bluuebunny/crossref_metadata_2025 huggingface dataset:
The global regulator GacA of Pseudomonas fluorescens CHA0 is required for suppression of root diseases in dicotyledons but not in Gramineae
Authors: SCHMIDLI‐SACHERER P. , KEEL C. , DÉFAGO G.
Published: November 2003

Pseudomonas fluorescens strain CHA0 suppresses various plant diseases caused by soil‐borne fungi. The pseudomonad produces the antimicrobial metabolites 2,4‐diacetylphloroglucinol (Phl), pyoluteorin (Plt) and hydrogen cyanide, which are important for disease suppression, as well as the siderophores pyoverdine (Pvd), salicylic acid (Sal) and pyochelin (Pch). In the current work, a derivative of CHA0 with a mutation in the global regulator gene gacA (GacA−), which is unable to produce Phl, Plt and HCN, failed to protect the dicotyledonous plants cress and cucumber against damping‐off caused by Pythium ultimum. In contrast, the GacA− mutant could still protect the Gramineae wheat and maize against damping‐off mediated by the same strain of P. ultimum, and wheat against take‐all caused by Gaeumannomyces graminis. However, the GacA− mutant overproduced Pch and Pvd. To gain more insight into disease protection afforded by the GacA− mutant, a GacA− Pvd− double mutant (strain CHA496) was constructed by gene replacement. Strain CHA496 overproduced Pch and Sal compared with CHA0 and protected wheat against P. ultimum and G. graminis, whereas cress and cucumber were not protected. Addition of FeCl3 repressed Pch and Sal production by strain CHA496 in vitro and impaired the protection of wheat in soil microcosms. In conclusion, a functional gacA gene was necessary for the protection of dicotyledons against root diseases, but not for that of Gramineae. Results indicated also that Pch and/or Sal were involved in the ability of the GacA− Pvd− mutant of CHA0 to suppress root diseases in Gramineae.
The effects of ultraviolet‐B (UV‐B: 290–320 nm) radiation on blister blight disease of tea (Camellia sinensis)
Authors: GUNASEKERA T. S. , PAUL N. D. , AYRES P. G.
Published: November 2003

Sunlight is known to reduce the incidence of blister blight disease of tea, caused by Exobasidium vexans. The effects of the ultraviolet‐B (UV‐B: 290–320 nm) component of solar radiation on the disease and the development of E. vexans were studied in the field in Sri Lanka using UV‐screening filter materials held over a commercial crop. Exclusion of UV‐B radiation by Polyester, which reduces fluxes in the UV‐B region by approximately 75–85%, increased both the number of translucent spots (immature sites of infection) and number of sporulating blisters (mature sites of infection), but it had little or no effect on the sporulation of the pathogen. When basidiospores were artificially inoculated on leaves, and were exposed to full or filtered solar radiation, their survival and germination increased when UV‐B wavelengths were removed; significant differences were found 62 h after inoculation. However, UV‐B did not affect the extension of germ tubes. This study indicates that the UV‐B component of solar radiation plays an important role in the natural regulation of blister blight disease in the field.
Identification and expression of the gene Yr2 for resistance to Puccinia striiformis in the wheat differential cultivars Heines Kolben, Heines Peko and Heines VII
Authors: CALONNEC A. , JOHNSON R. , De VALLAVIEILLE‐POPE C.
Published: November 2003

Genetic data showed that a common gene was present in the three differential wheat cultivars Heines VII (Yr2), Heines Peko (Yr2, Yr6) and Heines Kolben (Yr6) expressed against the race 109E9 of Puccinia striiformis which possesses virulence for Yr6 and avirulence for Yr2. This supported the hypothesis that Heines Kolben carries the gene Yr2 in addition to Yr6 for resistance to yellow rust in a genetic background in which Yr2 is weakly expressed in seedlings. F1, F2 and F3 progenies from the cross Heines Kolben × Peragis confirmed the monogenic segregation of Yr2 in Heines Kolben and demonstrated the variation of its expression with environmental conditions.
Antifungal activity of bergenin, a constituent of Flueggea microcarpa
Authors: PRITHIVIRAJ B. , SINGH U.P. , MANICKAM M. , SRIVASTAVA J.S. , RAY A.B.
Published: November 2003

The antifungal activity of bergenin against some plant pathogenic fungi, namely, Alternaria alternata, A. brassicae, A. carthami, Fusarium udum, F. oxysporum f.sp. ciceri, Curvularia lunata and Erysiphe pisi, was studied. Bergenin as its monosodium salt was effective against all the fungi and the effective dose for complete inhibition of spore germination varied from 15 μg mL−1 for F. udum to 125 μg mL−1 for E. pisi. Experiments on the effect of bergenin on powdery mildew development under glasshouse conditions revealed that it can control powdery mildew of pea at 2000 μg mL−1 by postinoculation treatment, the results being comparable with those of carbendazim (1000 μg mL−1) and wettable sulfur (2000 μg mL−1). It affected hyphal elongation and the number of primary and secondary branches.
Solarization and natural heating of irrigated soil amended with cruciferous residues for improved control of Macrophomina phaseolina
Authors: LODHA S. , SHARMA S. K. , AGGARWAL R. K.
Published: November 2003

