bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and analysis of inter‐ and intraspecific diversity of Pratylenchus spp. using isozyme markers
Authors: Andrés M. F. , Pinochet J. , Hernández‐Dorrego A. , Delibes A.
Published: March 2003

The root‐lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus, P. goodeyi, P. penetrans, P. scribneri, P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis. A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.
The effect of fungicide dose on the composition of laboratory populations of barley powdery mildew
Authors: O'hara R. B. , Nielsen B. J. , Østergård H.
Published: March 2003

The effect of the use of different doses of the fungicide fenpropimorph on populations of barley powdery mildew Blumeria (Erysiphe) graminis f. sp. hordei was investigated in a laboratory selection experiment. A sample from the Danish aerial population of powdery mildew was split into populations, and these were kept separately for 31 generations on susceptible barley seedlings treated with fungicide at two concentrations, as well as on a control. Samples from these populations were tested for their resistance to fenpropimorph and their virulence spectra. There was a large amount of environmental variation in the ED50 values used to measure fungicide resistance. In both treated populations, the average level of fungicide resistance increased, this increase being faster and greater in the population treated with the high dose. The diversity of pathotypes of the treated populations decreased, with the decline being more rapid in the population treated with the high dose, where one pathotype dominated the population after 31 generations. This pathotype was apparently not the fittest in the population treated with the low dose. This implies that knowledge of ED50 is not sufficient to predict pathotype evolution under different fungicide treatments. The dominant pathotype in the high‐dose treatment may not have been clonal, as there was evidence of two levels of fungicide resistance. The large environmental variation observed in estimated ED50 values for resistance towards fenpropimorph may help to explain why this resistance has evolved at a slower rate than resistance towards other fungicides.
Analysis of the spatiotemporal structure of rice sheath blight epidemics in a farmer's field
Authors: Savary S. , Castilla N. P. , Willocquet L.
Published: March 2003

The spatiotemporal structures of two rice sheath blight epidemics were studied during two consecutive (rainy and dry) rice cropping seasons in a farmer's field in the Philippines. The amount of primary inoculum in the soil measured at transplanting was higher during the dry than during the rainy season, and showed nonaggregated spatial patterns in both seasons. Disease incidence (percentage tillers infected per hill) was monitored at weekly intervals on a (14 × 17) grid of rice hills 1 m apart, at the centre of adjacent, nonoverlapping elementary quadrats of 5 × 5 hills. The rate of disease increase at the onset of the epidemics was higher in the dry than in the rainy season, but was afterwards higher in the rainy than in the dry season. A C(α) test was used to test for overdispersion, and the beta‐binomial parameter θ was used to assess disease aggregation in quadrats consisting of one, two, four, eight and 16 elementary quadrats. Aggregation was detectable at all quadrat sizes throughout the epidemic, according to the C(α) test. θ was highest at epidemic onset and at the smallest (elementary) quadrat size, indicating a very strong aggregation of disease at the individual hill scale. θ increased until disease incidence reached 40%, then declined over time. θ values were generally smaller in the dry season. Semivariograms for both epidemics showed strong initial discontinuities, that is, large variance of disease incidence at the elementary quadrat scale. Some aggregation in the spatial distribution of disease incidence was indicated at higher scales in the rainy season, coinciding with the highest rate of disease increase. Semivariograms did not suggest any structure in the dry season, except for one date in the early epidemic stage. Spatiotemporal autocorrelation of disease incidence at 45 days after transplanting yielded very few variables that contributed significantly to describing the disease incidence and its increase in a given hill. In the rainy season these variables were disease incidence (or increase) in the nearest sampled neighbours, and disease incidence (or increase) observed 1 week earlier in the same hill. In the dry season only disease incidence (or increase) observed 1 week earlier in the same hill was significantly correlated with the current incidence (or its increase). The development of a spatial structure in distribution of disease is hypothesized to coincide with the spread of the pathogen from one hill to another, and with the shift from monocyclic to predominantly polycyclic processes in sheath blight epidemics. These results concur with previous reports in suggesting that management of rice sheath blight should first be directed against the leafborne, polycyclic phase of epidemics, rather than their initial phase.
Vegetative compatibility and heterokaryon stability in Fusarium oxysporum f.sp. radicis‐lycopersici from Italy
Authors: Di Primo P. , Cartia G. , Katan T.
Published: March 2003

