate of the spruce bark beetle Ips typographus. Four vigorous, mature Norway spruce trees were mass‐inoculated with C. polonica at a density of four inoculations per dm2 within a 120‐cm‐wide band on the lower bole. Two other trees were inoculated with sterile agar and served as controls. Sap flow of the six trees was measured by the thermal dissipation method from three weeks prior to inoculation until five months after inoculation. In three trees that had been inoculated with the fungus, sap flow density decreased dramatically within the first weeks after inoculation until no sap flow was measurable 4–6 weeks after inoculation. No marked changes in sap flow density were recorded in the fourth tree that had been inoculated with C. polonica, and this tree survived the inoculation. Inoculation with sterile agar did not have any apparent influence on the sap flow of the control trees. The results confirm the ability of C. polonica to kill mature Norway spruce trees. Sap flow measurements by the thermal dissipation method provided a direct and nondestructive method for monitoring the effects of fungal infection on sapwood functionality. Its application for further studies on vascular wilt and vascular stain diseases of trees is therefore recommended.
Cytokine Induction by Mycoplasma arthritidis‐Derived Superantigen (MAS), but not by TSST‐1 or SEC‐3, is Correlated to Certain HLA‐DR Types
Authors: Alvarez‐Ossorio  , Johannsen  , Alvarez‐Ossorio  , Nicklas  , Kirchner  , Rink
Published: March 2003

Superantigens bind to major histocompatibility complex (MHC) class II molecules on antigen presenting cells and T cells in a Vβ‐restricted manner. Both cell types are activated resulting in cytokine production. Although the MHC‐II binding site for superantigens has been well described, little is known as to whether this binding complex has an influence on cytokine induction. In order to assess superantigen induced cytokine production and its correlation to HLA‐DR types, the authors stimulated peripheral blood from 40 subjects with superantigens toxic shock syndrome toxin‐1 (TSST‐1), staphylococcal enterotoxin C‐3 (SEC‐3) and Mycoplasma arthritidis‐derived superantigen (MAS), and measured cytokine levels thereafter. The HLA‐DR type was determined in each subject. A statistical evaluation was carried out between the highest superantigen cytokine induction and the presence of certain HLA‐DR types. Whereas MAS presented a statistical association between the highest cytokine production with HLA‐DR4, DR7 and DR12, no such associations were observed for TSST‐1 and SEC‐3. These results demonstrate that T cell stimulation, and consequently its cytokine production by MAS but not by TSST‐1 and SEC‐3, depends on the presenting HLA‐DR type. Because the diverse HLA‐DR specificities are given according to the variability of the β chain of the HLA‐DR molecule, the data suggest the participation of the human MHC‐II β chain in the MAS/MHC‐II binding.
Do Chickens Immunized with Bacterial Immunoglobulin G‐Binding Proteins Produce Rheumatoid Factor‐Like Antibodies?
Authors: Boyle  , Vogel  , Sisson  , Raeder
Published: March 2003

An association between the production of rheumatoid factor (RF)‐like antibodies in animals immunized with bacterial immunoglobulin (Ig)G‐binding proteins has been noted. Three potential explanations have been proposed: (1) altered host IgG due to binding of the immunogen; (2) B‐cell superantigenic properties of the binding proteins; and (3) idiotype‐anti‐idiotype response leading to an antibody which acts as an antigen mimic. In order to distinguish among these possibilities, it is necessary to carry out studies in animals whose IgG does not react with the IgG‐binding protein immunogen. Consequently, we have determined the effects of immunizing chickens with a purified group C streptococcal IgG‐binding protein, FcRc, a bacterium expressing this protein, and appropriate control immunogens. The results of these studies provided evidence for production of specific antibodies to FcRc in groups of chickens immunized with either the pure protein or bacteria expressing the protein. No significant association with production of RF‐like antibodies was noted, favouring the altered IgG‐binding explanation for the association between RF‐like antibodies and immunization with the bacterial IgG‐binding proteins.
Identification of a C → T Mutation in the Reactive‐Site Coding Region of the C1‐Inhibitor Gene and its Detection by an Improved Mutation‐Specific Polymerase Chain Reaction Method
Authors: Nielsen  , Fure  , Winge  , Mollnes
Published: March 2003

