bluuebunny/crossref_metadata_2025 huggingface dataset:
Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees
Authors: Rhim  , Völksch  , Gardan  , Paulin  , Langlotz  , Kim  , Geider
Published: March 2003

Bacteria from necrotic branches of Asian pear trees (Pyrus pyrifolia) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear (Pyrus communis) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia, but were different from Erwinia amylovora, the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2‐D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora. No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP‐PCR also produced bands differing for the two organisms.
Analysis of the Italian population of Ceratocystis fimbriata f.sp. platani using RAPD and minisatellite markers
Authors: Santini A. , Capretti P.
Published: March 2003

To test the genetic variability of the Italian population of Ceratocystis fimbriata f.sp. platani, a large sample of different isolates was analysed by the RAPD and direct amplification of minisatellite‐region DNA (DAMD) PCR techniques and compared with a number of Ceratocystis fimbriata isolates from various hosts growing in different parts of the world. The results indicated a high level of homogeneity in the Italian population of the fungus, whereas a certain variability was recognized in isolates from other hosts. The Italian population appears to be clonal, with the same genotype throughout the country. The pathogen was certainly introduced from abroad, so it is conceivable that it does not have the genetic variability in Italy that it had in its area of origin.
Effects of growing leafy daikon (Raphanus sativus) on populations of Plasmodiophora brassicae (clubroot)
Authors: Murakami H. , Tsushima S. , Akimoto T. , Murakami K. , Goto I. , Shishido Y.
Published: March 2003

Control of some soilborne pathogens may be achieved by use of decoy or catch crops. These stimulate the germination of resting spores, resulting in limited expression of disease symptoms. Results achieved using this approach are reported here using leafy daikon (radish, Raphanus sativus var. longipinnatus) for control of Plasmodiophora brassicae, the cause of clubroot disease of Brassicaceae. Disease indices of Chinese cabbage plants grown in pots that had previously contained leafy daikon were lower compared with pots where no plants had been grown before (control pots). Numbers of resting spores of P. brassicae in soil in pots after cultivation with leafy daikon were reduced by 71% compared with control pots when resting spores were recovered and counted directly. In a field experiment, numbers of resting spores were reduced by 94% compared with the start of the experiment when leafy daikon was grown in advance of Chinese cabbage, but there was no reduction in disease severity in the Chinese cabbage. Plasmodiophora brassicae infected the root hairs of leafy daikon and those of Chinese cabbage, but no clubs were found on leafy daikon roots. The results from pot trials indicate that leafy daikon may be useful as a decoy crop for the control of clubroot disease in field crops.
Diversity of phytoplasmas in northern Australian sugarcane and other grasses
Authors: Tran‐Nguyen L. , Blanche K. R. , Egan B. , Gibb K. S.
Published: March 2003

Plant pathogenic phytoplasmas found in grasses in northern Australia have the potential to adversely affect sugarcane production. To begin assessment of this threat, the number of grass species with phytoplasmas, the identity of those phytoplasmas, and their relationship with disease symptoms were determined. Sugarcane with and without symptoms of yellow leaf syndrome was included in the surveys. Molecular methods were used to detect and characterize phytoplasmas in grass species exhibiting symptoms typical of phytoplasma disease. Sugarcane samples were from the Ord River Irrigation Area, Western Australia, and Samford, Queensland. Samples of other grasses were from Wyndham, Kununurra and Broome, Western Australia, and Darwin, Northern Territory. Our survey identified four new phytoplasma host species and confirmed four known previously. Counting phytoplasmas, phytoplasma variants, and mixtures of phytoplasmas and variants, these eight host species had 33 different infections. Two phytoplasmas were new, cenchrus bunchy shoot which is related to Candidatus phytoplasma australiense, and sorghum bunchy shoot which is not closely related to any described phytoplasma. Twenty‐five phytoplasma isolates were detected in sugarcane. Of these, tomato big bud phytoplasma was the most common. In most cases no clear association between phytoplasmas and symptoms could be determined. None of the phytoplasmas in Australian sugarcane, but two in other grasses, were closely related to phytoplasmas associated with white leaf and grassy shoot diseases in Asian sugarcane. This study demonstrates that diversity of phytoplasmas and grass host species in northern Australia is greater than previously thought, and that symptoms alone are not always reliable indicators of phytoplasma presence or absence. It provides the groundwork to improve future field surveys, and for initiation of transmission trials to determine whether insect vectors capable of transmitting phytoplasmas from native grasses to sugarcane are present in the region.
Recovery and pathogenicity of Phytophthora species associated with a resurgence of ink disease in Castanea sativa in Italy
Authors: Vettraino A. M. , Natili G. , Anselmi N. , Vannini A.
Published: March 2003

Three species of Phytophthora, P. cambivora, P. citricola and P. cactorum, were found to be associated with a recent outbreak of ink disease causing high mortality of chestnut trees in central Italy. Phytophthora cambivora was isolated from 11·6% of the soil samples taken around symptomatic trees, and was mainly associated with heavily diseased trees. It was the most aggressive species to Castanea sativa, but survived poorly in the soil. Phytophthora citricola and P. cactorum showed a limited ability to induce disease on chestnut, but could be recovered from soil during most of the year. A fourth species, P. gonapodyides, was recovered only from mud of stream beds within the chestnut stands. Involvement of these species in the development of disease is discussed.
Fungicidal effects of azoxystrobin and epoxiconazole on phyllosphere fungi, senescence and yield of winter wheat
Authors: Bertelsen J. R. , De Neergaard E. , Smedegaard‐Petersen V.
Published: March 2003