Fusarium crown and root rot, caused by Fusarium oxysporum f.sp. radicis‐lycopersici (Forl), is one of the most destructive soilborne diseases of tomato in Italy. Chlorate‐resistant, nitrate‐nonutilizing (nit) mutants were used to determine vegetative compatibility among 191 isolates of Forl collected in five geographic regions (Calabria, Emilia‐Romagna, Liguria, Sardinia, Sicily) in Italy. The isolates were assigned to five vegetative compatibility groups (VCGs): 65 isolates to VCG 0090; 99 to VCG 0091; 23 to VCG 0092; two to VCG 0093; and two to VCG 0096. The population structure of Forl in Italy is similar to that reported for Israel, and differs from that found in North Atlantic European countries, where VCG 0094 is predominant. The stability of prototrophic heterokaryons originating from hyphal anastomosis between compatible complementary nit mutants was assessed through conidial analysis and mycelial mass transfer. Most monoconidial cultures (84%) recovered from 117 prototrophic heterokaryons were nit mutants, indicating that heterokaryons generally do not proliferate well through conidiation; most of the 177 prototrophic heterokaryons examined were unstable, and only 9% sustained prototrophic growth through the tenth mycelial transfer upon subculturing. The prototrophic growth is proposed to be maintained through restoration of the heterokaryotic state by continual anastomosis between adjacent homokaryotic hyphae. Since heterokaryosis is a prerequisite for parasexual recombination, we speculate that this mechanism is unlikely to play a major role in generating the VCG diversity found among Forl or other strains of F. oxysporum.
Studies on amoebae and cysts associated with the isolation of Spongospora subterranea f.sp. subterranea in vitro
Authors: Qu X.‐S. , Kavanagh J. A. , Egan D. , Lahert H.
Published: March 2003

New evidence is presented to support the contention that the amoeba/cyst colonies isolated from surface‐sterilized Spongospora subterranea f.sp. subterranea‐infected potato tubers and spore balls have a saprophytic phase but are contaminants and not S. subterranea. Amoebae isolated from infected tissues and spore balls formed colonies associated with bacteria on 1% water agar at 18°C and encysted after 5–7 days. These cysts were morphologically distinct from the resting spores of S. subterranea and were formed singly or in a layer, unlike the spore ball (cystosorus) of S. subterranea. Amoebae, cysts and mixtures of amoebae and cysts in primary, secondary and tertiary subcultures failed to infect tomato roots. PCR amplification of DNA from amoebae, cysts and spore balls using the S. subterranea‐specific primer pair SsF/R generated a 434‐bp product from S. subterranea spore balls only and not from amoebae or cysts. When an amoeba/cyst‐specific primer pair AmF/R was designed and used for PCR amplification, a single 411‐bp product was generated from DNA of amoebae and cysts, but not from DNA of S. subterranea spore balls. These results are discussed in relation to earlier reports claiming the successful isolation of S. subterranea and other plasmodiophorids in vitro.
Differential effect of nematicide treatments on tuber yields in early‐ and late‐maturing potato cultivars
Authors: Kimpinski J. , Arsenault W. J. , Sturz A. V.
Published: March 2003

A 3‐year field study examined the effects of 1,3‐dichloropropene or oxamyl on tuber yields in four early‐ and five late‐maturing potato cultivars. The nematicide treatments increased total tuber yield by ≈ 12% for early‐maturing cultivars, but by less than 2% for the late‐maturing cultivars. The treatments reduced the numbers of root lesion nematodes (primarily Pratylenchus penetrans), which were the dominant plant parasitic nematodes at the site. Covariance analysis indicated that treatment effects on total tuber yield were no longer significant after adjusting for root lesion nematodes in soil at harvest, confirming that these nematodes had a direct effect on the host. As expected, there were significant inverse relationships (P = 0·05) between total tuber yields and numbers of root lesion nematodes in soil and in roots. However, the application of nematicides in late‐maturing cultivars was found to be of no practical value. Crop production systems designed to reduce or optimize pesticide applications can form one criterion in the development of more environmentally sensitive management strategies. The merit of utilizing chemicals for root lesion nematode control are considered with regard to cultivar maturity.
Effect of coinoculation by Verticillium dahliae and Colletotrichum coccodes on disease symptoms and fungal colonization in four potato cultivars
Authors: Tsror (Lahkim) L. , Hazanovsky M.
Published: March 2003

Interactions between Verticillium dahliae and Colletotrichum coccodes, major causal agents of potato early dying (PED) syndrome, were studied in four potato cultivars that differ in their susceptibility to these pathogens. Aseptic plantlets of Nicola, Desiree, Alpha and Cara were inoculated with identical concentrations of each pathogen or with a mixture of the pathogens, and grown for 4 weeks in a monitored growth chamber. Coinoculation of Nicola with both pathogens caused more severe foliar disease symptoms and crown rot and greater C. coccodes colonization, than inoculation with each pathogen separately. Significant reductions in weight and height were also observed in plants coinoculated with both pathogens, as compared with plants inoculated with each pathogen separately or noninoculated plants. In Desiree, more roots were covered with C. coccodes sclerotia and disease symptoms were significantly more severe in plants inoculated with both pathogens together. However, plant weight and height were similar to those of plants inoculated with C. coccodes only. In Alpha, disease symptoms and levels of sclerotia in the roots were not affected by simultaneous inoculation with both pathogens. Weight and height of all plants were similar, whether inoculated with each pathogen separately or with both pathogens together. In Cara, plants inoculated with the mixture or either pathogen alone were smaller than the noninoculated control. Disease symptoms and occurrence of sclerotia were similar in plants inoculated with the combination and with a single pathogen. Compared with the effects of inoculation with either pathogen, simultaneous inoculation with both pathogens can, in some cultivars, increase the incidence of PED syndrome and thus severely decrease yields.
Characterization of agrobacteria from weeping fig (Ficus benjamina)
Authors: Zoina A. , Raio A. , Peluso R. , Spasiano A.
Published: March 2003