The efficacy of summer irrigation and soil solarization combined with cruciferous residues was tested against the dry root rot pathogen Macrophomina phaseolina in an arid climate. In irrigated amended soil, polyethylene mulching during May increased the soil temperature to 57°C and 50°C at depths of 0–15 and 16–30 cm, respectively. As a result, within l5 days the population of M. phaseolina was almost eradicated (93–99%) at both soil depths. A considerable reduction (75–96%) was also achieved by natural heating of irrigated soil (46–53°C) for l5 days after amending with cruciferous residues. Mulching alone was only partially effective (69–89% reduction). These results suggest a new approach to controlling soil‐borne pathogens in hot, arid regions by combining summer irrigation with soil amendment. Amendment with residues alone or in conjunction with soil solarization also increased the population of lytic bacteria against M. phaseolina.
Comparative host reactions and Frankliniella occidentalis transmission of different isolates of tomato spotted wilt tospovirus from Spain
Authors: ROCA E. , ARAMBURU J. , MORIONES E.
Published: November 2003

The abilities of different isolates of tomato spotted wilt tospovirus (TSWV) collected from northeastern and eastern Spain to infect 10 host species, and to be acquired and transmitted by the western flower thrips (WFT), Frankliniella occidentalis were compared. Two isolates of TSWV from a single source plant could be separated according to the different type of local lesions they induced in Nicotiana glutinosa. Host ranges of the studied TSWV isolates were very similar, but differences were found in the symptoms induced and in their capacity to infect specific hosts systemically. Lycopersicon esculentumDatura stramonium were evaluated for their potential as virus acquisition host species. The proportion of transmitter adult thrips obtained from WFT larvae fed on L. esculentum was greater than from D. stramonium. No differences were detected between TSWV isolates in their ability to be acquired and transmitted by WFT. No evidence was obtained of alterations in TSWV particles which could affect WFT transmissibility due to the repeated mechanical transfers used to clone the isolates. Our findings do not support the existence of pathological effects of TSWV on WFT.
Differentiation and quantification of the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis using a PCR assay
Authors: NICHOLSON P. , REZANOOR H. N. , SIMPSON D. R. , JOYCE D.
Published: November 2003

Isolates of Tapesia yallundae and Tapesia acuformis were subjected to Random Amplified Polymorphic DNA (RAPD) assay. Amplification products common to isolates of either species were cloned and primers were generated from each sequence for use in conventional PCR. The primer pair derived from a T. yallundae specific RAPD marker amplified a product only from DNA of T. yallundae isolates and not from DNA of a range of other fungal species associated with the stem base disease complex of cereals. Similarly, the primer pair generated from a T. acuformis‐specific RAPD marker amplifed a product only from DNA of T. acuformis isolates. Quantitative assays were developed for both species of Tapesia from these primer pairs, using competitive PCR. Competitive PCR was used to determine the level of colonization of seedlings by each species in glasshouse‐ and field‐inoculated cereal hosts and results compared to those for conventional seedling disease assessment.
Detection of citrus psorosis‐ringspot virus using RT‐PCR and DAS‐ELISA
Authors: GARCIA M. L. , SANCHEZ DE LA TORRE M. E. , DAL BO E. , DJELOUAH K. , ROUAG N. , LUISONI E. , MILNE R. G. , GRAU O.
Published: November 2003

Psorosis, sometimes also associated with ringspot symptoms, is a widespread and damaging disease of citrus in many parts of the world including South America and the Mediterranean basin. We describe the application of RT‐PCR and DAS‐ELISA diagnostics to an isolate of citrus ringspot virus (CtRSV‐4) and other virus isolates associated with this disease. Fragments of cDNA from bottom‐component RNA of CtRSV‐4 were cloned and sequenced, and PCR primers were designed, 5′ACAATAAGCAAGACAAC upstream, and 5′CCATGTCACTTCTATTC downstream. RT‐PCR experiments using these primers allowed detection of CtRSV‐4 in infected citrus leaves down to a tissue dilution of 1/12 800 representing 2 μg of tissue, and less sensitive detection of the related citrus psorosis‐associated virus (CPsAV90‐1‐1) and four other psorosis isolates from Argentina and the USA. In addition, CtRSV‐4 particles were partially purified from local lesions in Chenopodium quinoa, and the preparations used to raise a rabbit antiserum. The antiserum was absorbed with extracts of healthy C. quinoa leaves, and a DAS‐ELISA kit was prepared and tested for detection of CtRSV‐4, CPsAV90–1‐1, and other psorosis isolates from Argentina, the USA, Italy and Spain. The ELISA detected CtRSV‐4 down to a tissue dilution of 1/1600, and most other psorosis isolates down to dilutions of 1/200–1/800. Three of a total of 20 heterologous isolates were consistently negative. Comparison of the PCR and ELISA results suggests that both methods can be used for detection of a range of psorosis isolates, but that variation of the viruses in the field might cause problems for any one diagnostic test.
Quantifying the effect of reduced doses of propiconazole (Tilt) and initial disease incidence on leek rust development
Authors: CLARKSON J. P. , KENNEDY R. , PHELPS K. , DAVIES J. , BOWTELL J.
Published: November 2003