Mutations in the C1‐inhibitor (C1‐INH) gene, leading to low functional levels of C1‐inhibitor protein, cause hereditary angioedema (HAE). The disease is characterized by episodic edema in a number of organs. Typically, swellings occur in extremities and face, often accompanied by crampy abdominal pain. Laryngeal edema may lead to suffocation. Type II HAE patients have low functional C1‐INH values stemming from only one normal allele. Antigenic C1‐INH values, however, are normal or increased owing to the presence of a dysfunctional protein from the mutated allele. The mutations are usually found in exon 8 coding for the amino acids near the reactive centre (P1). Previously, no mutations in the C1‐INH gene had been published from the Scandinavian countries. In this work, exon 8 of the C1‐inhibitor gene was sequenced in members of two different kindreds, from western and northern Norway, who were suffering from HAE type II. A common point mutation was found within the bait region encoding the reactive centre. The codon CGC was converted to TGC at position 17 970, corresponding to an Arg → Cys replacement which reportedly is the second most frequent type II HAE mutation. This information was utilized to develop a mutation‐specific polymerase chain reaction (PCR) for the identification of affected family members. The antisense 17‐mer primer (5′‐AAGACCAGCAGGGTGCA‐3′) was successfully applied and AmpliTaq Gold was used in the PCR.
Cytokine Response to Group B Streptococcus Infection in Mice
Authors: Rosati  , Fettucciari  , Scaringi  , Cornacchione  , Sabatini  , Mezzasoma  , Rossi  , Marconi
Published: March 2003

This study was undertaken to better understand the complex relationship between specific and non‐specific host defence mechanisms and group B streptococci (GBS). A comprehensive kinetics analysis of cytokine mRNA expression was performed, by Northern blot assay, in peritoneal exudate cells (PEC) and spleen cells (SC) recovered from CD‐1 mice at various times during the course of an intraperitoneal infection with a lethal dose (5 × 103 microorganisms/mouse) of type Ia GBS, reference strain 090 (GBS‐Ia). Analysis of cytokines involved in the development of a specific TH response shows that GBS‐Ia in PEC induce only a weak increase of IL‐2 mRNA expression and in SC a cytokine pattern characterized by IL‐2, IFN‐γ and IL‐12 in the absence of IL‐4, IL‐5 and IL‐10. This selected cytokine pattern could provide appropriate conditions for the development of a TH1 response. Analysis of inflammatory cytokines, which are usually induced early during an in vivo infection, shows that there is a significant expression of mRNA specific for IL‐1β, TNFα and IL‐6, both in PEC and SC only at 24 h which persists at a high level until 36 h. This delayed cytokine induction, accompanied by the contemporary activation of splenic phagocytic cells, occurs only when the number of GBS‐Ia is extremely high. In fact, at 24 h GBS‐Ia have heavily colonized all organs. In vitro infection of thioglycollate‐elicited peritoneal macrophages confirms that the ability of GBS‐Ia to induce a strong inflammatory cytokine response depends strictly on the number of infecting microorganisms. Indeed, macrophages respond to GBS‐Ia with a very rapid induction of IL‐1β and TNFα mRNA when infected at a ratio of 1:10, but not at 100:1. Two major observations emerged from this study: (1) GBS‐Ia, by inducing a cytokine pattern which seems to favour development of a TH1 response, could evade antibody production essential for resistance to GBS; and (2) inflammatory cytokine response is induced when a heavy microbial invasion of the host has already occurred. These novel features of GBS‐Ia could contribute to the development and progression of lethal infection in mice.
Chlamydia pneumoniae‐Specific Cell‐Mediated and Humoral Immunity in Healthy People
Authors: Halme  , Von Hertzen  , Bloigu  , Kaprio  , Koskenvuo  , Leinonen  , Saikku  , Surcel
Published: March 2003

In this work, cell‐mediated immunity to Chlamydia pneumoniae was studied in 157 healthy individuals using lymphoproliferative assay and serum antibodies were analysed by microimmunofluorescence techniques. The C. pneumoniae‐specific IgG antibodies were elevated more frequently and the geometric mean titres for IgG (67.5 versus 44.1; P = 0.05) and IgA (14.9 versus 11.3; P = 0.025) antibodies were significantly higher in males than in females. However, no gender‐dependent differences were observed in cellular reactivity to C. pneumoniae, since the median cellular responses were similar (stimulation indices 7.5) in men and women. Although the cell‐mediated and humoral responses to C. pneumoniae did not correlate clearly, elevated IgG antibodies were associated with slightly higher lymphocyte proliferation in comparison to all subjects (15.5 versus 7.5) and significantly stronger in comparison to those with persistently elevated IgA (> 80) antibodies (15.5 versus 3.5; P = 0.023). Further studies are needed to evaluate a possible role of reduced cellular reactivity in the cause of chronic C. pneumoniae infection.
Oligoclonality of TCRint Cells with a Low Diversity of TCR Complementarity‐Determining Region 3 in Mice with Graft‐ Versus‐Host Disease
Authors: Sugahara  , Kuwano  , Sato  , Hasegawa  , Yamagiwa  , Kawamura  , Yoshida  , Asakura  , Abo
Published: March 2003

