bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of phormium yellow leaf phytoplasma in New Zealand flax (Phormium tenax) using nested PCRs
Authors: Andersen M. T. , Beever R. E. , Gilman A. C. , Liefting L. W. , Balmori E. , Beck D. L. , Sutherland P. W. , Bryan G. T. , Gardner R. C. , Forster R. L. S.
Published: March 2003

A reliable diagnostic method was developed for use in studying the relationship between phormium yellow leaf disease of New Zealand flax (Phormium tenax) and its associated phytoplasma (phormium yellow leaf phytoplasma: PYL). Diagnosis involved a nested PCR (polymerase chain reaction) technique targeting the 16S rRNA gene. DNA was extracted from woody rhizome tissues of NZ flax plants using CTAB and a high salt precipitation step. This method effectively eliminated polysaccharides, gum‐like material and other compounds inhibitory to PCRs that occur at high concentrations in diseased NZ flax rhizomes. PCR competence of each DNA preparation from both healthy and yellow leaf diseased plants was assessed using the general prokaryotic 16S rRNA gene primers, Gd1/Berg54. These primers amplified DNA from both diseased and healthy plants. PYL 16S rDNA sequences were not detected consistently following amplification by PCR (35 cycles) using the ‘universal’ phytoplasma‐specific primer pairs R16F2/R16R2 or P1/P6. By contrast, PYL was consistently detected in diseased, but not healthy, NZ flax plants, following nested PCR of the products of the above three primer pairs. Nested PCRs involve the primers NGF/NGR, which were designed to hybridize with all phytoplasmas for which published sequences were available. The most sensitive level of detection by nested PCR was achieved using primers R16F2/R16R2, rather than primers P1/P6 or Gd1/Berg54, for the primary amplification step. The consistent association found in this study between yellow leaf disease and PYL further substantiates this phytoplasma as the causal agent. PCR products of the expected size were also amplified by nested PCR using the primers R16F2/R16R2 followed by NGF/NGR from C. roseus tissues infected with five other phytoplasmas representing three distinct phytoplasma groups. Therefore nested PCRs with these pairs of primers should be useful for detecting other phytoplasmas, in particular those occurring at low concentrations or in recalcitrant tissues.
Effect of metalaxyl on formation and germination of oospores of Phytophthora infestans
Authors: Hanson K. , Shattock R. C.
Published: March 2003

Oospores of Phytophthora infestans were produced in potato leaf discs floating on metalaxyl solution (100 μg mL−1 a.i.) and inoculated with all combinations of two metalaxyl‐sensitive and two ‐resistant parental isolates. Numbers of oospores produced varied between different matings, depending on parents, in the absence of the fungicide and when metalaxyl was added 0, 7, 14 and 21 days after inoculation. Oospores were not produced when metalaxyl was added at the time of inoculation (0 days) when either one or both parents were sensitive to metalaxyl. In two of three such matings further oospore formation was arrested when metalaxyl was added either 7 or 14 days after inoculation. Oospores extracted from leaf discs 14, 21 and 28 days after inoculation were assessed for germination on water agar after 21 days. Germination of oospores from water control treatments varied between 6 and 30% depending on the cross. Germination was significantly reduced in oospores of metalaxyl‐sensitive parents extracted 28 days after inoculation of leaf discs treated with metalaxyl 0, 7 and 14 days after inoculation compared with the 21‐day treatment. Minimal differences in germination were observed for oospores from the mating of resistant parents irrespective of metalaxyl treatment, although germination was generally low, not exceeding 8.5%.
Characterization and partial sequence of a new furovirus of wheat in China
Authors: Ye  , Zheng  , Chen  , Diao  , Adams  , Yu  , Antoniw
Published: March 2003