Ficus benjamina plants, galled both at epi‐ and hypogeous parts, were observed in Italy and in The Netherlands, and these were the first records of the appearance of weeping fig crown gall in Europe. A total of 241 Agrobacterium isolates was obtained from 41 tumours and studied for their morphological, physiological and phytopathological characters. Two main groups of agrobacteria were distinguished by their colony morphology and through classical biovarietal tests that allowed allocation of 86 isolates into biovar 1 and 155 into an intermediate biovar rather different from any of the three biovars defined for agrobacteria. Most of the isolates were unable to utilize mannopine, nopaline or octopine as C and N sources; only 62 strains utilized nopaline. However, when nonopine‐utilizing strains were inoculated into F. benjamina, only nopaline was detected in the developing tumours. BIOLOG ML 1™ system analysis applied to 50 representative strains allowed identification of the biovar 1 isolates as Agrobacterium tumefaciens and most of the intermediate biovar isolates as the newly proposed species Agrobacterium fici. Analysis of sensitivity to a set of 14 antibiotics confirmed the allocation of the 50 strains into two well defined main clusters matching the BIOLOG identification. Out of 141 tumorigenic isolates, 66 were sensitive in vitro to agrocine 84, but four of these strains showed scarce or no sensitivity to the antagonist A. radiobacter K84 when tested in fig plants. The two types of agrobacteria could usually be isolated from the same tumours. Tumorigenic strains were able to induce tumours in six herbaceous plant species, in eight to 10 out of 12 woody plants and in six to eight out of nine Ficus species, indicating a wide host‐range Ti plasmid. Agrobacteria were able to survive and move in the vascular system of galled ficus plants and to induce tumour growth in stem‐cutting propagated plants. Moreover, agrobacteria were detected in many healthy F. benjamina plants as part of the endophytic microflora. These findings suggest potential for spread of the disease through latently infected plant propagation material produced as cuttings or by tissue culture.
The possibility of factors other than phytoalexin accumulation preventing fungal growth in the incompatible interactions of soybean with Phytophthora megasperma f. sp. glycinea
Authors: Abe K. , Fujikawa E. , Takeuchi Y. , Takada Y. , Yamaoka N.
Published: March 2003

Soybean (Glycine max) cv. Harosoy 63 is resistant to race 1 and susceptible to race 9 of Phytophthora megasperma f. sp. glycinea (Pmg). In detached primary leaves inoculated with zoospores, growth of race 1 was completely suppressed 16 h after inoculation, while race 9 was unaffected. The amount of the phytoalexin glyceollin that accumulated, however, was not significantly different in either the incompatible or compatible interaction 16 h after inoculation. At the circumference of the inoculated area, a slight accumulation of phytoalexin was observed only in the incompatible interaction 20 h or more after inoculation. Tolerance of race 9 to the phytoalexin was significantly higher than that of race 1 when the phytoalexin was added to agar. Moreover, race 9 degraded glyceollin faster than race 1. On leaves inoculated at separate points with either race, the lesion associated with race 9 never colonized areas inoculated with race 1. These results suggest that factor(s) other than the accumulation of phytoalexin in soybean tissue might cause cessation of growth of Pmg.
Antifungal vapour‐phase activity of allyl‐isothiocyanate against Penicillium expansum on pears
Authors: Mari M. , Leoni O. , Iori R. , Cembali T.
Published: March 2003

The fungitoxicity of allyl‐isothiocyanate (AITC) vapour against Penicillium expansum, the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L−1 AITC‐enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 103 conidia mL−1 to almost 80% at 1 × 106 conidia mL−1. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole‐resistant strain of P. expansum, reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum.
Identification of cold‐tolerant Pseudomonas viridiflava and P. marginalis causing severe carrot postharvest bacterial soft rot during refrigerated export from New Zealand
Authors: Godfrey S. A. C. , Marshall J. W.
Published: March 2003

In 1999, New Zealand carrots (Daucus carota) exported to the Middle East incurred substantial damage due to bacterial soft rot that resulted in major financial loss to farmers. A modified carrot tissue bioassay was developed under standard conditions to provide an economical and rapid means of monitoring export carrots for bacteria able to cause severe bacterial soft rot. Using this bioassay, two bacterial isolates causing severe degradation were detected and subsequently identified as Pseudomonas viridiflava (designated NZCX09) and P. marginalis (NZCX27), using the Biolog system and 16S rRNA phylogenetic analysis. Investigation of disease epidemiology of NZCX09 and NZCX27 at low temperatures showed that tissue degradation occurred at temperatures approaching 0°C. These findings emphasize the importance of postharvest sanitization, and the efficacy of refrigeration methods used in controlling soft rots in carrots stored over time.
Prepenetration stages in infection of clematis by Phoma clematidina
Authors: Van De Graaf P. , Joseph M. E. , Chartier‐Hollis J. M. , O’Neill T. M.
Published: March 2003