Conventional T cells (i.e. TCRhigh) are generated by the main stream of T‐cell differentiation in the thymus. However, primordial T cells (i.e. TCRint) are generated by extrathymic pathways and an alternative intrathymic pathway. Since TCRint cells contain self‐reactive clones, the diversity of the T‐cell antigen receptor (TCR) complementarity‐determining region (CDR) 3 was examined. The predominant Vβ8.2+ clones among TCRint cells were selected for DNA sequencing. Thymectomized, irradiated mice subjected to bone‐marrow transplantation (BMT) were used; graft‐versus‐host disease (GVHD), B6→(B6 × C3H/He)F1 and syngeneic BMT, B6→B6. In these combinations, only TCRint cells were generated. Vβ8.2+ cells with a low diversity of CDR3 of V‐gene expanded in GVHD mice. Vβ8.2+ cells of TCRint and TCRhigh cells in normal mice were polyclonal, showing that the former has a lower diversity of CDR3 than the latter. The clonality of activated TCRhigh cells was examined, in which CD3high cells (bm12 mice) were injected into 1 Gy‐irradiated B6 nude mice. Some Vβ8.2+ clones among TCRhigh cells were expanding but the diversity of CDR3 was greater than that of CD3int cells, despite the fact that the recognition site of the H‐2 difference was smaller. Taken together with invariant usage of Vα14, these results suggest that TCRint cells have a low diversity of CDR3 of Vβ genes.
Identification of Arthritogenic Adjuvants of Self and Foreign Origin
Authors: LORENTZEN
Published: March 2003

The lack of defined triggers for human inflammatory joint diseases warrants efforts to identify candidate molecules. For this task, it may be an important lead that nonspecific activation of the immune system can precipitate arthritis in rats. Consequently, arthritis‐prone rat strains were used to search for disease‐triggering factors among molecules which initially induce innate defence reactions rather than specific immune responses. A variety of immunological adjuvants were investigated by intradermal injection into DA and LEW.1AV1 rats and monitoring of clinical signs for 30 days. Several arthritogenic cell‐wall structures from yeast and bacteria were identified, such as β‐glucan, lipopolysaccharide and trehalosedimycolate. The test procedures also revealed arthritogens of chemical origin, such as dioctadecyldiammoniumbromide (DDA = C38H80NBr) and heptadecane (C17H36). Furthermore, it allowed the precise definition of arthritogenic determinants of lipids, since C16H34 induced arthritis, whereas the closely related linear hydrocarbons C16H32, C16H33Br and C15H32 did not. The observed pathogenicity of organic lipids raised the question of whether endogenous lipids can also precipitate arthritis. Indeed, this was true for the cholesterol precursor squalene (C30H50). In conclusion, this article describes the rational use of arthritis‐prone rat strains to identify arthritogenic factors of both foreign and self origin. Although structurally unrelated, the pathogenic molecules defined here share the feature of being nonspecific triggers of the immune system. This consolidates a general principle for the induction of adjuvant arthritis which may provide clues to the aetiology of human arthritides, including rheumatoid arthritis.
Genetic Analysis of the Maternally Induced Affinity Enhancement in the Non‐Ox1 Idiotypic Antibody Repertoire of the Primary Immune Response to 2‐Phenyloxazolone
Authors: LANGE  , KOBARG  , YAZYNIN  , SOLTERBECK  , HENNINGSEN  , HANSEN  , LEMKE
Published: March 2003

