bluuebunny/crossref_metadata_2025 huggingface dataset:
An evaluation of the evidence for, and implications of, cytoplasmic nitrate homeostasis
Authors: Siddiqi M. Y. , Glass A. D. M.
Published: March 2003

A review of literature, reporting values of cytoplasmic/cytosolic [NO3–] in plant cells, identified two major areas of disagreement: (1) disparity in the absolute values within the same system, and (2) constancy versus variability in cytoplasmic/cytosolic [NO3–] with varying [NO3–]o.These differences are related to the techniques used by the different authors. Estimates of cytoplasmic [NO3–] by compartmental analysis and by cell fractionation were consistently higher than the estimates by NO3–selective microelectrodes and by techniques based uponin vivoandin vitronitrate reductase activity (NRA). A model recognizing more than one cytoplasmic ionic pool would satisfactorily reconcile the differences in both aspects, i.e. absolute values and constancy. Compartmental analysis and cell fractionation techniques may measure the amount of NO3–in the cytoplasm as a whole (including organelles); by contrast, NO3–selective microelectrodes and NRA estimate only the cytosolic NO3–and, hence, may result in lower estimates. Thus, variable organellar pool(s) may maintain a constant cytosolic pool as estimated by microelectrodes. However, certain observations remain at odds with the notion of a constant cytosolic [NO3–].
Identification of a phytoplasma causing yellows of Monarda
Authors: Wang K. , Hiruki C. , Chen M. H.
Published: March 2003

Monarda yellows occurring in southern Alberta was found to be associated with a phytoplasma. Using two pairs of universal primers, 16S ribosomal DNA fragments (about 1.5 and 1.2 kb) were amplified separately by polymerase chain reaction (PCR) from DNA samples that had been extracted from infected monarda. No such DNA bands were observed using DNA samples from uninfected monarda. The DNA fragment (1.2 kb) amplified by nested‐PCR was analysed and compared with western aster yellows (AY27, Canada), eastern aster yellows (EAY, USA), French hydrangea aster yellows (AYHF), Belgium hydrangea aster yellows (AYHB), clover proliferation (CP, Canada) and potato witches'‐broom (PWB, Canada) by means of restriction fragment length polymorphism (RFLP) using endonucleases AluI, MseI, HpaII, Sau3AI, KpnI and RsaI. The results showed that monarda yellows phytoplasma belongs to the aster yellows subclade and is different from CP and PWB. This is the first report of aster yellows phytoplasma infecting monarda.
Characterization of a new cultural type (LP) of Rhizoctonia solani AG2‐2 isolated from warm‐season turfgrasses, and its genetic differentiation from other cultural types
Authors: Hyakumachi M. , Mushika T. , Ogiso Y. , Toda T. , Kageyama K. , Tsuge T.
Published: March 2003

Isolates of Rhizoctonia solani AG2‐2 obtained from turf with symptoms of large‐patch disease of warm‐season turfgrasses were compared with known AG2‐2 isolates belonging to cultural types IIIB and IV. Some isolates that were previously identified as type IV have been separated here and named LP isolates. Comparisons among isolates were based on cultural morphology, hyphal growth rate, pathogenicity and restriction fragment length polymorphism (RFLP) analysis in the nuclear encoded ribosomal DNA (rDNA) genes. The cultural characteristics of LP isolates varied from those of types IIIB and IV. LP isolates did not show distinct sclerotial formation and zonation, and the colour of their mycelia and pigment deposition was dark brown. LP isolates had slower hyphal growth rates than types IIIB and IV, with an optimum temperature of 25°C compared with 28°C for types IIIB and IV. LP isolates were less virulent on radish but highly virulent on zoysia grass when compared with isolates of types IIIB and IV. Genomic DNA was digested separately with Eco RI, Ban III, Xba I and Sal I, and probed with cloned rDNA from Alternaria alternata in Southern hybridizations. LP isolates had one RFLP pattern, while both IIIB and IV possessed four different patterns each. Cluster analysis of RFLPs showed that R. solani AG2‐2 is divided into three genetic subgroups, consisting of the IIIB, IV and LP isolates, respectively. The polymerase chain reaction (PCR) amplified rDNA internally transcribed spacer (ITS) regions of the IIIB, IV and LP isolates had the same length but produced different restriction patterns when digested with Msp I and Taq I. These results indicate that there are three cultural types in R. solani AG2‐2, namely IIIB, IV and LP.
Characterization of a new potyvirus isolated from peanut (Arachis hypogaea)
Authors: Cook G. , Rybicki E. P. , Pietersen G.
Published: March 2003

During a survey of viruses of peanuts in South Africa a mechanically transmissible virus was isolated from a plant exhibiting chlorotic ringspots and blotches on the leaves. Typical potyvirus‐like flexuous particles were detected by electron microscope examination. Pinwheel‐shaped and laminated inclusions in ultrathin sections, reaction with a monoclonal antibody directed to a potyvirus common epitope, a single 33 kDa coat protein and aphid transmission using Myzus persicae all confirmed that the virus was a subdivision II member of the Potyviridae. Host range studies suggested that the virus was none of the previously reported potyviruses of peanuts or of subdivision II potyviruses. The serological relationships of the virus were studied using a range of 17 antisera to potyviruses in ELISA and immunosorbent electron microscopy (ISEM). The isolate reacted weakly with antisera to plum pox virus and bean yellow mosaic virus in ISEM only. Nucleotide sequence of a 624 bp DNA product was obtained following immuno‐capture with a potyvirus common epitope antiserum, cDNA synthesis and PCR amplification with potyvirus specific primers which amplify the 3′ untranslated region and a part of the coat protein gene. The sequence was only distantly related to a number of potyviruses, whether amino acid or nucleotide sequences were used for comparisons. It is proposed that the virus be named peanut chlorotic blotch virus and be accepted as a new member of the genus Potyvirus in the family Potyviridae.
Refinement of PCR‐detection of Fusarium avenaceum and evidence from DNA marker studies for phenetic relatedness to Fusarium tricinctum
Authors: Turner A. S. , Lees A. K. , Rezanoor H. N. , Nicholson P.
Published: March 2003