A detailed study of conidial germination, germ‐tube growth and the formation of infection structures in Phoma clematidina, the causal agent of clematis wilt, is described for two clematis varieties differing in disease resistance. On both the resistant and susceptible varieties, the fungus entered leaves and stems by direct penetration of the cuticle, often, but not always, following the formation of infection structures. More germ tubes per conidium were formed on the susceptible host, but these germ tubes were on average shorter than on the resistant host. Although germ tubes regularly entered the plant via trichomes, stomata were not found to be sites of entry. Following penetration of the cuticle of resistant plants, germ‐tube growth was sometimes restricted to the subcuticular region, and halo formation occurred at the sites where penetration was attempted. Subcuticular growth and halo formation were not observed on susceptible plants. These observations may partly explain the resistance of small‐flowered clematis varieties to P. clematidina.
Evaluation of resistance to Phytophthora cinnamomi in seed‐grown trees and clonal lines of Eucalyptus marginata inoculated in lateral branches and roots
Authors: Hüberli D. , Tommerup I. C. , Colquhoun I. J. , Hardy G. E. St J.
Published: March 2003

Seed‐grown trees and six clonal lines of 3·5–4·5‐year‐old Eucalyptus marginata (jarrah) growing in a rehabilitated bauxite mine site in the jarrah forest were underbark‐inoculated on lateral branches (1995) or simultaneously on lateral branches and lateral roots (1996) with isolates of Phytophthora cinnamomi in late autumn. Individual seedlings from which the clonal lines were derived had previously been assessed as either resistant (RR) or susceptible (SS) to P. cinnamomi. At harvest, the acropetal lesion and colonization lengths were measured. Overall, the length of colonization in roots and branches was more consistent as a measure of resistance than lesion length, because colonization length recorded the recovery of P. cinnamomi from macroscopically symptomless tissue ahead of the lesion which, on some occasions, was up to 6 cm. In both trials, one RR clonal line was able to contain the P. cinnamomi isolates consistently, as determined by small lesion and colonization lengths in branches and roots. In contrast, the remaining two RR clonal lines used in both trials were no different from the SS line in their ability to contain lesions or colonization. These latter two RR lines may therefore not be suitable for use in rehabilitation of P. cinnamomi‐infested areas. Differences in lesion and colonization lengths among P. cinnamomi isolates occurred only in the 1995 trial. Colonization and lesion lengths in branches were up to eight times greater in 1996 than in 1995, but the relative rankings of clonal lines were consistent between trials. Although colonization was always greater in branches than roots, the relative rankings of the lines were similar between branch and root inoculations. Branch inoculations are a valid option for testing the resistance and susceptibility of young jarrah trees to P. cinnamomi.
Quantitative relationships between inoculum of Melampsora larici‐epitea and corresponding disease on Salix
Authors: Pei M. H. , Ruiz C. , Hunter T. , Arnold G. M. , Bayon C.
Published: March 2003

Six Salix clones were inoculated with urediniospores of four isolates of Melampsora larici‐epitea at five inoculum levels using a leaf‐disc method. Disease reactions were recorded using a digital camera; the number and size of uredinia were examined using image analysis software; and spore yield per leaf disc was measured. In three Salix/Melampsora combinations, S. × mollissima‘Q83’/Q1 (LET4); S. viminalis‘78183’/V1 (LET1); and S. × stipularis/V1, pustule numbers increased as inoculum density became higher. In the remainder, S. viminalis‘Mullatin’/V1; S. × calodendron/DB (LET3); and S. burjatica‘Korso’/K (LR1), pustule numbers initially increased, then decreased as inoculum densities exceeded 140–360 spores per disc. Calculated infection efficiency ranged from 0·11 to 0·20 on the three willows inoculated with V1: 0·16–0·68 for S. × calodendron/DB; 0·20–0·55 for ‘Q83’/Q1; and 0·07–0·48 for Korso/K. In single‐spore inoculations, up to 10% of spores produced single uredinia. Infection efficiency increased sharply between inoculum densities of 1–40 spores per leaf disc. Spore yield was more closely correlated to pustule area (accounting for 61·2% variance for the combined data) than to the number of pustules (42·7% variance). For spore yields in relation to pustule numbers, clone‐specific individual lines having different intercepts and slopes fitted significantly better than either a single line for all the tested willows, or parallel lines fitted to each clone (P < 0·001). For spore yields in relation to pustule area, clone‐specific individual parallel lines were significantly better than a single line (P < 0·001).
The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34+ Bone Marrow and Peripheral Blood Stem Cells
Authors: Croockewit  , Raymakers  , Preijers  , Vierwinden  , De Witte
Published: March 2003