A soil‐borne wheat virus causing severe mosaic and stunting symptoms on wheat in China has been characterized. It had been considered to be soil‐borne wheat mosaic virus (SBWMV) because of its rod‐shaped virions and similarities to epidemiology and host range. In this study, the virions purified from infected wheat tissue were approximately 20 nm in diameter and of two lengths (140–160 nm and 280–300 nm), with a coat protein of 19 kDa and two RNA components of approximately 7 and 3.5 kb. A rabbit antiserum was produced against the virus and a serological relationship to SBWMV from the USA (Oklahoma) was demonstrated. However, the coat protein was not recognized by most monoclonal antibodies against Oklahoma SBWMV in either ELISA, ISEM or Western blot analysis, indicating epitope differences. In RT‐PCR experiments the viral nucleotide sequences were significantly different from those of SBWMV, and this was confirmed by partial sequencing of the cloned PCR fragments generated from RNA1 (c. 1100 nt) and RNA2 (c. 1400 nt), which showed homologies of about 79 and 63%, respectively, to corresponding regions of SBWMV. Because of these significant differences in serology and nucleotide sequence it is suggested that it is a new furovirus for which the name Chinese wheat mosaic virus (CWMV) is proposed.
Detection and recovery of Rhizoctonia solani in naturally infested glasshouse soils using a combined baiting, double monoclonal antibody ELISA
Authors: Thornton  , O'Neill  , Hilton  , Gilligan
Published: March 2003

A combined baiting, double monoclonal antibody immunoassay was developed that allows specific and sensitive detection of the economically important soil‐borne plant pathogen Rhizoctonia solani in naturally infested soils. The assay is quick, taking only three days to complete from receipt of soil samples and the immunoassay format allows recovery of Rhizoctonia isolates from colonized baits for determination of anastomosis group (AG) affiliation and pathogenicity. The assay was tested on naturally infested soils from commercial glasshouses used to grow lettuce. Using the immunoassay, conventional anastomosis tests against known AG isolates, and pathogenicity tests, it was shown that R. solani isolates recovered from soil samples were pathogenic towards lettuce and belonged to AG4. Furthermore, those isolates that exhibited strong pathogenicity towards lettuce were recovered from sites that had experienced severe Rhizoctonia damage in previous lettuce crops. The possibility of developing a preplanting test to predict damage to specific crop plants due to the presence of particular AGs in the soil is discussed.
The occurrence of two phytoplasmas associated with stunted Rubus species in the UK
Authors: Davies
Published: March 2003

Polymerase chain reaction using universal primers to sequences in the 16S rRNA gene, and group‐specific primers to sequences in the 16S/23S spacer region, revealed two distinct phytoplasmas occurring in Rubus plants showing symptoms of rubus stunt. One phytoplasma appeared similar to phytoplasmas in the elm yellows group; the other appeared to fall into the X disease group. This finding was confirmed by RFLP analysis of PCR products. This is the first identification of phytoplasmas from either of these groups occurring in the UK, and the first report of a phytoplasma belonging to the X disease group in Rubus.
Characterization of new sources of resistance to Uromyces viciae‐fabae in a germplasm collection of Vicia faba
Authors: Sillero J. C. , Moreno M. T. , Rubiales D.
Published: March 2003

A collection of 648 accessions of Vicia faba was screened for resistance to faba bean rust (Uromyces viciae‐fabae). Two distinct types of resistance were identified, both resulting in reduced disease severity (DS) and area under the disease progress curve (AUDPC), but differing in the expression of hypersensitivity. One should be regarded as incomplete nonhypersensitive resistance and the other as incomplete resistance with late hypersensitivity. The hypersensitive resistance, which has not been reported before, was not dependent on temperature or plant age. These two types of resistance were characterized by three macroscopic components of resistance: increased latent period (LP), decreased colony size (CS) and a relatively reduced infection frequency (IF), both on seedlings and on adult plants. LP and CS were the components of nonhypersensitive resistance most highly correlated with DS and AUDPC measured under field conditions. The presence of necrosis was an additional component in the hypersensitive resistant response.
Incidence and natural spread of apple mosaic ilarvirus in hazel in north‐east Spain
Authors: Aramburu J. , Rovira M.
Published: March 2003