Existing PCR‐based assays for the detection of the cereal pathogen Fusarium avenaceum were found to cross‐react with F. tricinctum. An investigation into the phenetic relationship between these two species using two different marker systems revealed a close relationship between them despite their being from separate taxonomic sections. Whilst RFLP differences in the ITS regions surrounding the nuclear 5.8 S rDNA were insufficient to discriminate between isolates of the two species, they were clearly differentiated by RAPD profiling. RAPD fragments from F. avenaceum isolates were screened for hybridization to F. tricinctum DNA on Southern blots. One of 12 selected RAPD fragments showed no hybridization to genomic DNA from F. tricinctum. This fragment was cloned and sequenced, and the sequence obtained was used to design PCR primers. The primers were found to be specific for F. avenaceum, with no cross‐reactions obtained with F. tricinctum or any other wheat pathogen assayed. The primers were able to differentiate between the two species in infected plant material, in contrast to the earlier assays.
Detection of Fusarium oxysporum f.sp. vasinfectum in cotton tissue by polymerase chain reaction
Authors: Moricca S. , Ragazzi A. , Kasuga T. , Mitchelson K. R.
Published: March 2003

A polymerase chain reaction assay was developed for the detection of Fusariumoxysporum f.sp. vasinfectum (FOV), a serious wilt pathogen of cotton in many parts of the world. Based on small nucleotide differences in internal transcribed spacer sequences between 18S, 5.8S and 28S ribosomal DNAs, primers Fov1 (5′‐CCCCTGTGAACATACCTTACT‐3′) and Fov 2 (5′‐ACCAGTAACGAGGGTTTTACT‐3′) were selected. These primers unambiguously amplified a 400‐bp DNA fragment of all the FOV isolates tested (from Angola, Brazil, China and the USA) but did not amplify any other isolates of mycoflora associated with cotton, such as F. moniliforme, Verticillium albo‐atrum, V. dahliae, Aspergillus sp., F. oxysporum, F. sambucinum or F. solani. A control PCR assay was developed employing the universal primer pair ITS1 and ITS2 which amplified a fragment of approximately 220 bp from all isolates tested. This control assay demonstrated that all fungal DNAs were readily amplifiable, thus confirming that the lack of amplification with Fov1 and Fov2 primers was a result of primer specificity and not of other possible causes, such as DNA degradation or the presence of PCR inhibitors. The assay was effective on samples from the stems, leaves, roots and calli, and from plant tissues both with and without symptoms. This detection system proved to be accurate and sensitive and could aid not only diagnosis but also disease monitoring and forecasting.
Movement of barley powdery mildew within field plots
Authors: O'Hara R. B. , Brown J. K. M.
Published: March 2003

Movement of barley powdery mildew (caused by Erysiphe graminis f.sp. hordei) within fields was investigated by sowing the barley cultivars Tyra and Jupiter side by side in two field plots, and trapping spores along transects within the plots. The trapped spores were tested for virulence on the two cultivars. The epidemic on Tyra developed quickly, and a gradient in the proportion of spores with virulence on Tyra was detected in the Jupiter half‐plots. In the Jupiter half‐plots, the epidemic was much less severe; and no mildew could be found in one plot. Movement of spores from one half of the plot to the other usually declined steeply in the first 4 m from the boundary, and was not detectable beyond 12 m. There were exceptions where the gradient was much shallower, and these were consistent with differences in wind direction.
The effects of beet yellows virus on the growth and physiology of sugar beet (Beta vulgaris)
Authors: Clover  , Azam‐Ali  , Jaggard  , Smith
Published: March 2003

The effect of beet yellows virus (genus Closterovirus, BYV) on sugar beet growth was studied in a series of field and glasshouse experiments. Infection reduced total plant weight by 20%, primarily through a 25% reduction in storage root growth. Sugar extraction efficiency was depressed by an increase in root impurities. BYV had little effect on above‐ground yield or total crop cover but did decrease green cover significantly. Infection did not reduce water extraction depth in field experiments despite decreasing lateral root growth in the glasshouse. The growth reduction in infected plants resulted from both a decrease in net photosynthesis and an increase in the proportion of light intercepted by yellow leaves. Damage to the photosynthetic mechanism at least partly caused the reduction in net photosynthesis.
The effect of fungal density on fungicide dose–response curves in barley powdery mildew (Erysiphe graminis f.sp. hordei)
Authors: Damgaard  , Nielsen
Published: March 2003