Positive selection of CD34+ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34+ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that ± 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti‐CD34, 561, was compared with the class I epitope anti‐CD34, BI‐3C5, both coupled to M450 Dynabeads. The yield of CD34+ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95‐fold increase (1.3–2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34+ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti‐class III CD34 epitope are more efficient in isolating CD34+ cells from bone marrow, probably due to a broader distribution of the class III epitope.
Susceptibility and Resistance to Leishmania amazonensis in H‐2q Syngeneic High and Low Antibody Responder Mice (Biozzi Mice)
Authors: Lima  , Puel  , Decreusefond  , Bouthillier  , Mevel  , Abrahamsohn  , Mouton
Published: March 2003

H‐2 syngeneic H and L (Biozzi) mice provide a model to study Leishmania infections in which polar resistant and susceptible phenotypes are independent from H‐2 differences. High‐Ab‐responder (H) and low‐Ab‐responder (L) mice syngeneic at the H‐2 locus (H‐2q) were, respectively, susceptible and highly resistant to Leishmania amazonensis infection. L‐mice resistance was associated with high IFN‐γ and transient IL‐4 production by lymph node (LN) cells, in contrast with sustained IL‐4 and decreasing IFN‐γ production by susceptible H mice. IL‐12 production could be detected only in LN from resistant mice. The cytokine production pattern was consistent with preferential progression to a Th1‐type response in resistant L‐mice, and to a Th2‐type response in susceptible H‐mice. We also investigated whether this shift towards Th1‐ or Th2‐type cytokine responses was dependent upon H or L antigen presenting cells' (APC) intrinsic ability to preferentially stimulate either T‐cell subset. To this end, LN‐derived T‐cell lines were grown from 12‐day infected mice, when both strains produced IFN‐γ and IL‐4. L‐derived T‐cell lines developed a Th2 cytokine pattern whereas H‐derived T‐cell lines produced IFN‐γ, IL‐4 and IL‐10 whatever the APC origin (H or L) used for their derivation. This work constitutes the first characterization of cellular immune responses to the intracellular parasite, L. amazonensis in H‐2 syngeneic mice, an infection model in which polar resistant and susceptible phenotypes are determined by non‐MHC genes.
Identification of a Gliadin T‐Cell Epitope in Coeliac Disease: General Importance of Gliadin Deamidation for Intestinal T‐Cell Recognition
Authors: Sjöström  , Lundin  , Molberg  , Körner  , Mcadam  , Anthonsen  , Quarsten  , Norén  , Roepstorff  , Thorsby  , Sollid
Published: March 2003

Coeliac disease probably results from a T‐cell response to wheat gliadin and is associated to HLA‐DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut‐lesion‐derived DQ2‐restricted T cells from coeliac disease patients were used to identify an epitope within a purified γ‐type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T‐cell recognition. Other gut‐derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut‐derived DQ2‐restricted T cells isolated from several patients. Several DQ2‐restricted T‐cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.
Apoptotic Cells Actively Inhibit the Expression of CD69 on Con A Activated T Lymphocytes
Authors: Sun  , Zhang  , Zeng  , Ge  , Zhao  , Gao
Published: March 2003

Although apoptosis is commonly viewed as a silent cell death without damage to adjacent tissues, the effect of apoptosis on immunity has been unclear. We have investigated the influence of apoptotic cells on T‐cell activation. The K562 or HL‐60 human leukemia cell lines that had been induced apoptosis by FTY720 or cycloheximide (CHX) were added into the culture of mouse spleen cells stimulated with Con A. Six to 20 h later, the expression of CD69, an early T‐cell activation antigen, was detected using flowcytometry. Living cells and necrotic cells served as control groups. Apoptotic K562 or HL‐60 cells induced by either FTY720 or CHX unanimously inhibited CD69 expression on the CD3+ mouse T cells while living and necrotic cells did not. The inhibition was proportional to the number of apoptotic cells and was different in the T‐cell subsets, showing a rapid and transient inhibition on the CD3+CD8+ T‐cell activation but with a slow and continuous inhibition on CD3+CD8− T‐cell activation. In conclusion, the apoptotic cells actively inhibit a T‐cell activation that is independent of the cell lines or the apoptotic inducers, indicating that the apoptotic cells dominantly regulate T‐cell immunity.
Differences in Lymphocyte Subsets in the Wall of High Endothelial Venules and the Lymphatics of Human Palatine Tonsils
Authors: Zidan  , Jecker  , Pabst
Published: March 2003