Apple mosaic ilarvirus (ApMV) was detected in all (160) hazel (Corylus avellana) cv. Negret orchards assayed in Catalonia, north‐east Spain, during 1997 and 1998. In a preliminary survey, using enzyme‐linked immunosorbent assay (ELISA), only 9 of the 320 samples collected appeared free from ApMV and three of these individuals indexed ELISA positives when re‐tested. The spread of ApMV was studied in a plantation of Negret hazel in which virus‐free trees were randomly interspersed with graft‐inoculated individuals. When tested by ELISA 10 years after planting, 15% of the ungrafted trees contained ApMV. At that time, 8% of seeds on ungrafted and virus‐free trees and 54% of seeds on ApMV‐inoculated trees indexed ApMV‐positive. These results are discussed in terms of pollen‐mediated inoculation.
Reduced sensitivity of Cercospora beticola isolates to sterol‐demethylation‐inhibiting fungicides
Authors: Karaoglanidis G. S. , Ioannidis P. M. , Thanassoulopoulos C. C.
Published: March 2003

In a survey conducted during October 1995, single‐lesion isolates of the sugar beet leaf‐spot fungus, Cercospora beticola, were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf‐spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED50 values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED50 values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL−1, respectively (significantly different at P = 0·05). For bitertanol the mean ED50 values for the Serres and Imathia populations were 0·72 and 0·81 µg mL−1, respectively, which were not significantly different at P = 0·05. The mean ED50 value of the Amyndeon population was 0·48 µg mL−1, which was significantly lower than those of the other two populations (P < 0·05). A cross‐resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED50 values of 60 isolates were subjected to a linear regression analysis (r = 0·81).
Comparative analysis of powdery mildew development on leaves, seedlings and flower panicles of different genotypes of cashew
Authors: Sijaona M. E. R. , Clewer A. , Maddison A. , Mansfield J. W.
Published: March 2003

Resistance of cashew (Anacardium occidentale) genotypes to powdery mildew (Oidium anacardii) was assessed on detached leaves, seedlings and flower panicles. Leaves were more susceptible before the development of a waxy cuticle, which confers a dull appearance. Differences in susceptibility/resistance were observed on detached leaves inoculated in the laboratory. Some genotypes supported dense growth of mildew, whereas on others only slight infection was observed under comparable conditions. Mature leaves were immune to colonization. In the field the rate of mildew infection, as estimated from disease progress curves on both seedlings and flower panicles, varied significantly between genotypes allowing their categorization into susceptible and partially resistant clones. A similar rank order for degree of susceptibility/resistance was determined from studies on detached leaves, seedlings and flower panicles. Brazilian introductions ofA. occidentaleand the AZA2 and AC6 genotypes proved to be partially resistant, while AM6, ATA19 and AIN62 were highly susceptible. The relationship between leaf and flower mildew infection showed positive and highly significant correlations. It is concluded that each of the three approaches to the assessment of disease susceptibility can be used to screen genotypes against mildew attack. Screening in the laboratory provides a rapid indication of potential resistance providing that leaves are compared at the same stages of maturity.
Emergence of silver scurf (Helminthosporium solani) as an economically important disease of potato
Authors: Errampalli D. , Saunders J. M. , Holley J. D.
Published: March 2003

During the 1990s, silver scurf (causal agent Helminthosporium solani) emerged as an economically important disease of table stock and processing potatoes (Solanum tuberosum). The pathogen attacks the periderm of the potato tuber causing blemishes. The disease cycle of silver scurf has both field and storage phases. Primary infection occurs in the field and high relative humidity favours the spread and increase of silver scurf in potato stores. Control of the disease by chemical and cultural practices remains difficult. Increase in disease has been attributed to H. solani isolates resistant to the postharvest fungicide thiabendazole (TBZ). Polymerase chain reaction (PCR)‐based detection methods for H. solani and TBZ‐resistant isolates are rapid and more specific than traditional identification. This review discusses the biology of the pathogen, epidemiology of the disease, detection of the pathogen and integrated control measures for the management of silver scurf in both field and potato tuber stores.
Heterothallism in Plasmopara viticola
Authors: Wong F. P. , Burr H. N. , Wilcox W. F.
Published: March 2003