The effect of reduced doses of propiconazole (Tilt) and different initial inoculation levels on leek rust caused by Puccinia allii was quantified by three parameters: (i) rate of plant‐to‐plant spread (b), (ii) the time taken for 50% plant infection (m) and, (iii) rate of appearance of new pustules. All propiconazole doses tested reduced values for b compared with an unsprayed treatment but lower doses had little effect on new pustule appearance. Although b values were different for the same propiconazole dose at two field sites with different epidemics, values compared with the unsprayed treatments were similar. Fungicide efficacy was therefore measured independently of disease level in the field. The parameter b for rust spread was also directly related to marketable yield at harvest. Initial rust incidence levels of < 0.1% infected plants reduced b compared with higher levels (0.5–5%). This suggests that rust spread occurs at a maximum rate unless incidence is extremely low. Reduced doses of propiconazole also suppressed spore germination of P. allii and the appearance of rust pustules in inoculated plants in the glasshouse. The value of the parameters identified is discussed in relation to leek rust control, epidemiology and the effect of weather.
The effect of component number on Rhynchosporium secalis infection and yield in mixtures of winter barley cultivars
Authors: NEWTON A. C. , ELLIS R. P. , HACKETT C. A. , GUY D. C.
Published: November 2003

Mixtures of winter barley cultivars containing up to six components were grown over three years with and without fungicide treatment. Yield increases were recorded for mixtures compared with the mean of their monoculture components and there was a significant trend towards greater benefit from increased number of components. These benefits were partially attributable to a corresponding increase in control of Rhynchosporium secalis as component number increased. The potential for exploitation of mixtures in cereals for control of splash‐dispersed pathogens is discussed.
Variability amongst strains of Fusarium poae assessed by vegetative compatibility and RAPD polymorphism
Authors: KERÉNYI Z. , TÁBORHEGYI É. , POMÁZI A. , HORNOK L.
Published: November 2003

Vegetative compatibility tests and random amplification of polymorphic DNA (RAPD) were used to assess genetic relationships amongst 54 strains of Fusarium poae obtained from various geographical regions. Twenty‐seven strains were assigned to eight multiple member vegetative compatibility groups (VCGs), while the other 27 isolates were found to form single‐member VCGs. There was a partial correlation between VCG and geographical origin, but the relationship was not always clear. However, no correlation was observed between the VCG and the host plant of origin. RAPD patterns were closely associated with VCGs in all cases. Members of VCGs that were interconnected by bridging isolates formed common branches in the phenogram constructed on the basis of the RAPD patterns, while strains that belonged to single‐member VCGs were scattered throughout the phenogram. These data demonstrate that the combination of traditional and molecular methodologies allows reliable intraspecific subdivisions in an asexual fungus, which is a secondary invader of a wide range of host plants, and so has never been subject to the intense selection pressure of a single host species and lacks pathogenic subgroups.
Survival characteristics of oospore populations of Peronospora viciae f.sp. pisi in soil
Authors: VAN DER GAAG D. J. , FRINKING H. D.
Published: November 2003

Survival and germinability of oospore populations of Peronospora viciae f.sp. pisi in soil were investigated. The percentage survival was assessed using the vital stain tetrazolium bromide. Germinability was defined as the percentage of oospores that germinated in water. Oospores 1–3 weeks old, embedded in plant tissue, were incorporated in a loamy sand or silt‐loam soil and incubated at 3, 10 or 20°C or stored dry at 20°C and 30% RH. The percentage of surviving oospores in soil decreased rapidly with or after decomposition of the surrounding plant tissue at 10 and 20°C. After 29 weeks less than 10% of the oospores had survived. At 3°C, survival was 25% or more after 29 weeks. Germinability of the oospores was 3% at time of incorporation and had increased to a maximum of 50% after 4 weeks in the loamy sand soil. Increase in germinability of the dry‐stored oospores was significantly later than that of the soil‐incubated oospores. In soil, the initial increase in germinability was followed by a decline after decomposition of the surrounding plant tissue. In order to investigate survival of oospores under natural conditions, ground tissue or pod pieces of pea plants containing oospores 7 or 5 months old, respectively, were incorporated in two loamy sand soils or a silt loam soil in field plots. The surrounding plant tissue decomposed within 4 weeks and the percentage of surviving oospores decreased to less than 6% after a year. Survival of oospore populations was generally well described by the lognormal model, indicating that the risk of oospore death initially increased before decreasing later. Germinability of the oospore populations from ground tissue and pod pieces, 61 and 62% at incorporation, decreased rapidly after incorporation in soil. Oospores extracted from the silt‐loam soil germinated poorly in water but caused high disease incidences in a bioassay, suggesting that oospores had become dependent on the host plant for germination.
Histaminergic Regulation of Natural Killer Cell‐Mediated Clearance of Tumour Cells in Mice
Authors: ASEA A. , HERMODSSON S. , HELLSTRAND K.
Published: November 2003