Lymphocyte trafficking plays a critical role in disseminating specifically primed lymphocytes all over the body. Most concepts on the interaction of adhesion molecules on lymphocyte subsets and specialized endothelia such as those in high endothelial venules (HEV) are based on animal experiments as kinetic studies cannot be performed in humans. We therefore characterized lymphocyte subsets in the wall of HEV and in the lumen of lymphatics of 18 human palatine tonsils by immunohistology. All subsets studied were found in the wall of HEV (% of lymphocytes): 32% CD20+, 50% CD3+, 14% CD4+, 32% CD8+ and also 21% CD45RA+ and 39% CD45RO+. In the lymphatics, used to indicate lymphocytes emigrating from the tonsils, a different composition was found; e.g. many more T cells and three times more CD45RA+ than RO+ lymphocytes. Thus, HEV are not a selective entry site nor lymphatics an exit for specific lymphocyte subsets only, at least in these tonsils with chronic stimulation.
Immune Complex‐Induced Chronic and Intense IL‐4 Independent IgG1‐Rheumatoid Factor Production in NZB Mice
Authors: Nordström E. , Abedi‐Valugerdi M. , Möller E.
Published: March 2003

Immune complexes from the synovial fluid of rheumatoid arthritis (RA) patients, or artificial rheumatoid factors (RF)‐like (antibody–antibody) immune complexes, induce a remarkably intense, sustained and selective immunoglobulin (Ig)G1 response under certain experimental conditions in mice. Because the IgG1 antibody response is extraordinarily strong, the role of interleukin (IL)‐4, important for IgG1 synthesis, was investigated. Both C57BL/6 and NZB IL‐4‐deficient mice produced IgG1‐RF antibodies after injection with RF‐like immune complexes, although the antibody levels were slightly delayed compared to wild type mice. This shows that IL‐4 is not obligatory in RF‐like immune complex induced IgG1‐RF production. A discrepancy in the decline of serum IgG1–RF was noted between NZB and C57Bl/6 mice. Serum IgG1–RF declined 43 days postinjection (p.i.), in C57BL/6 mice whereas high serum levels of IgG1–RF were maintained more than 100 days in the NZB mice, indicating different regulatory mechanisms in these mice. To study if the affinity for mouse IgG increased with time in NZB mice and thus become more directed against self, the cross‐reactivity of the IgG1–RF antibodies with IgG from other species was investigated early and late after injection. It was, however, found that the cross‐reactivity with IgG of human, goat and rabbit origin did not change between the two time points.
Cytokine Production in Arthritis Susceptible and Resistant Rats: A Study with Arthritogenic and Non‐arthritogenic Lactobacillus Cell Walls
Authors: Šimelyte E. , Isomäki P. , Rimpiläinen M. , Zhang X. , Toivanen P.
Published: March 2003

The basis of the different susceptibility to bacterial cell wall‐induced arthritis between Lewis and Fischer rats is unclear. Likewise, it is not known why cell walls of some species of Lactobacillus are arthritogenic and those of others are not. With these two questions in mind, we investigated the role of anti‐inflammatory (interleukin (IL)‐10, IL‐4) and proinflammatory (tumour necrosis factor (TNF)‐α, IL‐1β) cytokines in Lewis and Fischer rats injected intraperitoneally with cell walls from arthritogenic or nonarthritogenic species of Lactobacillus. Cytokine levels in the serum and in vitro production by peritoneal macrophages and splenocytes were studied. The results obtained indicate that the differences in the production of IL‐10, IL‐4, TNF‐α or IL‐1β do not explain the difference in the arthritis susceptibility between Lewis and Fischer rats. Likewise, the arthritogenicity of different Lactobacillus cell walls appears not to be dependent on their capacity to stimulate cytokine production.
Elevated Antibody Responses in Patients with Crohn's Disease against a 14‐kDa Secreted Protein Purified from Mycobacterium avium subsp. paratuberculosis
Authors: Olsen I. , Wiker H. G. , Johnson E. , Langeggen H. , Reitan L. J.
Published: March 2003

Patients with Crohn's disease (CD) (n = 10) and ulcerative colitis (UC) (n = 10) were tested for immune responses against various antigens from Mycobacterium avium subsp. paratuberculosis; alkyl hydroperoxide reductase C (AhpC) and alkyl hydroperoxide reductase D (AhpD), which are constitutively expressed in this species as opposed to other mycobacteria, a 14‐kDa secreted antigen and PPD‐J. The CD patients had significantly elevated antibody levels against the 14 kDa protein (P < 0.05) that were negatively correlated with the duration of the disease (rs = − 0.85). They also seemed to have increased antibody levels against AhpC and AhpD, but the differences between the two groups were not significant. However, taken together, the antibody responses to three individual mycobacterial antigens in CD patients strengthen the possibility that the observed responses are caused by mycobacterial infection. No significant differences in the interferon (IFN)‐γ production, the interleukin (IL)‐10 production and the ability to proliferate upon stimulation with these antigens were observed. These results show that measuring antibody responses against purified specific antigens is a suitable and simple approach when assessing the connection between CD and mycobacteria in patients with clinical CD. Another important aspect in such studies is to have well defined patient groups tested at the onset of clinical symptoms.
Anti‐Idiotype Antibodies Neutralize In vivo the Blistering Effect of Pemphigus foliaceus IgG
Authors: Alvarado‐Flores E. , Avalos‐Díaz E. , Díaz L. A. , Herrera‐Esparza R.
Published: March 2003