The effects of the fungicides azoxystrobin (a strobilurin) and epoxiconazole (a sterol biosynthesis inhibitor) on phyllosphere fungi, senescence and yield were studied in winter wheat in field trials free of visible disease and under controlled environmental conditions. In two field trials, treatments with each of the two fungicides prolonged green leaf area retention and increased yield compared with untreated control plots. Azoxystrobin maintained green leaf area for longer than epoxiconazole and, in one trial, treatments with azoxystrobin gave a greater yield response than epoxiconazole. Mycelial growth on leaf surfaces, mainly originating from saprophytic fungi, was reduced by each of the fungicides. Papilla formation and hypersensitive reactions, almost exclusively against infection attempts by Mycosphaerella spp. (most probably M. graminicola), occurred with high frequency in the leaves. These defence reactions presumably incurred a significant energy cost, accelerating plant senescence. Fewer defence reactions were recorded in azoxystrobin‐treated leaves than in epoxiconazole‐treated and untreated leaves. Inoculation in a glasshouse experiment with the saprophytic fungi Alternaria alternata and Cladosporium macrocarpum accelerated wheat senescence. Control of the saprophytes by azoxystrobin or epoxiconazole treatments caused a delay in the accelerated senescence, but without significant increase in above‐ground biomass and yield. Neither fungicide influenced senescence, above‐ground biomass or yield in noninoculated wheat plants. In growth chamber experiments azoxystrobin inhibited spore germination and mycelial growth of A. alternata and C. macrocarpum. Epoxiconazole had little inhibitory effect on spore germination, but strongly inhibited mycelial growth of both saprophytes. Both fungicides reduced A. alternata‐induced papilla formation in wheat leaves, with epoxiconazole being more effective. Inoculation with either of the two saprophytes did not significantly increase wheat leaf respiration, in contrast to inoculation with the nonhost pathogen Erysiphe graminis f.sp. hordei. Treatment with azoxystrobin did not affect this latter increase in respiration whereas it was reduced by epoxiconazole treatment. It is proposed that the greater inhibition of infection attempts from Mycosphaerella spp. by azoxystrobin, compared with epoxiconazole, may account for the greater yield given by azoxystrobin in field plots.
Support for a stepwise mutation model for pathogen evolution in Australasian Puccinia striiformis f.sp. tritici by use of molecular markers
Authors: Steele K. A. , Humphreys E. , Wellings C. R. , Dickinson M. J.
Published: March 2003

Since its initial detection in Australia in 1979, wheat yellow (stripe) rust (Puccinia striiformis f.sp. tritici) has evolved in Australia and New Zealand into more than 20 pathotypes with assorted virulence characteristics. This evolution is believed to have occurred in a stepwise fashion from an original single pathotype, with no subsequent new introductions. A combination of random amplified polymorphic DNA (RAPDs) and amplified fragment length polymorphisms (AFLPs) was used to examine the level of molecular variation in Australian and New Zealand isolates, and to compare this with variation amongst other isolates of P. striiformis. Using 60 RAPD primers on seven Australian isolates representing seven different pathotypes collected between 1979 and 1991, more than 300 potentially polymorphic loci were analysed and no polymorphisms were detected. Using the same primers on two UK isolates, 3% of loci showed a polymorphism. A similar level of polymorphism was found between UK isolates using AFLP primers, and between 5 and 15% of fragments were polymorphic between an isolate from the UK, an isolate from Denmark, and one from Colombia. However, no AFLP polymorphisms were found amongst 14 Australian and New Zealand isolates tested, at over 100 potentially polymorphic loci. The lack of molecular variation in the Australian and New Zealand collection is consistent with the stepwise mutation theory of pathotype evolution from a single introduction.
Association of aster yellows subgroup 16SrI‐B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea
Authors: Přibylová J. , Špak J. , Fránová J. , Petrzik K.
Published: March 2003

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra‐thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle (Catharanthus roseus). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI‐B) in naturally infected samples of R. glutinosa var. purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI‐B. This is the first report of natural occurrence of phytoplasma‐associated disease in R. glutinosa var. purpurea.
Clonality and long‐distance migration of Puccinia striiformis f.sp. tritici in north‐west Europe
Authors: Hovmøller M. S. , Justesen A. F. , Brown J. K. M.
Published: March 2003

The biotrophic fungus Puccinia striiformis f.sp. tritici, a basidiomycete that causes yellow rust on wheat, is spread by wind‐dispersed spores. Analysis of amplified fragment length polymorphism (AFLP) variation showed that the fungus frequently migrates between the UK, Germany, France and Denmark. There is no biological evidence for sexual or parasexual reproduction under natural conditions, and this was supported by the lack of recombination, as revealed by AFLP, over the time and area represented by the samples in this study. A phylogeographic analysis revealed that there was effectively a single, clonal population in the four countries, up to 1700 km apart, consistent with a ‘continent‐island’ model in which Denmark is the recipient of migrants from other countries. In five cases, specific pathogen clones were dispersed between the UK and Denmark, and on at least two recent occasions clones were also spread from the UK to Germany and France, causing outbreaks of yellow rust on wheat cultivars that were previously resistant to the disease in these countries. The agronomic consequences of migration were enhanced because of the limited genetic diversity for yellow rust resistance in wheat cultivars in the area. These results demonstrate that long‐distance migration of pathogen clones, coupled with low diversity in the host species, may cause previously useful resistance genes to become ineffective for disease control on a continental scale.
Colonization of winter oilseed rape tissues by A/Tox+and B/Tox0Leptosphaeria maculans(phoma stem canker) in France and England
Authors: West J. S. , Balesdent M.‐H. , Rouxel T. , Narcy J. P. , Huang Y.‐J. , Roux J. , Steed J. M. , Fitt B. D. L. , Schmit J.
Published: March 2003