The effect of inoculation density on fungicide dose–response curves and the estimated ED50 value (the fungicide concentration needed to halve the infected leaf area relative to the infected area of leaves that had not been treated with a fungicide) were investigated theoretically, and predictions were tested experimentally using powdery mildew of barley (Erysiphe graminis [syn. Blumeria graminis] f.sp. hordei). A host leaf was assumed to consist of a number of independent compartments, in each of which only a single fungal spore could germinate successfully. The number of fungal spores landing in a compartment was assumed to be Poisson‐distributed. The spores were assumed to vary in their sensitivity toward a fungicide, and the sensitivity of the spore population was assumed to be normally distributed around a mean sensitivity. Under these assumptions, the ED50 value was shown to be a positively biased estimate of the mean sensitivity in the fungal population, and the bias increased with inoculum density. Consequently, the estimated ED50 value is expected to vary between experiments and laboratories if the inoculation density varies over a considerable range. The correlation between inoculum density and estimated ED50 value is expected to be strongest when the assayed fungal population consists of several genotypes differing in sensitivity. This expectation was tested by estimating the ED50 values for one barley powdery mildew isolate at different inoculation densities for three different fungicides. These ED50 values were positively correlated strongly with the inoculum density for fenpropimorph and more weakly for triadimenol, whereas no correlation was observed for propiconazole.
Genetic relationship between the adult plant resistance gene Lr12 and the complementary gene Lr31 for seedling resistance to leaf rust in common wheat
Authors: Singh  , Park  , McIntosh
Published: March 2003

A resistant phenotype similar to that conferred in wheat by the complementary genes Lr27 + Lr31 was produced in the progeny of intercrosses of cultivars carrying Lr27 and a line possessing Lr12. This confirms that Lr12 is either completely linked with Lr31 or is the same gene. On the basis of these findings and that Lr31 is located on chromosome 4BS, it is concluded that Lr12 must also be located on 4BS. Adult‐plant genetic tests confirm that the Australian wheat cultivar Timgalen carries Lr12, and stocks with Lr12 alone were established from this cultivar.
Establishment of a relationship between grapevine leafroll closteroviruses 1 and 3 by use of monoclonal antibodies
Authors: Seddas  , Haidar  , Greif  , Jacquet  , Cloquemin  , Walter
Published: March 2003

Five stable hybridoma cell lines secreting monoclonal antibodies (Mabs) specific for GLRaV‐1, one of the agents involved in the aetiology of grapevine leafroll disease, were produced by fusing a nonsecreting myeloma cell line with spleen cells from BALB/c mice immunized with purified GLRaV‐1. The Mabs were characterized for their recognition of virus coat protein by DAS‐ELISA and Western blotting. Mab (1G10) reacted specifically in ELISA, immuno‐electron microscopy and immunoblotting with both GLRaV‐1 and GLRaV‐3 coat proteins. Mab 1C4 detected 25 of the 33 GLRaV‐1 isolates, while Mab 1B7 reacted with 32 isolates including the eight isolates not recognized by Mab 1C4. Two of these hybridoma lines (2F11 and 2F3) are now used routinely for the immunodiagnosis of GLRaV‐1.
Contribution of uneven distribution of genomic RNA variants of citrus tristeza virus (CTV) within the plant to changes in the viral population following aphid transmission
Authors: D'Urso F. , Ayllón M.A. , Rubio L. , Sambade A. , Hermoso de Mendoza A. , Guerri J. , Moreno P.
Published: March 2003

The population of genomic RNA sequence variants of citrus tristeza virus (CTV) isolates was characterized by single‐strand conformation polymorphism (SSCP) analysis of complementary DNA (cDNA) of the genes p18 and p20. Comparison of field and aphid‐transmitted isolates showed that aphid transmission frequently altered the single‐strand conformation polymorphism (SSCP) pattern of both genes, indicating changes in the population of genomic RNA variants. SSCP analysis of the cDNA of RNA extracted from small pieces of tissue sampled at different sites of the same plant sometimes yielded different patterns, indicating uneven distribution of the genomic RNA variants within the infected plant. Different SSCP patterns were also obtained when the RNA extracted from individual aphids probing in the same infected leaf was used as a template. Uneven distribution of the genomic RNA variants within the infected plant and sorting of some of these variants by individual aphids probably contribute to changes observed in the CTV population following aphid transmission.
Effects of inoculum concentration, wetness duration and plant age on development of early blight (Alternaria solani) and on shedding of leaves in tomato plants
Authors: Vloutoglou I. , Kalogerakis S. N.
Published: March 2003

Effects of inoculum concentration, wetness duration and plant age on the development of tomato early blight were evaluated in relation to host susceptibility under controlled environmental conditions. The main effect of early blight was premature defoliation, which was linearly related to the percentage of leaf area showing symptoms. As ln(inoculum concentration, conidia mL−1) increased from 6·2 to 11·5, the percentages of leaf area affected and of defoliation increased linearly. Four h of leaf wetness after inoculation were sufficient to initiate the disease on plants of hybrid Skala RZ but not on those of cv. Rio Rojo, for which at least 6 h leaf wetness were needed. As wetness duration increased up to 24 h, there was an increase in the percentage leaf area showing symptoms and in the percentage of defoliation, but thereafter there was no significant increase in either parameter. Tomato plants were susceptible to Alternaria solani at all growth stages, but susceptibility increased as plants matured. There were no significant differences in susceptibility between tomato cultivars and hybrids.
Resistance of wheat cultivars and breeding lines to septoria tritici blotch caused by isolates of Mycosphaerella graminicola in field trials
Authors: Brown J. K. M. , Kema G. H. J. , Forrer H.‐R. , Verstappen E. C. P. , Arraiano L. S. , Brading P. A. , Foster E. M. , Fried P. M. , Jenny E.
Published: March 2003