Treatment of Swiss albino mice with histamine enhanced the clearance of natural killer (NK)‐cell sensitive YAC‐1 lymphoma and B16/F10 melanoma cells from lung tissue in vivo, but did not affect the elimination of NK‐cell‐insensitive P815mastocytoma cells. The effect of histamine was apparently mediated by H2‐type histamine receptors (H2R) since it was blocked by ranitidine, an H2R antagonist. Histamine did not affect clearance of tumour cells in animalsdepleted of NK cells in vivo by treatment with antibodies to asialo‐GM1 or NK1.1. The effect of histamine was time‐dependent: pretreatment with histamine for 3 h significantly augmented the clearance of YAC‐1 cells, whereas, pretreatmentwith histamine for 5 min was ineffective. Histamine potentiated the anti‐tumour properties of NK‐cell activators such as interleukin‐2 (IL‐2) or interferon‐α (IFN‐α) in vivo. None of these lymphokines significantly affected theclearance of YAC‐1 cells unless animals were concomitantly treated with histamine. Treatment with ranitidine alone reduced the in vivo clearance of YAC‐1 cells from lungs but did not affect the clearance of NK‐cell‐insensitive P815 cells. Effects of ranitidine on NK‐cell function in vivo were not shared by a chemical control to ranitidine, AH20239AA, thus indicating that the inhibition of NK‐cells results from H2R antagonism rather than non‐specific toxicity. It is concludedthat histaminergic mechanisms may be involved in the regulation of NK cell function in vivo
Nordihydroguaiaretic Acid Blocks IL‐2‐Independent Lymphocyte Proliferation and Enhances Responses to PPD
Authors: ESA A. H. , CONVERSE P. J.
Published: November 2003

The authors examined the mechanism by which the non‐specific lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibits human lymphocyte proliferative responses and compared its effects with those of cyclosporine (CsA). It was found that CsA‐resistant proliferative responses induced by direct PK‐C activators such as mitogenic concentrations of the phorbol ester TPA or the putative anti‐tumour agent bryostatin 1 (bryo 1) were inhibited in a concentration‐dependent manner by NDGA (IC50 = 2 μM). In contrast, CsA‐sensitive IL‐2‐dependent proliferative responses induced by PHA, anti‐CD3 or the purified protein derivative (PPD) of M. tuberculosis were not significantly inhibited by NDGA concentrations as high as 8 μM. The expression of the IL‐2R by lymphocytes was also resistant to NDGA concentrations that effectively blocked the mitogenic effects of TPA or bryostatin, but could be inhibited by higher concentrations of NDGA (IC50 = 8 μM). In addition, NDGA, but not CsA, blocked the production of IL‐6 by human mononuclear cells. Furthermore, PPD‐induced proliferation was significantly enhanced by NDGA. These data would suggest that NDGA at concentrations below 8 μM selectively inhibits IL‐2‐independent proliferation. NDGA’s ability to inhibit IL‐6 while enhancing the proliferative response to PPD may indicate an anti‐inflammatory therapeutic potential of anti‐oxidants in mycobacterial infections.
Conformation of the T‐Cell Antigen Receptor‐β Chain C‐Domain Contributes to Vβ3 Epitope Recognition by Monoclonal Antibody KJ25
Authors: LI ZHAN‐GUO , KEMP O. , LONGHURST T. , MANOLIOS N.
Published: November 2003

The clonotypic T‐cell antigen receptor (TCR)‐β chain contains two extracellular intrachain disulfide bonds. It belongs to the immunoglobulin gene superfamily and is subdivided into variable (V), joining (J), diversity (D) and constant (C) region. Monoclonal antibody (MoAb) KJ25 is believed to recognize an epitope in the V‐domain of TCR‐β (Vβ3) chain, but its epitope requirements are unknown. In this study of TCR‐αβ chain interactions using chimeric recombinant TCR‐β chains, the authors found that partial substitution of the Cβ‐domain with that of interleukin‐2 receptor α chain (Tac) sequences led to the loss of TCR‐Vβ3 epitope recognition by KJ25. These results suggest that epitope recognition of the TCR‐Vβ3 by KJ25 MoAb is dependent not only on the V‐domain, but also on the close contact with the extracellular C‐domain which influences the conformation and epitope recognition of the Vβ3‐region. This may not be unique to Vβ3 and may be a general feature of TCR‐β protein folding.
Soluble CD4: a Link Between Specific Immune Mechanisms and Non‐Specific Inflammatory Responses?
Authors: GOTO H. , GIDLUND M.
Published: November 2003

The CD4 molecule is an important cell surface molecule expressed on many cell types including T‐helper cells. CD4 can be shed from cells, and the truncated soluble form of CD4 (sCD4) has been found elevated in serum in several diseases. In this study the authors show that migration of polymorphonuclear neutrophils (PMN) is induced by sCD4. Histopathology of the site of injection of recombinant sCD4 in the skin shows local infiltration of PMN. In vitro, in Boyden chamber, sCD4 can rapidly give rise to a dose‐dependent migration of PMN. The authors speculate that sCD4 can be another chemotactic factor and, as such, constitutes a link within the immune system between specific and non‐specific elements of inflammation.
The Human Mast Cell Line HMC‐1 Expresses C5a Receptors and Responds to C5a but not to C5a(desArg)
Authors: WERFEL T. , OPPERMANN M. , BUTTERFIELD J. H. , BEGEMANN G. , ELSNER J. , GÖTZE O. , ZWIRNER J.
Published: November 2003