The colonization of winter oilseed rape plants and epidemiology of phoma stem canker differed between A/Tox+and B/Tox0Leptosphaeria maculans. In France and England, where plant colonization was investigated during two and three growing seasons, respectively, there was a difference in timing of leaf infection; A/Tox+L. maculanswas predominant on leaves in the autumn (October/November) but there was an increase in the incidence of B/Tox0in the winter (January/February). In May, June and July both species could be isolated from all external parts of the plant (root to the upper stem) and all crown (stem base) tissues, although they differed in their distribution. At the root and crown, A/Tox+L. maculanswas predominant and was located throughout the cortex, wood and pith tissues, but the rarer B/Tox0was located mainly in the cortex. Approximately equal numbers of A/Tox+and B/Tox0isolates were obtained from the upper stem – there was a greater proportion of B/Tox0isolates than at the crown. In England, after harvest in 1999 and 2000, pseudothecia on the lignified tap root and crown tissues produced predominantly A/Tox+ascospores (94%), while pseudothecia higher up the stem produced more B/Tox0ascospores (60%) than A/Tox+ascospores (40%). The timing of the onset of leaf spotting, earlier in the season for A/Tox+than B/Tox0L. maculans, and the predominance of mycelium of A/Tox+at the crown are consistent with the assumption that A/Tox+is more likely to cause the most damaging stem cankers than B/Tox0L. maculans. Identification as A/Tox+or B/Tox0by cultural characteristics differed only slightly (2·3%) from identification by molecular techniques.
Selection of fungal biological control agents of Sclerotium cepivorum for control of white rot by sclerotial degradation in a UK soil
Authors: Clarkson J. P. , Payne T. , Mead A. , Whipps J. M.
Published: March 2003

A range of fungal isolates was tested in a three‐stage screening system for their ability to degrade sclerotia of Sclerotium cepivorum on agar and in soil, and to reduce white rot disease on onion seedlings. Biological control agents (BCAs) were identified that could degrade up to 60% of sclerotia in soil and significantly reduce white rot disease on onion seedlings. The efficacy of the BCAs was enhanced when applied as wheat bran cultures compared with spore suspensions, and two of the best BCAs from the screening procedures were both identified as Trichoderma viride (L4, S17A). When L4 and S17A were fluid‐drilled in guar gum with bulb onion seed in the field white rot symptoms were significantly reduced, but stem base applications applied mid‐season had little effect. The strategy of selecting and using BCAs that degrade sclerotia of S. cepivorum and integration with other control methods is discussed.
Genetic analyses of resistance of the wheat differential cultivars Carstens V and Spaldings Prolific to two races of Puccinia striiformis
Authors: Calonnec A. , Johnson R. , De Vallavieille‐Pope C.
Published: March 2003

Genetic analysis of resistance of wheat seedlings to two races of Puccinia striiformis was conducted on F1, F2 and F3 generations from crosses Carstens V (CV) × Lee, Spaldings Prolific (SPA) × Lee and CV × SPA. F2 generations from crosses of CV and SPA with Strubes Dickkopf (SD) were also studied. The plants were classified into six resistance classes and analysed by factorial correspondence analysis and nonhierarchical classification. The two P. striiformis isolates tested were a French isolate of race 43E138 and a Lebanese isolate of race 2E16, selected for the differences in their virulence spectra for the common differential cultivars Strubes Dickkopf and Nord Desprez. Resistance of CV and SPA was recessive and dominant to races 43E138 and 2E16, respectively. CV possessed three or four resistance genes, one of them being expressed with both races. Two genes of CV had a cumulative effect for resistance to 43E138 and two or three gave dominant resistance to 2E16. SPA had three resistance genes, all of which gave resistance to 2E16 and two of which also gave resistance to 43E138. SPA had one gene in common with CV for resistance to both races. Furthermore, the gene for resistance to race 2E16 in CV and SPA was allelic with a gene in SD, and was probably Yr25.
Mice Deficient for the Complement Factor B Develop and Reproduce Normally
Authors: Pekna  , Hietala  , Landin  , Nilsson  , Lagerberg  , Betsholtz  , Pekny
Published: March 2003

Factor B is an essential component of the complement cascade which forms the C3 and C5 convertase of the alternative pathway. Factor B cleavage products also function as cofactors in antibody‐independent monocyte‐mediated cytotoxicity, macrophage spreading, plasminogen activation and proliferation of B lymphocytes. Several healthy kindreds heterozygous for the factor B null or non‐functional allele have been reported but the absence of homozygous factor B deficiency in humans or in animals has been speculated to be caused by the lethality of the phenotype. Here we report the generation of factor B‐deficient mice by gene targeting in vivo. These mice were born at the expected Mendelian ratio and they both develop and breed normally in a conventional animal facility. These mice represent a model of complete alternative pathway deficiency. This model enables the dissection of the complement cascade in vivo and the elucidation of the relative contribution of this complement pathway in the various physiological and pathological phenomena ascribed to the complement system.
Internalization of Mycobacterium bovis Bacillus Calmette‐Guérin into Osteoblast‐Like MC3T3‐E1 Cells and Bone Resorptive Responses of the Cells against the Infection
Authors: Hotokezaka  , Kitamura  , Matsumoto  , Hanazawa  , Amano  , Yamada
Published: March 2003