Isolate‐specific resistance of 71 cultivars and breeding lines of wheat (Triticum aestivum) to septoria tritici blotch was evaluated in six field trials in the Netherlands, Switzerland and the UK between 1995 and 1997. Each plot was inoculated with one of six single‐pycnidium isolates of the pathogen Mycosphaerella graminicola. There were strong interactions between wheat lines and M. graminicola and the line‐by‐isolate interactions were stable over the six trials. Lines with specific resistance or specific susceptibility to each of the isolates were identified. Specific resistance to isolate IPO323 was especially common, being carried by 22 lines from 10 countries. The results confirm that line‐by‐isolate interactions in septoria tritici blotch of wheat are effective in adult plants in field conditions, and are not simply confined to seedlings. Wheat lines with good, quantitative resistance to all or most isolates were identified, including lines from Brazil, the USA and seven European countries. These may be useful as sources of resistance in wheat breeding.
Induction of systemic disease resistance and pathogen defence responses in Asparagus officinalis inoculated with nonpathogenic strains of Fusarium oxysporum
Authors: He C. Y. , Hsiang T. , Wolyn D. J.
Published: March 2003

The ability of nonpathogenic isolates of Fusarium oxysporum (npFo) to induce systemic resistance and defence responses against subsequent challenge with a pathogenic strain of F. oxysporum f. sp. asparagi (Foa) was examined in Asparagus officinalis. In a split‐root experiment, roots inoculated with npFo exhibited a hypersensitive response and those subsequently inoculated with Foa displayed resistance. Induction of systemic resistance in npFo‐treated plants led to significantly fewer necrotic lesions (P = 0·05) and reduced Foa disease severity compared with plants not treated with npFo. In hyphal‐sandwich root inoculation experiments, activities of peroxidase and phenylalanine ammonia‐lyase and lignin content were higher in npFo‐treated plants and increased more rapidly than in npFo‐untreated plants after Foa inoculation. Antifungal activity (inhibition of fungal spore germination and germ‐tube growth) from exudates of roots inoculated with Foa were observed for npFo‐treated plants but not for npFo‐untreated plants. Thus, isolates of npFo may function as inducers of systemic acquired resistance (SAR) and defence responses against Foa invasion in A. officinalis.
Plant defence reactions against fusarium wilt in chickpea induced by incompatible race 0 of Fusarium oxysporum f.sp. ciceris and nonhost isolates of F. oxysporum
Authors: Cachinero J. M. , Hervás A. , Jiménez‐Díaz R. M. , Tena M.
Published: March 2003

Germinated seeds of ‘kabuli’ chickpea cv. ICCV 4 were inoculated with a conidial suspension of the incompatible race 0 of Fusarium oxysporum f.sp. ciceris (Foc) or of nonhost F. oxysporum resistance ‘inducers’, and 3 days later were challenged by root dip with a conidial suspension of highly virulent Foc race 5. Prior inoculation with inducers delayed the onset of symptoms and/or significantly reduced the final amount of fusarium wilt caused by race 5. However, the extent of disease suppression varied with the nature of the inducing agent; the nonhost isolates of F. oxysporum were more effective at disease suppression than the incompatible Foc race 0. Inoculation with the inducers gave rise to synthesis of maackiain and medicarpin phytoalexins in inoculated seedlings; these did not accumulate in plant tissues but were released into the inoculum suspension. Inoculation with inducers also resulted in accumulation of chitinase, β‐1,3‐glucanase and peroxidase activities in plant roots. These defence‐related responses were induced more consistently and intensely by nonhost isolates of F. oxysporum than by incompatible Foc race 0. The phytoalexins and, to a lesser extent, the antifungal hydrolases, were also induced after challenge inoculation with Foc race 5. However, in this case the defence responses were induced in both preinduced and noninduced plants infected by the pathogen. It is concluded that the suppression of fusarium wilt in this study possibly involved an inhibitory effect on the pathogen of preinduced plant defences, rather than an increase in the expression of defence mechanisms of preinduced plants following a subsequent challenge inoculation.
Identification of a major gene (Mex‐1) from Coffea canephora conferring resistance to Meloidogyne exigua in Coffea arabica
Authors: Noir S. , Anthony F. , Bertrand B. , Combes M.‐C. , Lashermes P.
Published: March 2003

Among the most damaging root‐knot nematode species, Meloidogyne exigua is especially common in Latin America and constitutes a major agronomic constraint in all major coffee‐growing (Coffea arabica) areas. Growing nematode‐resistant coffee represents the most promising option for control of the pest. The present study aimed to determine the mode of inheritance of the M. exigua resistance transferred into C. arabica from a related species, Coffea canephora, and to identify associated molecular markers. Segregation data analysis of F2 progeny derived from a cross between the resistant introgression line T5296 and the susceptible accession Et6 showed that the resistance to M. exigua is controlled by a simply inherited major gene (designated the Mex‐1 locus). The gall index distribution exhibited by the F2 individuals suggested incomplete dominant expression. Fourteen AFLP markers were found associated with the resistance to M. exigua and a localized genetic map of the chromosome segment carrying Mex‐1 was constructed. Furthermore, the association of the identified AFLP markers with Mex‐1 was confirmed by analysis of a set of genotypes involving 28 introgression Arabica lines either resistant or susceptible to M. exigua in field conditions. These results represent an important starting point to enhance backcross breeding programmes and to perform an early selection of resistant seedlings.
Participation of the CD69 Antigen in the T‐Cell Activation Process of Patients with Systemic Lupus Erythematosus
Authors: Crispin  , Martínez  , De Pablo  , Velasquillo  , Alcocer‐Varela
Published: March 2003