The early phases of ontogeny are decisive for the development of the B‐cell repertoire. Here, we demonstrate that maternal tertiary immunization of BALB/c mice with 2‐phenyloxazolone (phOx) caused a drastic alteration of the primary antigen‐specific repertoire of the offspring. Maternal tertiary immunization or quaternary antibodies, which exhibited an extremely weak cross‐reactivity with the major Oxl idiotype (IdOx1), induced a change in the proportion of IdOx1/non‐IdOx1 antiphOx antibodies in the F1 and F2 primary repertoire. The observed variability in the level of IdOx1 expression (10–90%) exceeded even the seemingly genetically based differences between various mouse strains. In comparison with the non‐IdOxl of control mice, half of the non‐IdOxl antibodies showed a 5–100‐fold enhanced affinity. Sixty per cent of these antibodies exhibited an affinity identical to that of IdOx1 antibodies, which are normally of the highest affinity, while the remaining 40% exceeded even that of IdOx1 by a factor of 10. The non‐IdOx1 were encoded by VH/VL genes and/or combinations thereof which are either new, hitherto unobserved in the antiphOx response, or typical of memory responses in normal mice. The significance of these data is discussed with respect to the possibility that maternal antibodies, which are acquired through multiple immune maturation processes, may have an epigenetic (non‐Mendelian) inheritable potential for the offspring.
An Ultrastructural Study of Amyloid Intermediates in Aβ1–42 Fibrillogenesis
Authors: NYBO  , SVEHAG  , HOLM NIELSEN
Published: March 2003

Many attempts have been made to define early stages and intermediates in amyloid fibrillogenesis that may be susceptible to inhibition. We have developed an in vitro system, based on the use of Aβ1–42 peptides, in which the development of prestages of protofilaments and protofilament and fibril formation could, for the first time, be followed by electron microscopy, supported by fluorescence spectrometry. The first recognizable ultrastructures after incubation of Aβ1–42 peptides at 37 °C were globular subunits (4–5 nm in diameter) that gradually became organized into short protofilaments (30–100 nm), which in turn formed fibrils mainly by lateral association. At this stage, part of the protofilaments were seen first as collaterals protruding from the fibrils and then, as they were gradually incorporated, as buds on the fibril surface. A continuous growth of Aβ1–42 fibrils was observed, seemingly originating from a nucleus, which appeared to consist of aggregates of amyloid intermediates. That protofilaments are intermediates also in the in vivo formation of amyloid was supported by the finding that AL fibrils isolated from amyloid tissues also exhibited radiating protofilaments. The demonstrated globular subunits and early formed protofilaments may be attractive targets for inhibition of fibril formation.
Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis‐Specific RD1 Region Gene
Authors: Ahmad  , Amoudy  , Thole  , Young  , Mustafa
Published: March 2003

A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guérin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF‐10, ORF‐14 and ORF‐15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX‐4T vector system and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti‐GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST‐ORF‐14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF‐14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST‐ORF‐14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF‐14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG‐vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long‐term contacts of TB patients also had antibodies reactive to the ORF‐14 protein. These results suggest that the ORF‐14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.
A Trypanosoma cruzi Alkaline Antigen Induces Polyclonal B‐Cell Activation of Normal Murine Spleen Cells by T‐Cell‐Independent, BCR‐Directed Stimulation
Authors: Montes  , Zuñiga  , Minoprio  , Vottero‐Cima  , Gruppi
Published: March 2003

We have previously reported that a cytosolic alkaline fraction (FI) obtained from epimastigotes of Trypanosoma cruzi promotes the activation, proliferation and differentiation of normal murine B cells into antibody‐secreting plasmocytes. Neither the mechanism nor the cells involved in the FI‐induced polyclonal B‐cell activation were established. In this work we report that accessory cells are required for FI‐induced polyclonal B‐cell activation as no proliferative responses were obtained following treatment of normal spleen mononuclear cells (NSMC) with
l‐leucine methyl ester. Furthermore, FI did not induce the expression of CD25 on T cells and it promoted the proliferation of a T‐cell‐depleted population, indicating that it acts in a T‐independent manner. We observed that NSMC were stimulated in vitro by FI‐released cytokines, such as interleukin (IL)‐4, IL‐6 and IL‐10, which are involved in B‐cell proliferation and differentiation. Interestingly, while significant amounts of interferon‐γ (IFN‐γ) were found in culture supernatants we did not observe detectable levels of IL‐2. Additionally, we found that B‐cell receptor (BCR) and major histocompatibility complex (MHC) class II antigens were involved in the proliferative response induced by FI because antibodies directed against cell‐surface immunoglobulin M (IgM), CD45 and MHC class II molecules inhibited the FI‐induced B‐cell proliferation. CD40 ligand (CD40L) did not participate in such a phenomenon.
Molecular Cloning and Phylogenetic Analysis of the Atlantic Salmon Immunoglobulin D Gene
Authors: Hordvik  , Thevarajan  , Samdal  , Bastani  , Krossøy
Published: March 2003