Mycobacterium bovis BCG (BCG) is a live vaccine used worldwide against tuberculosis. However, it has unfavourable side effects such as osteitis or osteomyelitis, and these sometimes lead to vertebral caries in some patients as a result of bone resorption. Osteoblasts might play a role in the bone resorption caused by BCG infection, because they are central cells in bone metabolism. Cultured osteoblast‐like cell lines (MC3T3‐E1) derived from C57BL mice susceptible to BCG infection cells were infected with BCG at several doses. Interestingly, internalization of BCG‐enveloped phagosome‐like membrane in osteoblast‐like cells were observed by transmission electron microscopy (TEM). Owing to infection, the proliferation and alkaline phosphatase activity of the osteoblast‐like cells were reduced in a dose‐dependent manner. On the other hand, interleukin (IL)‐6 production was considerably enhanced by infection. These results suggest that BCG infects osteoblasts, suppressing their proliferation and differentiation and inducing bone resorption, which may be related to osteitis/osteomyelitis and bone caries caused by BCG infection.
Adjuvant is the Major Parameter Influencing the Isotype Profiles Generated During Immunization with a Protein Antigen, the Schistosoma mansoni Sm28‐GST
Authors: Comoy  , Capron  , Thyphronitis
Published: March 2003

We have previously shown that immunization of mice with the vaccine candidate, the 28‐kDa glutathione‐S‐transferase of Schistosoma mansoni (Sm28‐GST), in alum or complete Freund's adjuvant, or with recombinant Salmonella typhimurium expressing Sm28‐GST, induced type 2, mixed, or type 1 immune responses, respectively. In the present study we examined whether the genetic background, the dose and the route of antigen administration could modulate the profile of the immune response induced during these immunizations. Our results show that the nature of the adjuvant is the major factor that determines the profile of the response. Surprisingly, the genetic background did not influence the response, while the route of immunization, and to a lesser extent the dose of the antigen, weakly modulated the adjuvant‐dependent orientation of the immune response.
Proliferative Responses of Peripheral Blood Leucocytes of Sheep Infected with Trypanosoma evansi
Authors: Onah  , Hopkins  , Luckins
Published: March 2003

The effects of Trypanosoma evansi on the proliferative responses of ovine peripheral blood leucocytes (PBL) were examined in in vitro cell culture systems. Sheep were vaccinated against pneumonic pasteurellosis with a monovalent Pasteurella haemolytica vaccine and then infected with T. evansi TREU 2143. From 1 week post‐infection (p.i.), the PBL were separated and stimulated in cultures with either Concanavalin A (Con A), bacterial lipopolysaccharide (LPS), pasteurella antigen (P.ag), or homologous trypanosome antigen (T.ag). The proliferative responses of the cells to Con A and LPS were significantly (P < 0.001) suppressed by the infection. This suppression was associated with active infection, as treatment of the sheep with a trypanocide restored the proliferative ability of the cells to both mitogens. Similarly, active infection significantly (P < 0.001) suppressed specific responses to P.ag and T.ag but although treatment resulted in full specific proliferative responsiveness to the homologous trypanosome antigen, the same was not true of P.ag, in which the responsiveness of cells from uninfected vaccinated sheep to it were still significantly higher (P < 0.001) than those of cells from infected sheep.
Antigenic Phenotype of Early Intra‐Embryonic Lymphoid Progenitors in the Chicken
Authors: Lampisuo  , Katevuo  , Lassila
Published: March 2003

The stem cells for the definitive haematopoiesis are derived from intra‐embryonic sources originally described in an avian model and later also in mammals. However, the molecular make‐up of the early embryonic haematopoietic progenitors is not yet clearly defined. We have recently characterized the phenotype of prethymic intra‐embryonic progenitors capable of thymic colonization. Here we studied the ontogeny of cell‐surface antigens HEMCAM, α2β1 integrin, thrombomucin, chL12 and c‐kit and their co‐expression on prethymic T‐cell progenitors. The early intra‐embryonic expression of avian B‐cell antigen chB6 was also demonstrated on cells derived from the intra‐embryonic areas. We suggest that in the chicken, embryonic B‐cell progenitors segregate earlier than T‐cell progenitors in the differentiation of multipotent haematopoietic stem cells to committed progenitors.
Differential Oncogene and TNF‐α mRNA Expression in Bone Marrow Cells from Systemic Lupus Erythematosus Patients
Authors: ALVARADO‐DE LA BARRERA  , ALCOCER‐VARELA  , RICHAUD‐PATIN  , ALARCÓN‐SEGOVIA  , LLORENTE
Published: March 2003