Accumulating evidence has implicated T cells in the pathogenesis of systemic lupus erythematosus (SLE). The CD69 antigen is an integral membrane protein rapidly induced on the surface of activated lymphocytes. We obtained CD4+ and CD8+ T cells from normal subjects and patients with SLE. The percentage of CD69 expression in freshly isolated cells and after in‐vitro incubation with mitogens was quantified by three‐colour immunofluorescent staining. Expression of this protein was increased in both CD4+ and CD8+ T‐cell subsets from SLE patients when compared with normal cells, although the difference was significant only in the CD8+ T‐cell subset (P = 0.05). Cellular activation increased CD69 expression. When stimulated with anti‐CD2/CD2R or phytohaemagglutinin (PHA), the percentage and absolute numbers of CD69+ cells were lower in patients than in controls. Addition of anti‐interleukin (IL)‐10 monoclonal antibody (MoAb) increased the percentage of in‐vitro CD69 expression in SLE cells. These results suggest that the peripheral blood lymphocytes from patients with SLE have an intrinsic defect that alters their activation process, including the expression of CD69, and might explain some of the T immunoregulatory abnormalities observed in these patients.
Increased PGE2 Production Mediates the In Vitro Inhibitory Effect of the Human Immunodeficiency Virus P24 Immunosuppressive Heptapeptide Ch7
Authors: Giacomini  , Giordani  , Di Modugno  , Chersi  , Luzzati
Published: March 2003

Previous work from our laboratory demonstrated that a synthetic heptapeptide (Ch7), corresponding to a conserved sequence of human immunodeficiency virus (HIV) core protein p24 (amino acids 232–238), was able to specifically abrogate antigen‐induced responses in cultures of normal human peripheral blood lymphocytes (PBL). Addition of recombinant human interferon‐γ (IFN‐γ) to Ch7‐suppressed cultures restored the capacity to mount an antigen‐specific antibody response, suggesting that a cytokine imbalance may be at the basis of the Ch7 immunosuppressive activity. In the present paper we show that the Ch7‐dependent in vitro immunosuppression was accompanied by a significant up‐regulation of prostaglandin E2 (PGE2) production and induction of interleukin‐10 (IL‐10)‐secreting cells. In the presence of the PGE2 inhibitor indomethacin, IL‐10 up‐regulation was prevented and the induction of a specific antibody response was partially restored. PGE2 is indeed an important regulator of immune responses with the ability to differentially affect cytokine production. Thus, our results demonstrate that the Ch7 immunosuppressive epitope may primarily act by up‐regulating PGE2 production and, through this mediator, by causing a cytokine dysregulation, finally responsible for immune response suppression.
Human Serum Amyloid A has Cytokine‐Like Properties
Authors: Patel  , Fellowes  , Coade  , Woo
Published: March 2003

Human serum amyloid A (SAA) proteins are a group of 12–14 kDa apolipoproteins found predominantly in the high‐density lipoprotein (HDL) fraction of plasma. Several functions have been proposed for SAA, but its primary physiological function remains elusive. In this report, we used the monocytic cell line THP‐1 to investigate whether recombinant SAA1 (rSAA) or the HDL–rSAA protein complex can affect the capacity of these cells to produce inflammatory cytokines in vitro. Incubation of rSAA, plasma HDL (which contains ≤ 30 μg/ml of SAA) or HDL–rSAA complex with THP‐1 cells induced synthesis of IL‐1β, IL‐1ra and sTNFR‐II protein and mRNA. The induction of cytokine synthesis was not due to endotoxin contamination since the effect was abrogated by protein denaturation. The rSAA and HDL–rSAA complex did not induce detectable levels of IL‐6 or TNFα protein or mRNA. In contrast 10 μg/ml LPS stimulated secretion of the inflammatory cytokines, IL‐1β, IL‐6 and TNFα, as well as IL‐1ra and sTNFR‐II from THP‐1 cells. We confirmed that rSAA has chemoattractant properties in vivo, by subcutaneous injections into mice and examined the histology of the injection site at 72 h, however, the HDL–rSAA complex has a substantially reduced effect.
A Longitudinal Analysis of Alteration in Lecithin‐Cholesterol Acyltransferase and Paraoxonase Activities Following Laparoscopic Cholecystectomy Relative to Other Parameters of HDL Function and the Acute Phase Response
Authors: Kumon  , Nakauchi  , Kidawara  , Fukushima  , Kobayashi  , Ikeda  , Suehiro  , Hashimoto  , Sipe
Published: March 2003

The composition of high‐density lipoprotein (HDL) changes during inflammation; however, potential changes of HDL function during inflammation and the effects of acute phase proteins that are either on the HDL particles or in the serum have not been clarified. The concentrations of C‐reactive protein (CRP), serum amyloid A protein (apoSAA) isoforms, lipids and apolipoproteins, and the activities of lecithin‐cholesterol acyltransferase (LCAT) and paraoxonase (PON) were measured before and after laparoscopic cholecystectomy, in 12 patients with cholecystolithiasis to clarify the function of acute‐phase HDL and the relationship between acute‐phase proteins and HDL functions. Both acute‐phase apoSAA (A‐apoSAA) and CRP increased, reached their maximum levels 3–6 days after the operation, and then returned to preoperative levels after 2 weeks. In contrast, apolipoproteins and LCAT decreased reciprocally, reached their minimum levels 3–6 days after the operation, and returned to preoperative levels after 2 weeks. However, PON decreased 3–6 days after the operation, and remained low even after 2 weeks. At the nadir the mean activities of LCAT and PON were 56 and 76% of the preoperative levels, respectively. HDL‐cholesterol or constitutive apoSAA did not change significantly. LCAT has been reported to be involved in reverse‐cholesterol transport and PON to be preventive for lipid peroxidation of low‐density lipoprotein in vitro. Thus, during the acute phase of inflammation, HDL may be altered to an atherogenic state due to a decrease in LCAT and PON activities. Therefore, this longitudinal analysis was carried out to determine whether HDL function is modified in a single episode of inflammation and thus may contribute to the occurrence of atherosclerotic disease in patients with chronic or recurrent acute inflammation.
Interleukin‐13 and Human Immunoglobulin E Production in Severe Combined Immunodeficiency Mice Transplanted with Human Peripheral Blood Lymphocytes
Authors: STEINSVIK  , AABERGE  , GAARDER  , LØVIK
Published: March 2003