A gene homologous to the IgD heavy chain (δ) gene in channel catfish (Ictalurus punctatus) was found 0.9 kb downstream of the IgM heavy chain (μ) gene in Atlantic salmon (Salmo salar). As in catfish, the first constant μ exon is spliced into the δ transcripts. In agreement with the tetraploid ancestry of the salmonid fish family there are two highly similar δ genes in Atlantic salmon. Characterization of these genes showed that they encode seven ‘unique’ Ig domains, three of which are tandem duplicated, i.e. like δ1–(δ2–δ3–δ4)*–(δ2–δ3–δ4)–δ5–δ6–δ7. Sequence analysis indicates that δ1–δ7 arose from two duplication events. Accordingly, salmon δ can be reduced to a unit of three Ig domains corresponding to the three C‐terminal domains of a prototypic Ig molecule. The ancestral three‐domain unit is apparently best conserved in δ1–δ5–δ6. Phylograms indicate a relationship between teleost and mammalian IgD mainly because of the similarity between the teleost δ5 and human δ2. The corresponding domain in mouse IgD has been deleted during evolution. The teleost δ1 and δ6 sequences are most similar to domains of other non‐IgM isotypes, including those in cartilaginous fishes.
Lymphocyte Stimulation by CD3‐CD28 Enables Detection of Low T Cell Interferon‐γ and Interleukin‐4 Production in Rheumatoid Arthritis
Authors: Verhoef  , Van Roon  , Vianen  , Glaudemans  , Lafeber  , Bijlsma
Published: March 2003

The analysis of cytokine production is increasingly important in defining the course of an immune response and in evaluating specific therapies of immune diseases. In rheumatoid arthritis (RA), a dysregulation in T1/T2 cell balance, as defined by the production of their specific cytokines, IFN‐γ and IL‐4, respectively, is suggested. A predominance of T1‐cell mediated macrophage activity in the joint plays a key role in the destruction of articular cartilage and subchondral bone, whereas local T2 cell activity, mitigating disease, fails. However, analysis of the cytokines defining both T cell subsets is difficult and spontaneous production is often below detection limits. Several stimuli are therefore used to increase cytokine production. In the present study we examined whether stimulation of peripheral blood T cells in the context of mononuclear cells (PB MNC) by CD3‐CD28 is a reliable method for assessing IFN‐γ and IL‐4 production and is representative for the spontaneous production of these cytokines. The production of IFN‐γ and IL‐4 following CD3‐CD28 stimulation of RA PB MNC correlated significantly in a ratio 1 : 1 with production following ionomycin‐PMA stimulation. In samples with detectable spontaneous production of IFNγ and IL‐4, production following CD3‐CD28 stimulation was significantly higher than in stimulated samples with undetectable spontaneous production. Moreover, in the case of spontaneous production there was a significant positive linear correlation between the CD3‐CD28 stimulated and spontaneous IFNγ and IL‐4 production, although production of both cytokines was not equally enhanced. Serial sampling did not show significant daily or weekly variation in stimulated cytokine production. The results demonstrate that a pecific T‐cell stimulation by CD3‐CD28 is a reliable way to enhance IFN‐γ and IL‐4 production above the detection limit and so measure the T1/T2 cell balance in RA.
Lipopolysaccharide Induces Expression of APO2 Ligand/TRAIL in Human Monocytes and Macrophages
Authors: Halaas  , Vik  , Ashkenazi  , Espevik
Published: March 2003

Monocytes express cytotoxic factors of the tumour necrosis factor (TNF) ligand superfamily, including TNF and Fas ligand, both on the cell surface and in secreted form. In this report, we show that human monocytes and monocyte‐derived macrophages stimulated with lipopolysaccharide (LPS) express APO2 ligand (APO2L, TRAIL), a recently discovered cytotoxic member of the TNF ligand superfamily. LPS increased the transcription of APO2L mRNA in monocytes and macrophages. Flow cytometric analysis showed low surface and high intracellular levels of APO2L, and LPS increased the expression of both. In addition, LPS increased the monocyte‐ and macrophage‐mediated cytotoxicity against the APO2L‐sensitive Jurkat and RPMI‐8226 cells. Addition of the APO2L‐binding decoy receptor 1 (DcR1)–Fc fusion protein inhibited the cytotoxicity by 30–70%. LPS also stimulated the release of soluble APO2L from the monocytes and macrophages. Monocytic phagocytosis of target cells was increased by LPS and partially inhibited by DcR1–Fc. Taken together, these data demonstrate a novel mechanism of cytotoxicity mediated by LPS‐activated human monocytes and macrophages.