The aim of the study was to investigate the bone marrow expression of genes involved in cell growth and apoptosis in patients with systemic lupus erythematosus (SLE). Spontaneous expression of bcl‐2, bax, c‐myc, c‐fos, c‐jun, p53, Fas and tumour necrosis factor (TNF)‐α by bone marrow cells was measured using either semiquantitative or quantitative reverse transcription polymerase chain reaction in SLE patients (n = 8) and in eight normal control subjects. The expression of bcl‐2 was found to be higher in SLE patients than in controls. Bone marrow cells from SLE patients showed significant down‐regulation of bax, c‐myc, c‐fos and p53 (P ≤ 0.05), as compared to normal controls. In both SLE patients and controls the expression of c‐jun and Fas was very low or undetectable. Finally, TNF‐α gene expression was higher in bone marrow samples from SLE patients than in those of controls (P = 0.01). The abnormal expression of genes regulating cell growth and apoptosis in bone marrow cells from SLE patients may help explain the presence of autoreactive cells in secondary lymphoid organs and peripheral blood of SLE patients.
Small Cytoplasmic Antigens from Pseudomonas aeruginosa Stimulate γδ T Lymphocytes
Authors: JuliÀ  , Serra  , Matamoros  , Raga  , MartÍnez
Published: March 2003

γδ T lymphocytes respond to different bacterial antigens and transformed cells. The antigenic molecules responsible for this activity have been studied extensively in antigenic preparations from Mycobacterium. We describe here the in vitro effect of Pseudomonas aeruginosa on γδ T lymphocytes and the properties of the implicated compounds. We found a preferential γδ T‐cell expansion when we used heat‐treated P. aeruginosa preparations and a dose‐dependent inhibition of proliferation of peripheral blood mononuclear cells (PBMC) when non‐heat‐treated antigens were studied. This expansion corresponded to a Vγ9‐positive subpopulation. In contrast to αβ T lymphocytes, the highest stimulatory activity was restricted to very small cytosolic compounds. This activity was protease resistant and phosphatase sensitive and always dependent on interleukin (IL)‐2 or αβ T‐cell activation. We concluded that the antigenic molecules from P. aeruginosa that activated γδ T lymphocytes were small, non‐peptidic, phosphorylated compounds, similar to those previously described from Mycobacterium.
Intravenous or Intranasal Administration of Gliadin is Able to Down‐Regulate the Specific Immune Response in Mice
Authors: Rossi  , Maurano  , Caputo  , Auricchio  , Sette  , Capparelli  , Troncone
Published: March 2003

The mucosal lesion in coeliac disease (CD) represents an immunologically mediated injury triggered by gliadin and is restricted by a particular assortment of major histocompatibility complex (MHC) class II genes. Therefore, immunomodulatory strategies to tolerize gliadin‐specific, class II‐restricted T‐cell responses could represent an alternative to current treatments of CD, which are based on a gluten‐free diet. In this study, BALB/c mice derived from a gluten‐free diet colony were tolerized by either intranasal (i.n.) or intravenous (i.v.) administration of single or multiple doses of gliadin. While a single dose failed to induce tolerance, a significant decrease in gliadin‐specific T‐cell proliferation was detected (P < 0.001) after multiple i.n. or i.v. administrations. No significant difference in antibody titre was detected for antigen‐specific immunoglobulin G (IgG) or the IgG1 subclass, but a lower IgG2a‐specific titre was observed. Both interferon‐γ (IFN‐γ) and interleukin (IL)‐2 expression, measured by enzyme‐linked immunosorbent assay (ELISA) and reverse transcription–polymerase chain reaction (RT–PCR), were reduced on antigen administration, both i.v. and i.n. Neither regimen showed a regulatory effect on IL‐4 production. As T helper 1 (Th1) cytokines seem to be important in the pathogenesis of CD, our data therefore highlight the potential of i.n. and i.v. routes for the design of useful immunomodulatory strategies for CD.
Why So Many Coinhibitory Receptors?
Authors: Sinclair
Published: March 2003

Receptors that display negative signalling functions on lymphocytes and other cells of the reticuloendothelial system now number about 30. These negative receptors are transmembrane glycoproteins activated by phosphorylation of a tyrosine residue in immunoreceptor tyrosine‐based inhibitory motifs that bind various phosphatases to induce dominant negative signals. Since these receptors are armed by the action of activating receptors and inhibit signalling by activating receptors, we have termed them coinhibitory receptors and the negative outcome is coinhibition. Coinhibitory receptors and some inhibitory mediators include FcγRIIB, CTLA‐4, CD5, CD22, p58/70/140 KIR, gp49B1/gp91, PIRB1‐5, LAIR‐1, NKB1, Ly49 A/C/E/F/G, NKG2‐A/B APC‐R, CD66, CD72, PD‐1, SHPS‐1, SIRP‐α1, ILT1‐5, MIR7,10, hMIR(HM18), hMIR(HM9), LIR1‐3,5,8, Fas (CD95), TGFβ‐R, TNF‐R1, IFNγ‐R (α and β chains), mast cell function Ag, H2‐M, HLA‐DM, CD1, CD1‐d, CD46, c‐cbl, Pyk2/FADK2, P130 Ca rel prot, PGDF‐R, LIF, LIF‐R, CIS, SOCS13 and 5, and others are being defined regularly. This long list suggests that coinhibitors are needed not only for self–nonself discrimination, but also for control of ongoing responses to foreign antigens so that infectious agents are ideally dealt with by an appropriate level of immune responses to nonself and an appropriate amount of immunopathology and sickness behaviour.
Inhibitory Effect of Haptoglobin on Granulocyte Chemotaxis, Phagocytosis and Bactericidal Activity
Authors: Rossbacher  , Wagner  , Pasternack
Published: March 2003