The expression of the receptor for the anaphylatoxin C5a (C5aR, CD88) on the human mast cell line HMC‐1 was studied with four anti‐C5aR monoclonal antibodies directed to the N‐terminal domain of the receptor. All antibodies bound to the human mast cell line HMC‐1. The binding could be blocked by recombinant C5a and by peptide EX‐1 representing amino residues 1–31 on the N‐terminal domain of the C5aR. In addition, FITC‐labelled C5a bound to HMC‐1, and this binding could be blocked by unlabelled C5a or C5aR antibodies. C5aR‐specific mRNA was detected in HMC‐1 cells by RT‐PCR which confirmed the expression of the C5aR gene made by these cells. Lymphocyte‐conditioned medium, interferon‐γ or phorbol esters which have been shown to induce a down‐regulation of C5aR on myeloid cells did not influence the expression of C5aR on HMC‐1. C5a let to a transient mobilization of intracellular calcium in HMC‐1 which could be inhibited by pre incubation of C5a with a C5a‐specific antibody. In contrast to findings with granulocytes, HMC‐1 did not respond to C5a(desArg), confirming previous findings with human skin mast cells. The findings show that (i) although HMC‐1 differ from granulocytes in their responsiveness to C5a(desArg), they express similar C5aR and (ii) HMC‐1 resemble skin mast cells in the expression and function of C5aR and may therefore serve as a model in future studies addressing the biology of this anaphylatoxin receptor on skin mast cells.
CD45 Engagement Induces L‐Selectin Down‐Regulation
Authors: STIBENZ D. , BÜHRER C. , LAUFER D. , OBLADEN M.
Published: November 2003

The CD45 glycoprotein isoforms exhibit a receptor‐like composition and display intracellular protein tyrosine phosphatase (PTPase) activity. The present study links CD45 to the regulation of L‐selectin (CD62L), a leucocyte glycoprotein important for extravasation and homotypic aggregation. Monoclonal antibodies (MoAbs) IOL1b and AICD45.2, but not GAP8.3, all of which are directed against common CD45 epitopes, were found to elicit lymphocyte L‐selectin down‐regulation. Lymphocyte L‐selectin down‐regulation in response to anti‐CD45 MoAbs was enhanced by high cell density and partially antagonized by the protein tyrosine kinase (PTK) inhibitor, herbimycin A. The MoAbs IOL1b, AICD45.2 and GAP8.3 recognized granulocyte‐expressed CD45 but did not induce loss of L‐selectin expression of granulocytes. In contrast, the CD45 PTPase inhibitor, vanadate, induced L‐selectin down‐regulation both in lymphocytes and granulocytes. The PTPase activation by nitric oxide (NO) or the NO‐generating compound, sodium nitroprusside, did not affect L‐selectin surface expression. Increased concentrations of soluble L‐selectin were detected after anti‐CD45 or vanadate‐induced down‐regulation of L‐selectin surface expression. While activation of protein kinase C (PKC) by phorbol 12‐myristate 13‐acetate (PMA) induces rapid L‐selectin down‐regulation of L‐selectin surface expression in both lymphocytes and granulocytes, the PKC inhibitor, H 7, was also found to down‐regulate lymphocyte and granulocyte L‐selectin surface expression. The inhibitor H 7 synergized with vanadate in down‐regulating lymphocyte L‐selectin surface expression, but partially inhibited vanadate‐induced granulocyte L‐selectin down‐regulation. The results suggest that in a cell type‐specific fashion the PKC system and tyrosine phosphorylation and dephosphorylation cascades are involved in the regulation of L‐selectin surface expression.
Early B‐Cell Factor (EBF) Down‐Regulates Immunoglobulin Heavy Chain Intron Enhancer Function in a Plasmacytoma Cell Line
Authors: ÅKERBLAD P. , SIGVARDSSON M. , LEANDERSON T.
Published: November 2003

The immunoglobulin heavy chain intron enhancer contains two potential binding sites for early B‐cell factor (EBF). To investigate the functional properties of these, EBF was expressed in the EBF non‐expressing S194 plasmacytoma cell line and found to down‐regulate the activity of a co‐transfected immunoglobulin heavy chain intron enhancer reporter construct. The expression of an unrelated reporter construct was unaltered. Dividing the immunoglobulin heavy chain intron enhancer into two subregions showed that the EBF mediated down‐regulation of expression was mediated by at least two independent sites. These data indicate a role for EBF in the regulation of immunoglobulin gene expression.
Kinetics, Localization and Isotype Profile of Antibody Responses to Immune Stimulating Complexes (Iscoms) Containing Human Influenza Virus Envelope Glycoproteins
Authors: SJÖLANDER A. , LÖVGREN BENGTSSON K. , JOHANSSON M. , MOREIN B.
Published: November 2003

The immune stimulating complex (iscom) is a particulate adjuvant formulation combining multimeric presentation of antigen with a built‐in adjuvant, Quillaja saponin. Iscoms induce strong serum antibody responses that are readily boosted. To further characterize this property of iscoms, the development and maturation of primary and secondary antibody responses to iscoms containing influenza virus antigen were investigated, in serum by ELISA and on single B‐cell level by ELISPOT. After a single subcutaneous injection, B cells secreting antigen‐specific IgG (IgG‐SC) were primarily observed in the draining lymph nodes (LN), showing peak numbers at day 7 which then declined rapidly. Serum IgG levels, as well as IgG‐SC in the spleen, persisted for several weeks and, with time, IgG‐SC cells also appeared in the bone marrow (BM). These results suggest that the IgG response to iscoms initially is located to the LN but that IgG‐SC are redistributed with time and may persist for a long time in other organs, including the spleen and BM. Moreover, a booster dramatically enhanced the frequency of IgG‐SC in LN, spleen and BM suggesting that iscoms induce a potent B‐cell memory. Comparisons of antibody responses to iscoms with those to influenza virus antigen in Freund’s complete adjuvant, TiterMaxTM or aluminium hydroxide suggest that the choice of adjuvant influences both the magnitude, kinetics, localization and isotype profile of antibody responses.
Degradation of Serum Amyloid A in Amyloid‐Susceptible and Amyloid‐Resistant Mouse Strains
Authors: ELLIOTT‐BRYANT R. , LIANG J.‐S. , SIPE J. D. , CATHCART E. S.
Published: November 2003