Idiotypes are molecular clues used to explore the specificity and diversity of immune response. In the present study, anti‐idiotype antibodies were used to neutralize the pathogenic effects induced by the injection of pemphigus immunoglobulin(Ig)G into BALB/c mice. To achieve our goal, antidesmoglein 1 IgG was obtained from a patient with pemphigus foliaceus with high titer of antiepithelial antibodies. The IgG was isolated by ion exchange chromatography, then digested by pepsin. F(ab′)2 fragments were purified in Sephacryl S‐300 and injected in rabbits to produce anti‐idiotype antibodies. The rabbit sera reacted with the pemphigus F(ab′)2 fragments. Eleven pemphigus foliaceus sera were recognized by the anti‐idiotype serum at the light or heavy chains whereas bullous pemphigoid and normal IgG were negative. Neonatal BALB/c mice injected with pemphigus IgG developed intraepidermal blisters, mimicking the clinical and immunopathological features of the pemphigus. In contrast, the animals treated with anti‐idiotype antibodies and pemphigus IgG did not develop blisters. Thus, anti‐idiotype antibodies neutralize in vivo the pathogenic effects of pemphigus IgG.
Differences in the Reactivity of CD4+ T‐Cell Lines Generated against Free Versus Nucleosome‐Bound SV40 Large T Antigen
Authors: Bredholt G. , Rekvig O.P. , Andreassen K. , Moens U. , Marion T.N.
Published: March 2003

Previous results have revealed a strong correlation between polyomavirus BK reactivation and disease activity and antinuclear auto‐antibody production in the human autoimmune disease systemic lupus erythematosus. BK virus establishes a latent infection in most humans, and reactivation requires the production of the DNA‐binding large T antigen. Experimentally induced expression of the polyomavirus SV40 large T antigen in mice induces both an immune response to large T antigen and autoimmune response to nuclear antigens and antinuclear antibody production. Previous results have indicated that human T‐antigen‐specific CD4+ T‐cell lines are stimulated equally by free, soluble and nucleosome‐bound T antigen. This study was designed to determine how antigen processing of nucleosomes containing bound SV40 large T antigen may affect the specificity and response characteristics of experimentally induced T‐antigen‐specific CD4+ T cells. The results indicated that CD4+ T‐cell lines generated from mice immunized with soluble, free T antigen responded very poorly in response to stimulation with T antigen bound to nucleosomes. CD4+ T‐cell lines generated from mice immunized with nucleosomes that had bound T antigen in situ responded to both free and nucleosome‐bound T antigen. The T‐antigen‐specific, CD4+ memory T cells induced by latent polyomavirus infections in humans may be uniquely suited to initiate autoimmunity to nuclear antigens upon virus reactivation.
An Investigation into the Heparin‐Binding Properties of a Synthetic Peptide Deduced from the Antigenic Domain 2 of Human Cytomegalovirus Glycoprotein B
Authors: Silvestri M. E. , Sundqvist V.‐A.
Published: March 2003

An investigation was performed into the heparin‐binding properties of a synthetic peptide deduced from the sequence of human cytomegalovirus glycoprotein B. The peptide, T7‐13:3, amino acids 69–78, which was previously shown to contain a neutralization epitope was able to bind heparin coated onto microtitre plates as well as immobilized on agarose beads. Conversely, labelled heparin could be used to specifically detect the immobilized peptide. The peptide bound to human cells in a manner which suggested an interaction with extracellular matrix, and binding of the peptide to human fibroblasts could be inhibited both by adding soluble heparin and by enzymatic pretreatment of the cells with heparinase. The sequence of T7‐13:3 shows similarity to several proteins with known or supposed ability to bind heparin, e.g. basic fibroblast growth factor, the heparin‐binding capacity of which could also be inhibited by T7‐13:3. The peptide was also found to bind DNA, probably due to the similarities between DNA and heparin in terms of structure and charge. Because heparin is a chemical homologue of heparan sulfate, the results strongly indicate that the sequence represented by T7‐13:3 is involved in the binding of virus to cell surface heparan sulfate. The described region of gB may have the potential to contribute to a subunit vaccine although possible hazards, such as the induction of auto‐antibodies to heparin, and thus also to DNA, need to be considered.
Identification and Design of p53‐Derived HLA‐A2‐Binding Peptides with Increased CTL Immunogenicity*
Authors: Petersen T. R. , Buus S. , Brunak S. , Nissen M. H. , Sherman L. A. , Claesson M. H.
Published: March 2003