As normal mice do not respond to interleukin‐13 (IL‐13), we have used mice with severe combined immunodeficiency transplanted with human peripheral blood lymphocytes (hu‐PBL‐SCID mice) as an in vivo model for studying human IL‐13. PBL from three donors (two allergic and one non‐allergic) were prestimulated with IL‐13 in vitro and thereafter transplanted into SCID mice. As evidenced by flow cytometry, IL‐13 in the in vitro cell cultures was physiologically active and suppressed CD14 expression, while it enhanced the expression of CD23 on human monocytes. In the in vivo experiments, SCID mice transplanted with cells from both allergic donors produced twice as high maximum levels of IgE when the cells were preincubated with IL‐13 in vitro before transplantation, as compared with mice receiving cells that had not been preincubated with IL‐13. Two succeeding intraperitoneal (i.p.) injections of IL‐13 resulted in a further increase of maximum IgE levels. Using cells from the non‐allergic donor, no enhancing effect of IL‐13 was observed. Transplanted human cells from one allergic donor examined were shown to migrate to the spleen and lungs of the recipient mice, while cells from the non‐allergic donor were found only in the peritoneal cavity. Altogether, our results indicate that IL‐13 enhances human IgE production in vivo and suggest that lymphocytes in allergic individuals are hyper‐reactive to this cytokine. Furthermore, the allergic status of the cell donor may affect migration and engraftment of cells transplanted into SCID mice.
Immunization with a Synthetic Peptide Conjugate Derived from the N‐Terminal Sequence of Either the β‐Chain of Haemoglobin or the Immunosuppressive Protein (reOLT 4) Reduces the Litter Size of Pregnant Rats
Authors: LORD  , GOTO  , VARI  , LONG‐PAN  , CHIANG  , CHEN  , SUNAGAWA
Published: March 2003

A synthetic peptide conjugate based on the N‐terminal sequence of a 10 000 MW immunosuppressive serum protein (reOLT 4) was used to immunize female Lewis rats prior to mating, in order to determine whether blocking this protein had an effect on pregnancy. The N‐terminal sequence of (reOLT 4) has close sequence homology to the β‐chain of rat haemoglobin so a peptide conjugate based on the N‐terminal sequence of this protein was also used to immunize female Lewis rats. Controls included animals that were not immunized and animals that received the peptide carrier, diphtheria toxoid (DT). No statistical differences were found in gestation time or litter sizes in these groups. Differences were, however, evident between these groups and animals that received DT–(reOLT 4) (group 4) or the DT–β‐chain haemoglobin (group 5). There were no statistical differences in litter size or gestation time for group 4 when compared with group 5. Enzyme‐linked immunosorbent assay and dot‐blot analysis revealed that rats from both groups also had strong responses against DT, the peptide conjugate they were immunized with and the corresponding full‐length protein. In both cases, animals from group 4 and group 5 had weak responses to the peptide that they did not receive, together with lower erythrocyte counts and haematocrits, and elevated heart to body weight ratios. Additionally, antibody purified on a (reOLT 4) immunoaffinity column was capable of binding to rat erythrocytes. A second investigation comparing anaemia prior to fertilization and maintained anaemia over the gestation period revealed that only the latter was capable of decreasing litter size to the same degree as obtained for groups 4 and 5. We conclude that for groups 4 and 5 it is the autoimmune effect of continual anaemia over the gestation period, mediated by autoantibodies, which results in the observed lower litter size.
Increased Frequency of Cells Secreting Interleukin‐6 and Interleukin‐10 in Peripheral Blood of Patients with Primary Sjögren's Syndrome
Authors: Halse  , Tengnér  , Wahren‐Herlenius  , Haga  , Jonsson
Published: March 2003

Sjögren's syndrome (SS) is a chronic autoimmune disease of exocrine glands. There is increasing evidence that interferon‐γ (IFN‐γ) plays a role in the pathogenesis of SS. It has also been suggested that other type 1 cytokines, as well as interleukin‐6 (IL‐6), IL‐10 and transforming growth factor‐β, are important in the induction and/or maintenance of SS. The aim of this study was to investigate the type 1/type 2 cytokine pattern in peripheral blood of primary SS patients. The enzyme‐linked immunospot (ELISPOT) assay was performed to quantify the number of mononuclear cells (MNC) secreting IFN‐γ, IL‐6 and IL‐10 in peripheral blood samples from 33 patients with primary SS and 12 healthy controls. The mean number of cells secreting IFN‐γ was 9/105 MNC in the SS patient group, and 4/105 MNC in the control group (P = 0.73). Fifteen of the SS patients had anti‐Ro 52 kDa antibodies in serum. In this patient group the mean number of cells secreting IFN‐γ was 4/105 MNC, while in the patient group without such antibodies the mean number of cells secreting IFN‐γ was 14/105 MNC (P = 0.04). The mean number of cells secreting IL‐6 was 12 000/105 MNC in the SS patient group, and 5000/105 MNC in the control group (P = 0.01). The mean number of cells secreting IL‐10 was 270/105 MNC in the SS patient group, and 180/105 MNC in the control group (P = 0.04). The SS patients had a significantly higher number of cells secreting IL‐6 and IL‐10 in peripheral blood than the healthy controls, which may facilitate B‐cell activation and production of autoantibodies.
Prophylactic Intravenous Immunoglobulin Treatment Influences Serum Immunoglobulin M Repertoire Development after Allogeneic Bone Marrow Transplantation
Authors: Näsman Björk  , Fesel  , Brissac  , Lundkvist
Published: March 2003