Degradation of serum amyloid A (apoSAA) by resident peritoneal cells (RPCS) and conditioned medium (CDM), prepared with RPCS, from amyloid‐susceptible CBA/J mice, amyloid‐resistant CE/J mice and their amyloid‐resistant CBA/J × CE/J F1 progeny was investigated in vitro. Serum amyloid A was derived from murine acute phase (AP) plasma and associated with high density lipoprotein (HDL). Degradation of apoSAA by RPCS and CDM from CBA/J mice was complete while degradation by RPCS and CDM from CE/J mice did not occur. Degradation of apoSAA by RPCS and CDM from CBA/J × CE/J F1 hybrid mice was indistinguishable from that by RPCS and CDM from the CBA/J parent. Intermediate fragments were not detected with either RPCS or CDM from CBA/J mice or CBA/J × CE/J F1 hybrid mice. Degradation of apoSAA was inhibited by phenylmethanylsulfonyl fluoride (PMSF) indicating that the enzyme, secreted into the fluid phase, was a serine esterase. Unlike apoSAA, HDL‐associated apoA‐1 remained intact. It was thus concluded that while selective degradation of HDL‐associated apoSAA (apoSAA‐HDL) by RPCS from the CBA/J and CE/J mice was significantly different, the genetic study did not support the hypothesis that there was direct linkage between impaired degradation of apoSAA‐HDL in the CE/J mouse strain and protection against amyloid fibril formation. As amyloid resistance in CBA/J × CE/J F1 hybrid mice is not attributable to failure to express the amyloidogenic isoform apoSAA2, the study supports the original hypothesis that amyloid resistance may be linked to expression of apoSAAcej
Recognition of Human Renal Cell Carcinoma and Melanoma by HLA‐A2‐Restricted Cytotoxic T Lymphocytes is Mediated by Shared Peptide Epitopes and Up‐Regulated by Interferon‐γ
Authors: Bernhard H. , Maeurer M. J. , JÄger E. , Wölfel T. , Schneider J. , Karbach J. , Seliger B. , Huber C. , Storkus W. S. , Lotze M. T. , Meyer zum Büschenfelde K.‐H. , Knuth A.
Published: November 2003

Cytotoxic T lymphocytes (CTL) have previously been isolated from peripheral blood of patients with renal cell carcinoma (RCC). The CD8‐positive CTL line MZ1257‐CTL‐5 (CTL‐5) has been shown to lyse autologous cultured RCC cells in an HLA‐A2 restricted fashion. Allogeneic, HLA‐A2‐matched RCC and melanoma cell lines were also lysed by CTL‐5, suggesting that melanoma and renal cancer share antigenic determinants. The aim of the study was to determine whether RCC and melanoma share peptide epitopes that are recognized by CTL‐5 in the context of HLA‐A2 molecules. Peptides were acid‐eluted from various cell lines, separated by reversed phase high performance liquid chromatography (RP‐HPLC), and assessed for their ability to reconstitute the CTL‐5‐defined epitope by pulsing the peptides on HLA‐A2 positive antigen‐processing mutant cell line CEM × 721.174.T2 (T2). Peptides eluted from allogeneic HLA‐A2‐matched RCC and melanoma cell lines exhibited the CTL‐5‐defined epitope in the same HPLC fractions as peptides derived from the autologous RCC line. Renal cancer and melanoma cells preincubated with interferon‐γ (IFN‐γ) resulted in an additional peak of reconstitution activity in both cell types. This second lytic peak was also observed when high amounts of autologous RCC cells were used for peptide preparation without IFN‐γ pretreatment, indicating that IFN‐γ increases the amount of MHC class I/peptide complexes per cell, rather than inducing a neo‐epitope.
Kinetics and Isotype Profile of Rheumatoid Factor Production During Viral Infection: Organ Distribution of Antibody Secreting Cells
Authors: Fazekas G. , Rosenwirth B. , Dukor P. , Gergely J. , Rajnavölgyi É.
Published: November 2003