Human haptoglobin (Hp) is synthesized at hepatic and extrahepatic sites as an acute‐phase reactant protein (APP). We investigated the effects of Hp on granulocyte function. The chemotaxis of freshly isolated human granulocytes and differentiated HL‐60 cells in response to the bacterial tripeptide, f‐met‐leu‐phe, was inhibited in the presence of a physiological concentration of Hp, but chemotaxis in the presence of the proinflammatory cytokine interleukin‐8 (IL‐8) was not inhibited. Phagocytosis of viable Escherichia coli, as well as fluoresceinated nonviable E. coli, was inhibited. Hp also reduced granulocyte intracellular bactericidal activity against E. coli. The observed inhibitory effects of Hp on granulocyte function are similar to those reported for C‐reactive protein and suggest that APPs dampen the acute inflammatory response.
Serum Gm Allotype Development During Childhood
Authors: Oxelius  , Aurivillius  , Carlsson  , Musil
Published: March 2003

Gm allotypes are genetic variants of the immunoglobulin heavy G chains (IGHG) of IgG molecules, coded from chromosome 14q32, characterized by differences in amino acid epitopes of the constant heavy G chains and inherited in the Mendelian manner. Gm allotypes have influence on IgG subclass levels, and serum Gm allotype levels have been given for different Gm genotypes in adults. Four hundred and thirty healthy children, aged 1–15 years, were examined for serum Gm allotypes and IgG subclasses from the six most common Gm genotypes and different age groups were measured using competitive enzyme‐linked immunosorbant assay and radial immunodiffusion methods. Quantities (in g/l) of G1m(a) and G1m(f) of IgG1, G2m(n) and G2m(–n) of IgG2 and G3m(g), and G3m(b) of IgG3 are given. Different maturation rates of the alternative Gm allotypes within IgG1, IgG2 and IgG3 were shown. G2m(n) development was strikingly retarded compared with G2m(–n) from the γ2 locus. This was found comparing IgG2 levels from homozygous G2m(–n–n) and G2m(nn) individuals, but was also seen in heterozygous G2m(n–n) genotypes. From the γ1 locus G1m(f) levels dominated significantly, but inconstantly, over G1m(a) levels in heterozygous G1m(af) individuals. In homozygous G1m genotypes, G1m(aa) compared with G1m(ff) of the same age, one or the other dominated, sometimes significantly. Serum levels of G3m(b) from the γ3 locus of homozygous G3m(bb) individuals were increased significantly compared with G3m(g) levels of homozygous G3m(gg) individuals, in ages over 3 years. However, in heterozygous G3m(gb) individuals G3m(b) dominance was not evident. There is a relatively rapid development of G1m(f) molecules and a retarded development of G2m(n) in the Gm(f;n;b) haplotype. In comparison, G1m(a) is retarded and G2m(–n) is enhanced in the Gm(a;–n;g) haplotype. The retarded serum G2m(n) development is comparable with serum IgA development during childhood. Different maturation rates of Gm allotypes within the same IgG subclass provide further explanation for the variation of the antibody response during childhood. Quantitative Gm allotype determinations give information of the activity from IGHG genes. The genetic variation constitutes an additional basis for evaluation of IgG antibodies in different diseases in childhood.
Expression and Analysis of Recombinant Salmon Parvalbumin, the Major Allergen in Atlantic Salmon (Salmo salar)
Authors: Van Do  , Hordvik  , Endresen  , Elsayed
Published: March 2003

The parvalbumin from white muscle of Atlantic salmon was previously found to be a major allergen, and designated Sal s1. Two distinct cDNAs, 14.1 and 24.1, which comprise the entire parvalbumin‐encoding regions, were cloned, revealing transcripts from two different parvalbumin genes. In the present study, the protein‐coding regions of these cDNAs were subcloned into an Escherichia coli expression vector (pET‐19b). Both proteins were expressed and the generated target proteins were localized in both soluble and insoluble fractions of the expression host. The recombinant products in the soluble fraction were purified using the His tag‐purification system and analysed on Western blots with anti‐salmon parvalbumin polyclonal rabbit sera and sera from patients allergic to fish. Both recombinant products (His10‐14.1 and His10‐24.1) reacted positively with salmon parvalbumin‐specific immunogloblin G (IgG) from rabbits, and with specific immunoglobulin E (IgE) from the sera of six fish‐allergic patients. The allergenicity of His10‐14.1 was confirmed using enzyme‐linked immunosorbent assay (ELISA). The 14.1 cDNA of salmon parvalbumin was shown to be the dominant type represented in a muscle cDNA library.
Impaired Human Ovarian Follicular Fluid Complement Function in Hereditary Angioedema
Authors: Perricone  , De Carolis  , Giacomello  , Giacomelli  , De Sanctis  , Fontana
Published: March 2003