The replacement of a suboptimal amino acid in a primary anchor position with an optimal residue improves human leucocyte antigen (HLA) binding and immunogenicity, while maintaining cytotoxic T lymphocyte (CTL) specificity. Using a neural network capable of performing quantitative predictions of peptide binding to HLA‐A2 molecules, we identified three p53 protein‐derived nonamer peptides with intermediate binding owing to suboptimal amino acids in the P2 anchor position. These peptides were synthesized along with the corresponding analogs, where the natural P2 residue had been replaced with the optimal leucine residue. All three modified peptides bound to and more efficiently stabilized HLA‐A2 molecules than the corresponding nonmodified peptides. The HLA‐A2 transgenic mice were used for immunization. Two of the epitopes were more immunogenic in their modified than in their natural versions. The CTLs raised against the modified peptides efficiently killed the target cells pulsed with the corresponding native peptide. In terms of sensitizing the targets cells for the CTL killing, the modified peptides were more efficient than native peptides. Finally, the CTLs induced by modified peptide killed HLA‐A2 transgenic mouse fibrosarcoma cells transfected with human p53 DNA. The data suggest that modified self‐peptides derived from overexpressed tumour‐associated proteins can be used in vaccine development against cancer, and that quantitative predictions of HLA binding is of value in the rational selection and improvement of target epitopes recognized by CTLs.
Reply: Some Afterthoughts – Pragmatic and Philosophical
Authors: Sinclair N. R. StC.
Published: March 2003

Conditions under which one searches for Fc‐signalling determine the success in finding it. A major condition is that one must have enough T cells to drive a T‐cell‐dependent response, but not enough T‐cell activity to obscure the Fc‐signalling. Antigen‐masking and destructive phagocytosis of antigen occur. Under conditions and in areas of the body where T‐cell activities are limiting and Fc‐signalling can not be blocked, Fc‐signalling may be observed. The balance between costimulation and coinhibition is a major determinant in immune responsiveness, because costimulation and coinhibition interfere with each other.
Diagnostic and Biological Significance of Anti‐p41 IgM Antibodies against Borrelia burgdorferi
Authors: Ulvestad E. , Kanestrøm A. , Sønsteby L. J. , Jureen R. , Omland T. , Edvardsen B. , Lundervik J. , Kristoffersen E. , Van Dam A. P.
Published: March 2003

We performed a prospective study to investigate the biological significance and diagnostic specificity of anti‐p41 immunoglobulin (Ig)M antibodies against Borrelia burgdorferi. During a 1‐year interval 2403 patients were referred to our department for B. burgdorferi serology. Sixty‐three patients had repetitive positive tests for IgM anti‐p41 antibodies and negative tests for anti‐p41 IgG antibodies. Ten of the 63 patients recently had symptoms of erythema migrans. A confirmatory IgM Western blot gave a positive reaction in 5 patients out of 53 patients with little or no clinical evidence of B. burgdorferi infection. The remaining 48 patients were negative in this test and were considered as false‐positives. Two whole cell enzyme‐linked immunosorbent assay (ELISAs), two immunofluorescence assays and Western blotting were not useful as confirmatory tests. Sera from 330 blood donors and 72 cord sera were also screened for anti‐p41 IgM. Five blood donor sera and five cord sera showed an IgM reactivity against p41. Based on our data we hypothesize that up to 1.5% of the population may have natural IgM antibodies against p41 in their sera. We observed that six out of nine sera with such antibodies could immobilize a B. afzelii reference strain in vitro. Whether anti‐p41 IgM antibodies are capable of inactivating infective spirochetes and thereby prevent infection in vivo is, however, not yet clarified. The paradoxical conclusion that anti‐p41 IgM antibodies may be a sign of resistance to infection rather than a sign of infection should be given consideration.
Recent Thymic Emigrants (CD4+) Continuously Migrate Through Lymphoid Organs: within the Tissue They Alter Surface Molecule Expression
Authors: Luettig B. , Sponholz A. , Heerwagen C. , Bode U. , Westermann J.
Published: March 2003

T‐cell progenitors migrate from bone marrow (BM) into the thymus. After maturation they are released as recent thymic emigrants (RTE) into the periphery ensuring the diversification of the T‐cell repertoire. Both the kinetics with which RTE migrate through the periphery and the surface molecules they express are still unclear. In 1‐ and 18‐month‐old Lewis rats CD4+ RTE were identified in blood, spleen, lymph node, and thoracic duct lymph by flow cytometry (CD45RC− and CD90+), were differentiated from CD4+ naive (CD45RC+) and memory T cells (CD45RC−CD90−), and were characterized regarding the expression of surface molecules. Both in 1‐ and 18‐month‐old animals the percentage of RTE among the CD4+ population in blood was comparable to that in all other compartments. Surprisingly, RTE expressed α4‐integrin, LFA‐1, and interleukin (IL)‐2 receptor at a significantly higher level than naive T cells and more comparable to memory T cells. Within lymphoid tissues RTE, naive, and memory T cells significantly upregulated the expression of CD44 and ICAM‐1, and downregulated the expression of l‐selectin. These changes were reversed before the cells re‐entered the blood. Thus, our data indicate that CD4+ RTE travel through the periphery of young and old rats like mature T cells, continuously modulating their surface molecule expression.