The kinetics and isotype profile of influenza virus‐specific IgG antibodies were studied in correlation with the serum titre of IgG‐reactive autoantibodies. An increased level of IgG isotype‐specific, rheumatoid factor‐type autoantibody secretion was observed in the late phase of the virus‐specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti‐viral antibody response. As revealed by a preparative immunosorbent technique combined with isotype quantitation the majority of IgG2a‐ or IgG1‐bound immunoglobulins isolated from the serum of virus‐infected mice belonged to the same subclass as the target antibody. Comparison of the kinetics of appearance and the number of IgM‐, IgG‐ and IgA‐type IgG2a‐reactive autoantibody secreting cells during the primary and memory anti‐viral antibody responses showed isotype switch of IgM rheumatoid factor secreting cells predominantly to IgA. Localization of IgM and IgA antibody secreting cells demonstrated the wide organ distribution of IgM‐type rheumatoid factor secreting cells. On the contrary, IgA rheumatoid factor production was observed only in Peyer's patches and at the site of the local virus‐specific immune response, i.e. in mediastinal lymph nodes and in the lung. These results demonstrate that B cells specific for self IgG are activated and differentiated in concert with the virus‐specific antibody response in similar microenvironments. The predominant involvement of the mediastinal lymph nodes and the spleen in the production of IgG2a‐specific IgM‐type autoantibodies suggest a regulatory function of this type of autoantibodies in modulating IgG2a production in both systemic and local anti‐viral immune responses. The results also suggest a strictly regulated rheumatoid factor production which, however, can be unbalanced by repeated viral infections resulting in the escape of high affinity, isotype‐switched autoantibodies.
Monocyte‐Dependent Costimulatory Effect of TGF‐β1 on Rat T‐Cell Activation
Authors: SCHIÖTT Å. , SJÖGREN H. O. , LINDVALL M.
Published: November 2003

TGF‐β1 is known to have suppressive effects on both T‐cell proliferation and effector functions, but costimulatory effects have also been reported. In the present investigation the effect of TGF‐β1 is studied in vitro on T‐cell proliferative responses of rat spleen cells and of lymph node cells to alloantigens (MLR), the superantigen Staphylococcal enterotoxin A (SEA) or IL‐2. Without addition of TGF‐β1, adherent, freshly isolated rat spleen monocytes have a suppressive effect on T‐cell activation, which upon addition of TGF‐β1 is reversed to a strong costimulatory effect. The costimulatory effect of TGF‐β1 is shown to be entirely dependent on the presence of fresh monocytes. Costimulation is demonstrated when TGF‐β1 is added to spleen cells at the start of the in vitro assays but not when added more than 24 h after the start. Costimulation is not demonstrable when TGF‐β1 is added to lymph node cells alone but is readily detectable after admixture of freshly isolated spleen monocytes to the lymph node cells. TGF‐β1 added at the end of culture induces suppression of T‐cell activation irrespective of the presence or absence of monocytes. When TGF‐β1 is added both at the start of an MLC and again after 4 days, the costimulatory effect is maintained, although somewhat moderated. The costimulatory effect of TGF‐β1 is demonstrated as an increase of the T blast cell population of both CD4+ IL‐2R+ and CD8+ IL‐2R+ T‐cell subsets, whereas the suppressive effect of TGF‐β1 is shown as reduction of the same parameters.
Immunosuppressive Properties of Plasma and Lymphocytes from the Human Retroplacental Blood
Authors: XIAO LINSHENG , NORMAN A. M. , ENEROTH P. H. E.
Published: November 2003

The retroplacental compartment in which maternal blood is in direct and continuous contact with trophoblasts derived from the fetus seems to be the place where maternal and fetal cells regulate each others' activities. To assess the role of the retroplacental compartment in the immune regulation of pregnancy, 10 healthy pregnant women were selected for evaluation of the immune activity of their lymphocytes and plasma from the retroplacental blood. The results showed that the proliferation and cytotoxic capacity of these lymphocytes, but not of maternal peripheral lymphocytes, against fetal cells were significantly inhibited in unidirect mixed lymphocyte culture and direct cell‐mediated cytotoxic assay, respectively. The plasma from retroplacental blood showed significant immunosuppressive properties and depressed the proliferation of maternal peripheral lymphocytes stimulated by fetal HLA as well as the cytotoxic activity of these cells against the fetal lymphocytes. The present data suggest that the retroplacental compartment seems to be an immunosuppressive barrier, protecting the fetus from maternal rejection.
Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells
Authors: SCHREIBER L. , SHARABI Y. , SCHWARTZ D. , GOLDFINGER N. , BRODIE C. , ROTTER V. , SHOHAM J.
Published: November 2003

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ‐irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non‐thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti‐CD2 and anti‐LFA‐1 antibodies. The immature CD3−/+dull CD4+CD8+ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC‐induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid‐induced thymocyte apoptosis is p53‐independent, glucocorticoids are conceivably not involved in TEC‐induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.
Cloning of Two Distinct cDNAs Encoding Parvalbumin, the Major Allergen of Atlantic Salmon (Salmo salar)
Authors: LINDSTRØM C. D‐V. , VAN DÔ T. , HORDVIK I. , ENDRESEN C. , ELSAYED S.
Published: November 2003

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin β (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal s1 ). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high‐performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal s1 was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST‐ and ELISA‐inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA‐library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the β‐lineage of parvalbumins and include the entire coding region.
Sendai Virus‐Induced IFN‐α Production Analysed by Immunocytochemistry and Computerized Image Analysis
Authors: ÅKERLUND K. , BJÖRK L. , FEHNIGER T. , POHL G. , ANDERSSON J. , ANDERSSON U.
Published: November 2003