We sought and detected functionally active complement in human ovarian follicular fluid obtained during the peri‐ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity, alternative pathway activity and C1 inhibitor function were present with values within the normal serum range. Active complement in follicular fluid is relevant for the function of the enzymatic multifactorial mechanism of ovulation. The presence in hereditary angioedema patients of both complement (C1 inhibitor deficiency and chronically consumed complement) and ovarian abnormalities (cystic ovaries), led us to study complement function in the follicular fluid of women of reproductive age affected with hereditary angioedema. In contrast to healthy women, hereditary angioedema patients showed dramatically reduced classical pathway activity and undetectable functional and antigenic C1 inhibitor. C4 was very low, while C3 and B were slightly reduced or within the normal serum range. This complement profile was also detected in patients' sera. Since hereditary angioedema patients often show cystic ovaries (polycystic or multifollicular), the presence of multifollicular ovaries in the two patients studied, along with complement dysfunction, may be relevant. These findings, as well as the normalisation of the ovaries found by us in hereditary angioedema patients and in the patients reported here who were undergoing danazol treatment, and the increase in C1 inhibitor and the improvement of clinical symptoms, suggest a further link between complement and ovarian function.
Human T‐Cell Response to Meningococcal Immunoglobulin A1 Protease Associated α‐Proteins
Authors: Jose  , Wölk  , Lorenzen  , Wenschuh  , Meyer
Published: March 2003

A unique feature of the immunoglobulin A1 (IgA1) protease from pathogenic Neisseriae, i.e. N. meningitidis and N. gonorrhoeae, is its co‐secretion with an amphipathic α‐protein. Polymerase chain reaction (PCR) analysis of the respective igaα gene region in 48 meningococcal strains revealed that this protein domain is conserved throughout all isolates in four different principal variants. Despite strain‐dependent size and sequence variations, sequence analysis showed common structural characteristics. More than 80% of the amino acid sequence of all α‐proteins is dependent on the five amino acids Q, E, A, K and R, resulting in a pI > 10. The sequences are highly conserved at the N‐terminus and the C‐terminus and contain long amphipathic α‐helical stretches. These stretches have a strong probability of forming coiled coil conformations and comprise short repetitive sequence modules with pronounced similarities to T‐cell epitopes. We therefore analyzed the T‐cell response of 20 volunteer blood donors to four peptides, representing such predicted epitopes, and a recombinant meningococcal α‐protein. Sixteen donors reacted against at least one peptide after culture of peripheral blood mononuclear cells in interleukin (IL)‐2‐rich medium, while two individuals showed a positive reaction only against an IgA1 protease‐derived control peptide. From one donor, we established and maintained T‐cell clones specific for purified α‐protein. Characterization of the T‐cell clones revealed a CD3‐ and a CD4‐positive phenotype and the secretion of IL‐2 and interferon‐γ (IFN‐γ), but not of IL‐4. This indicates an important role of α‐proteins during meningococcal carriage and neisserial infection.
Diminished Expression of CD59 on Activated CD8+ T Cells Undergoing Apoptosis in Systemic Lupus Erythematosus and Sjögren's Syndrome
Authors: Tsunoda  , Kawano  , Koni  , Kasahara  , Yachie  , Miyawaki  , Seki
Published: March 2003

The present study was undertaken to examine the phenotype of T cells undergoing in vitro apoptosis in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS). Compared with normal controls, we found diminished expression of CD59 antigen (one of the cell‐surface complement‐regulatory proteins) on CD8+ T cells, but not on CD4+ T cells, from patients with SLE and SS. Three‐colour immunofluorescence analysis revealed that these CD59dim CD8+ T cells were activated T cells, expressing both human leucocyte antigen (HLA)‐DR and CD45RO antigens. In addition, these CD59dim CD8+ T cells were more susceptible to in vitro apoptosis than CD59bright CD8+ T cells. In two patients with active lupus, the percentage of CD59dim CD8+ T cells was significantly decreased after steroid therapy. These findings suggest that decreased expression of CD59 antigen on in vivo‐activated CD8+ T cells may be correlated with disease activity and may be involved in activation‐induced apoptosis in patients with SLE and SS.
Protective DNA Immunization against Chlamydia pneumoniae
Authors: Svanholm  , Bandholtz  , Castaños‐Velez  , Wigzell  , Rottenberg
Published: March 2003

We have investigated the efficacy of the DNA vaccination using the heat shock protein 60 (HSP‐60) gene of C. pneumoniae, for protection of mice against infection with the bacteria. C57Bl/6 mice had a 5–20‐fold reduction of C. pneumoniae numbers in lungs when immunized intranasally (i.n.) with plasmids (p) encoding pHSP‐60. The reduction of the bacterial load coincided with a decreased severity of disease. No specific antibodies were detected after protective i.n. immunization. In contrast, mice immunized intradermally (i.d.) were not protected against challenge with C. pneumoniae, although specific humoral Immunoglobulin (Ig)G responses were generated. Co‐inoculation i.n. of pHSP‐60 with pIL‐12 but not with pGM‐CSF further increased protection of mice against infection with C. pneumoniae. Lungs from pHSP‐60 i.n. immunized and infected mice showed higher levels of interferon (IFN)‐γ mRNA, and spleen cells from these mice co‐cultured with r‐HSP‐60 released higher levels of IFN‐γ and displayed higher proliferative responses than nonimmunized and infected controls. pHSP‐60 immunized IFN‐γ receptor (R)−/− mice were not protected against infection with C. pneumoniae. Likewise, i.n. administration of pIFN‐γ alone induced significant protection. DNA vaccine‐induced protection was CD4+ and CD8+T‐cell dependent, as shown by DNA‐vaccination of MHC class II−/−, CD4−/−, CD8−/− and CD4−/−CD8−/−mice. Interestingly, DNA vaccine induced CD4+ T cells, in the absence of CD8+T cells, were involved in worsening the outcome of infection. This worsening was linked with a shift towards a Th2 cytokine pattern.
Resistance of Extrathymic T Cells to Stress and the Role of Endogenous Glucocorticoids in Stress Associated Immuno Suppression
Authors: Shimizu  , Kawamura  , Miyaji  , Oya  , Bannai  , Yamamoto  , Weerasinghe  , Halder  , Watanabe  , Hatakeyama  , Abo
Published: March 2003