Patients treated with allogeneic bone marrow transplantation (BMT) suffer from a deficient humoral immunity during the post‐transplant period. To prevent infections patients may receive prophylactic intravenous immunoglobulin (IVIG) therapy from 1 week before to 3 months after BMT. We have studied the effect of IVIG treatment on reconstitution of immunoglobulin repertoires in transplanted patients. Sera obtained from 13 IVIG‐treated and 31 non‐IVIG‐treated patients before and at different time points after BMT, ranging from 3 days to 3 years, and from 18 healthy controls, were analyzed using a quantitative immunoblot system. The average immunoglobulin (Ig)M and IgG reactivity profiles against antigens derived from human liver, muscle and skin as well as Staphylococcus epidermidis protein extracts were similar in both patient groups and in controls. Both IgG and IgM reactivity profiles are, however, less heterogeneous among the individuals in the IVIG‐treated patient group. Around 1 year after BMT the heterogeneity of the IgM reactivity profiles against allogeneic protein extracts is much lower in the IVIG‐treated group compared to the non‐IVIG‐treated group and the healthy controls. This effect remains months to years after the IVIG treatment has been completed. Our results suggest that IVIG influences selection of the natural antibody repertoire mediated by the variable (V)‐region during reconstitution after BMT.
Changes in T‐Lymphocyte Subsets in Lungs and Spleens of Mice with Slowly Progressive Primary Mycobacterium tuberculosis Infection: Involvement of Unconventional T‐Cell Subsets
Authors: Phyu  , Sørnes  , Mustafa  , Tadesse  , Jonsson  , Bjune
Published: March 2003

Our previous study showed that the cell‐activation responses and cytokine‐secretion patterns were different in lungs and spleens of mice with slowly progressive primary Mycobacterium tuberculosis infection. The aim of the present study was to characterize the T‐cell subsets in lungs and spleens of mice with a similar infection. The percentages of T‐cell subsets were determined by flow cytometry and the absolute numbers were calculated. Spleens of infected mice showed a threefold expansion of CD4+ cells but no change in CD8+ cells, whereas lungs had a threefold increase of both subsets. A significant expansion of CD4−CD8−αβ+ [double negative (DN)αβ+] subsets was observed in the lungs of infected mice compared with uninfected mice. This was not the case in the spleens of infected mice. In infected mice the CD4−CD8− (DN) population preferentially expressed αβ‐T‐cell receptors (TCR) in the lungs but γδ‐TCR in the spleens. The percentages of many T‐cell subsets were significantly higher in the lungs than in the spleens of both uninfected and infected mice. However, the percentages of CD4+ and CD4−CD8+TCR− subsets in the lungs were significantly lower than in the spleens of infected mice. We also observed some previously unreported T‐cell subsets: double positive‐TCR− (DPTCR−), DPαβ+ and DPγδ+. So far their functions are unknown.
The Standard Model of T‐Cell Receptor Function: A Critical Reassessment
Authors: LANGMAN  , COHN
Published: March 2003

The Standard Model of T‐cell receptor (TCR) function is the distillation of many views. Here we provide a summary that is intended to capture the flavour of the whole, without assigning particular blame, or credit, to any one part. The Standard Model is based on the notion of a single TCR‐combining site that sums the binding contributions of MHC and peptide to produce a single signal to the T cell. How this signal is interpreted can vary with the state of the T cell. A growing number of creaks in the tweaks needed to maintain the Standard Model suggest that it may be timely to make a critical reassessment of the facts and their interpretation. The result of this effort has been to uncover a long‐overlooked fact that T cells do not recognize hybrid class II major histocompatibility complex alleles; they recognize only those haplotypes directly associated with each α‐ or β‐ subunit of class II. Our attempts to tweak the Standard Model to deal with lack of recognition of hybrid class II alleles led us, by surprise, to a quite different framework with which to view TCR function.
Cytokine‐Mediated Regulation of Granulocyte Colony‐Stimulating Factor Production
Authors: Hannen  , Banning  , Bönig  , Kim  , Shin  , Lorenz  , Seeger  , Körholz
Published: March 2003

Granulocyte colony‐stimulating factor (G‐CSF) is an important regulator of granulopoiesis and an inducer of T helper 2 (Th2)‐related cytokines. In this study we investigated the mechanism of cytokine‐modulated G‐CSF production in lipopolysaccharide (LPS)‐stimulated peripheral blood mononuclear cells (PBMC) and bone marrow stromal cells (BMSC). In PBMC, LPS significantly induced G‐CSF and interleukin (IL)‐1, an inducer of G‐CSF. Both were partly inhibited by IL‐4, IL‐6 and IL‐10. None of these effects were the result of altered monocyte activation or proliferation. The effects of IL‐4 and IL‐10 appeared to be independent of IL‐1 suppression or IL‐1 receptor antagonist (IL‐1ra) induction, but rather seemed to involve a blockade of IL‐1 function, in addition to a blockade of IL‐1‐independent stimulatory effects on G‐CSF production. The effect of the IL‐6‐induced suppression of G‐CSF production differed from that of IL‐4 and IL‐10 in that it was much less pronounced and could be partially overridden by addition of functional IL‐1, yet it also appeared to involve the interference with IL‐1 function and the suppression of IL‐1‐independent mechanisms. In BMSC, G‐CSF synthesis was regulated differently. Here, IL‐1 was the main stimulator of G‐CSF release, and the effect of IL‐1 was neither affected by IL‐10 nor IL‐6, while IL‐4 had a stimulatory effect. Thus, G‐CSF production was found to be differently regulated in distinct cellular compartments, and a cross‐regulation between these might be facilitated by IL‐4‐, IL‐6‐ and IL‐10‐induced inhibition of IL‐1. These results could be important for the understanding of G‐CSF production in neutropenic patients during severe infection.
Gene Transfer of the Co‐Stimulatory Molecules B7‐1 and B7‐2 Enhances the Immunogenicity of Human Renal Cell Carcinoma to a Different Extent
Authors: Jung  , Hilmes  , Knuth  , Jaeger  , Huber  , Seliger
Published: March 2003