IFN‐α production in Sendai virus‐stimulated human buffy coat cultures could readily be demonstrated in individual cells at a protein level by the use of a novel immuno‐enzymatic staining procedure. A distinctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN‐α in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colour camera, which then transfers binary images directly into a computer‐controlled operating system. The characteristic appearance of the immunocytochemical staining enabled a computerized image‐analysis system to measure IFN‐α producing cells based on defined criteria set for morphology, intensity, colour and size. The automated system could accurately and reproducibly register a range of 0.1–7.0% of the total cell population as IFN‐α producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscopy and image analysis concerning the assessment of the incidence of IFN‐α producing cells in the total cell populations. All IFN‐α producing cells expressed surface HLA‐DR molecules and 95% of these cells belonged to the myelomonocytic lineage. The image analysis system provided, in contrast to conventional microscopy, an opportunity to assess and document differences of signal intensity and cell size of individual IFN‐α producing as well as non‐producing cells.
Comparison of Surface Properties of Human IgA, IgE, IgG and IgM Antibodies with Identical and Different Specificities
Authors: WINGREN C. , MICHAELSEN T. E. , MAGNUSSON C. G. M. , HANSSON U.‐B.
Published: November 2003

In this paper, the authors report the use of liquid‐liquid partition chromatography (LLPC) in an aqueous polyethylene glycol (PEG)/dextran two‐phase system to compare the surface properties (partition properties) of human antibodies and fragments thereof. The surface properties of all the monoclonal antibodies of different classes and subclasses investigated were within the same broad range as that observed for the polyclonal antibodies and no relationship was found between the exposed surfaces of the immunoglobulins (Ig) and their heavy chain isotype. Moreover, Fc fragments from various IgG1, 2 and 4 myeloma proteins were found to exhibit similar surface properties. Employing chimeric antibodies with identical variable regions the authors found that intact IgG1, 2 and 4 displayed identical surface properties, while the corresponding IgA1, IgA2, IgG3, IgE and IgM antibodies differed both from each other and from the IgGs. The surface properties of chimeric IgG3 could be made similar to those of the IgG1, 2 and 4 chimers by partially reducing the length of the hinge section, but new differences in surface properties appeared when their hinges were of similar length. Thus, LLPC can be used to detect differences or similarities in the surface properties of the antigen‐binding regions as well as the Fc part in the various isotypes. This can shed light on biological activities such as antigen binding and effector function.
The Age‐Associated Increase in Autoreactive Immunoglobulins Reflects a Quantitative Increase in Specificities Detectable at Lower Concentrations in Young Mice
Authors: NOBREGA A. , HAURY M. , GUERET R. , COUTINHO A. , WEKSLER M. E.
Published: November 2003

Serum immunoglobulins reactive with several autoantigens have been reported to increase with age. The authors have studied the reactivity of serum immunoglobulins from mice between 2 and 24 months of age with antigens present in lysates of syngeneic tissue extracts from young mice. The profile of immunoglobulin binding with the immunoblots of spleen and brain tissue increased progressively with age, showing only minor differences from mouse to mouse and, with one exception, revealing that the age‐associated increase in binding of immunoglobulins occurred with antigens with the same migratory position in the immunoblots detectable, at lower concentration, in sera from young mice. Not all sera from older mice had increased immunoglobulin binding when tested with extracts of skin, muscle and liver but those that did expressed increased binding with antigens in all three lysates and with the same profile shown by sera from young mice. These results are consistent with the hypothesis that the age‐associated increase in autoreactive immunoglobulins represents a selective increase in autoreactive specificities expressed by serum immunoglobulins from young animals at lower levels.
Binding Specificities of a Polyreactive and a Monoreactive Human Monoclonal IgG Rheumatoid Factor: Role of Oligosaccharides
Authors: AL‐BALAGHI S. , ABEDI‐VALUGERDI M. , MÖLLER E.
Published: November 2003

The immunological specificites of two human rheumatoid factor‐reactive IgG monoclonal antibodies derived from unstimulated rheumatoid synovial lymphocytes have been analysed. A malaria antigen‐reactive IgG monoclonal antibody from an immune donor served as a control. Purified IgG monoclonal antibody from one IgG‐RF hybridoma (L1), but not from the other IgG‐RF hybridoma (D1) or the anti‐malaria monoclonal antibody, exhibited dose‐dependent binding to multiple self and non‐self antigens such as ds‐DNA, cytochrome‐c, bovine thyroglobulin, transferrin, cellulose and lipopolysaccharide and therefore was considered polyreactive. The immunological specificity was confirmed by inhibition experiments using the same soluble antigens as inhibitors. The polyreactivity of the IgG‐RF MoAb was markedly inhibited by absorption with glycoproteins such as thyroglobulin, a commonly used target for xenoreactive natural antibodies, and cytochrome‐c, indicating that the monoclonal antibody is reactive with epitopes expressed on these ligands. Since some naturally occurring antibodies are carbohydrate specific, the authors tested the IgG‐RF MoAb for possible carbohydrate specificity. Absorption with certain polysaccharides containing only one or two different sugar moieties did not inhibit the binding reactivities to any of the tested antigens. Polyreactivity of the monoclonal antibody, unlike most xenoreactive natural antibodies, was not caused by reactivity with (galα1‐3gal) as indicated by the remaining binding reactivity after α‐galactosidase treatment of the antigen. Removal of the N‐linked glycosylation sites within the Fc portion of target IgG markedly reduced the antibody binding. The findings suggest that the carbohydrate content of the antigen is necessary for binding of the polyreactive IgG‐RF MoAb. Reactivity to carbohydrate antigens may readily explain the so‐called multispecificity of certain antibodies.