When mice were exposed to restraint stress for 12 or 24 h, severe lymphopenia was induced in all immune system organs, including the liver and the thymus. However, in adrenalectomized mice, this response was completely absent. Phenotypic characterization revealed that interleukin (IL)‐2Rβ+CD3int cells (i.e. extrathymic T cells) with CD4+ phenotype and the NK1.1+ subset of CD3int cells (i.e. NKT cells) in the liver as well as the mature conventional T cells in the thymus were resistant to such stress. In adrenalectomized mice, there was no significant change in the distribution of lymphocyte subsets in all tested organs before stress. Interestingly, the number of lymphocytes in the liver and spleen and the proportion of NKT cells in the liver rather increased after stress in these adrenalectomized mice. Therefore, endogenous steroid hormones were indicated to be important in the induction of immunosuppressive states after stress. Among stress associated cytokines, the secretion of tumour necrosis factor (TNF)‐α was completely suppressed while that of IL‐6 was partially suppressed in adrenalectomized mice. These results suggest that endogenous steroid hormones are important for the induction of the stress associated immunosuppression and that NKT cells are resistant to stress, namely, resistant to exposure to endogenous steroid hormones.
Serum ImmunoglobulinD in Infants and Children
Authors: Haraldsson  , Weemaes  , Jónasdóttir  , Ólafsson  , Van De Wiel  , Göertz  , Klasen
Published: March 2003

Serum immunoglobulinD (IgD) concentration is usually low in healthy individuals as compared to other immunoglobulin classes. Most studies on serum IgD are concerned with serum levels in healthy adults but reference values for young children and infants are not easily available. In order to establish age specific reference values we measured IgD levels in serum of 184 healthy Icelandic children, age 0–14 years and 60 healthy blood donors age 18–63, using the ELISA technique. Special attention was paid to the youngest age groups. Results showed low IgD values in infants and young children, gradually increasing until the age of 10 but then decreasing with age. We conclude that IgD gradually increases with age in childhood as other immunoglobulin classes but later declines. These findings can be of importance in revealing the function of IgD in the immune system as well as in the diagnosis of the hyper‐IgD syndrome.
Azathioprine Associated T‐Cell Mutations in Insulin‐Dependent Diabetes Mellitus
Authors: Falta  , Atkinson  , Allegretta  , Vacek  , Albertini
Published: March 2003

Somatic mutations arise regularly in human T lymphocytes. As these events occur at increased frequencies in several autoimmune disorders, presumably because of increased T‐cell proliferation, we investigated if this is also true for insulin‐dependent diabetes mellitus (IDDM). Mutations of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene measured by 6‐thioguanine (TG) selection were studied in 28 patients (60 determinations) enrolled in a prospective double‐blinded placebo‐controlled study of azathioprine immunosuppression: 17 patients (34 determinations) were receiving azathioprine and 11 (26 determinations) placebo. Mean hprt T‐cell mutant frequencies (MFs) were elevated in both patient groups, but only in the azathioprine group were elevations large and statistically correlated with the duration of the therapy. These results suggest that the organ‐specific antigenic stimulus of the T‐cell proliferation in IDDM does increase mutant cells in the peripheral blood, but this increase is relatively small. However, azathioprine, which is converted to 6‐mercaptopurine in vivo, selects and amplifies the hprt mutants that do arise. Clinical azathioprine resistance may be explained by hprt mutations arising in T cells relevant to the underlying autoimmune process. Monitoring for these mutations should allow more effective use of this immunosuppressive agent.
Characterization of Forssman and Other Antigen/Antibody Systems in Vascularized Mouse Heart to Rat Xenotransplantation
Authors: Gustavsson M. L. , Johnsson C. , Albertsson P. , Lukes D. , Steen L. M. , Johansson B. R. , Mjörnstedt L. , Norrby J. , Tufveson G. , Olausson M.
Published: March 2003

In the present study, the nature of hyperacute xenograft rejection was closely studied in a vascularized mouse‐to‐rat transplantation model. Antibodies against mouse heart, erythrocytes and lymphocytes and against the Forssman antigen were raised in the rat. Upon heterotopic heart transplantation the respective antisera were intravenously (i.v.) injected. Passive transfer of antiheart, antierythrocyte or antilymphocyte serum resulted in hyperacute rejection of the transplanted mouse heart. Subfractionation of the antiheart serum showed that the capacity to induce hyperacute rejection was carried by the immunoglobulin (Ig)G fraction. When antierythrocyte serum adsorbed with mouse erythrocytes was administered the cardiac grafts remained beating. To the contrary, antilymphocyte serum adsorbed with erythrocytes still had the capacity to induce hyperacute rejection. None of the rats that had previously been challenged with the Forssman antigen rejected their grafts hyperacutely. Subsequent investigations by electron microscopy revealed that the Forssman antigen is expressed on dendritic cells (DC) adjacent to the vessels, but not on the vascular endothelium, thus explaining the inability of the anti‐Forssman serum to induce hyperacute rejection. Taken together, we have demonstrated the existence of several xenoantigens that can be targets for antibody‐mediated rejection, suggesting that more than one relevant xenoantigen exists also in more distantly related combinations, such as the pig‐to‐human combination.