Stimulation of a specific antitumour immune response with recruitment and induction of T‐cell effector functions represents an attractive concept in human cancer therapy. Different cytokines and the B7 co‐stimulatory molecules are both able to provide proliferation and activation signals for T cells. In the present study, we first demonstrated the absence of both B7‐1 and B7‐2 expression in human renal cell carcinoma (RCC) cell lines. The lack of B7 expression was associated with a low or absent proliferative response of allogeneic and autologous T cells upon stimulation with tumour cells. In order to investigate the role of B7‐1 and B7‐2, the human RCC cell line, MZ1257RC, which expresses normal levels of adhesion molecules and major histocompatibility complex (MHC) class I surface antigens, was transfected with B7‐1 and B7‐2 expression vectors, respectively. The B7‐1‐ and B7‐2‐transduced MZ1257RC cells were potent stimulators of allogeneic and autologous T‐cell proliferation. B7‐2 transfectants were approximately two‐ to threefold more effective in the induction of primary T‐cell activation than B7‐1‐transduced cells. Interleukin (IL)‐12 synergized with the B7/CD28 interaction to enhance allogeneic T‐cell proliferation, independently of the B7 molecule transduced. In contrast, IL‐2 only co‐operatively increased T‐cell activation in the presence of B7‐2. Our results suggest the following: first, that co‐stimulatory molecules are required for efficient T‐cell responses directed against RCC; second, that B7‐2 appears to be a more potent stimulator of tumour immunity as compared to B7‐1; and third, that B7 molecules selectively co‐operate with different T‐cell stimulatory cytokines. The different activity of B7‐1 and B7‐2 molecules on the immunogenicity of RCC will have implications for the development and optimization of RCC‐specific cancer vaccines.
Enhanced Expression of Integrins and CD66b on Peripheral Blood Neutrophils and Eosinophils in Patients with Rheumatoid Arthritis, and the Effect of Glucocorticoids
Authors: Torsteinsdóttir  , Arvidson  , Hällgren  , Håkansson
Published: March 2003

The aim of this study was to elucidate signs of granulocyte activation by studying adhesion and phagocytosis receptors on peripheral blood granulocytes from patients with rheumatoid arthritis (RA), and to observe the effect of glucocorticoids. Analyses by flow cytometry showed elevation of the neutrophil and eosinophil expression of the α‐ and β‐chains of the β2‐integrin Mac‐1 (CD11b/CD18) and of the CEA‐gene family member 6 (CGM6, CD66b). Expression of the adhesion receptor antigens CD11a, CD29, CD49d, CD49f and CD44, and the Fcγ receptors II and III, was unaffected. Treatment with low‐dose prednisolone reduced the expression of CD11b on neutrophils and of CD11b, CD18 and CD66b on eosinophils to the same level as that found in healthy controls. Metyrapone treatment increased the surface expression of CD35 and CD49f on eosinophils, but did not affect surface expression on neutrophils. Activation of blood granulocytes may be important for the increased recruitment of neutrophils and eosinophils to the synovial cavity in RA. Treatment with low doses of glucocorticoids in RA normalizes the enhanced expression of the studied adhesion molecules in eosinophils but has minor impact on neutrophil activation. Endogenous glucocorticoid production seems to have minimal or no effect on the expression of adhesion and phagocytosis receptors on circulating granulocytes.
Quillaja Saponin Formulations that Stimulate Proinflammatory Cytokines Elicit a Potent Acquired Cell‐Mediated Immunity
Authors: Behboudi  , Morein  , Villacres‐Eriksson
Published: March 2003

We examined the ability of various Quillaja saponins in iscom‐matrix formulations to induce proinflammatory cytokines, such as interleukin (IL)‐1α and IL‐6, and to stimulate acquired immune responses to influenza virus envelope proteins. The A‐fraction of Quillaja saponins (QH‐A) was shown to stimulate antigen‐presenting cells (APC) to produce proinflammatory cytokines, and elicited a high primary antigen‐specific antibody response and potent cell‐mediated responses, as measured by T‐cell proliferation, production of cytokines and cytotoxic T‐lymphocyte (CTL) activity. The C‐fraction of Quillaja saponins (QH‐C) was shown to have a low capacity to stimulate proinflammatory cytokines and elicited low primary antibody and T‐cell responses. However, the QH‐C iscom‐matrix mediated a potent booster effect, resulting in a high secondary antibody response. The ability of APC to discriminate and to respond to QH‐A formulations more efficiently than to QH‐C with release of proinflammatory cytokines, which precedes a potent acquired immune response, identifies an important mechanism through which some adjuvants may exert their immunoenhancing activities.