Sexuality in the oomycete Plasmopara viticola, the causal agent of grapevine downy mildew, was studied using isolates from five populations from North America and Europe. Leaf discs of Vitis vinifera cv. ‘Chardonnay’ were inoculated with either individual single‐sporangiophore isolates, or in all possible pairwise combinations of 25 isolates from New York State, USA. The occurrence of oospores in leaf discs indicated that the pathogen was heterothallic with two mating types, P1 and P2 in a ratio of 11 : 14 for this population. Heterothallism was confirmed when three representative isolates of each mating type from New York were coinoculated with each of 40 isolates from populations of P. viticola from Michigan, Missouri (USA), Germany and Italy. For each isolate tested, oospores formed with either test isolates of P1 or test isolates of P2 mating types, indicating that the isolates were exclusively P1 or P2 only. For these same isolates, no oospores formed as a result of self‐crosses. The ratio of P1 : P2 mating types for all isolates in the study was 27 : 38, statistically equivalent to a 1 : 1 ratio according to χ2 analysis (P = 0·68).
Haustoria of microcyclic rust fungiUromyces ficariaeandPuccinia tumidaand other gall‐forming species,U. dactylidis(macrocyclic) andP. smyrnii(demicyclic)
Authors: Quilliam R. S. , Shattock R. C.
Published: March 2003

Cytological studies involving light and scanning electron microscopy examined the morphology of intracellular hyphae of various gall‐forming species of rust fungi.Uromyces ficariae(microcyclic) andU. dactylidis(macrocyclic, heteroecious) both produced vermiform and largely indeterminate intracellular hyphae typical of M‐haustoria, associated with telial and aecial galls, respectively, on their common hostRanunculus ficaria. Similar M‐haustoria were observed in telial galls ofPuccinia tumida(microcyclic) onConopodium majus, in host tissue affected by nine other microcyclic species and in aecial galls ofP. smyrnii(demicyclic, autoecious) onSmyrnium olusatrum. Telia ofP. smyrnii, however, infecting photosynthetically active mesophyll without gall formation, produced typical D‐haustoria with determinate growth, a narrow neck region and a bulbous clavate body. Gall formation was associated with hyphal infection of vascular tissue. The relationship between M‐haustoria, vascular infection and gall formation is discussed in relation to the evolution of microcyclic rusts and Tranzschel's law.
Expression of chL12 Surface Antigen is Associated with Cell Survival in the Avian Bursa of Fabricius
Authors: Lampisuo  , Arstila  , Liippo  , Lassila
Published: March 2003

During B‐cell development in the avian bursa of Fabricius most of the developing B cells die by apoptosis and only a minority survive to emigrate into the periphery. Recently, it has been shown that when developing bursal cells become mature and ready to migrate they start to express chL12 antigen. The expression of this cell‐surface molecule was found to be associated with the survival of the bursal cells both after in vitro culture and after in vivo cyclophosphamide (CY) treatment. The frequency of early apoptotic cells in freshly isolated bursal cells was found to be high. The high susceptibility of these cells to apoptosis is in line with the finding of low bcl‐2 mRNA expression. We conclude that expression of avian chL12 antigen is associated with the survival of bursal cells.
Flow Cytometric Analysis of Cellular Changes in Mice after Intradermal Inoculation with a Liposome–Iscom Adjuvanted Vaccine
Authors: San Gil  , Turner  , Mullbacher  , Walker  , Djordjevic  , Eamens  , Chin
Published: March 2003

As it is not known what changes to leucocyte homeostasis are mandatory for effective adjuvant action, the biological relevance of systemic changes elicited by different vaccine formulations can only be interpreted in the context of the immunological outcomes. We used flow cytometry to quantify the changes in leucocyte subsets induced in mice intradermally immunized with SAMA4 (adjuvant group), outer membrane proteins (OMP) purified from Actinobacillus pleuropneumoniae (OMP antigen group), SAMA4 adjuvanted OMP (OMP vaccine group), or phosphate‐buffered saline (PBS: control group). This approach allowed direct comparisons to be made between the effects of antigen, adjuvant or antigen–adjuvant complexes on immune effector cell populations. Antigens complexed with the liposome–iscom hybrid adjuvant, SAMA4, generated strong antibody responses and cytotoxic T‐cell activity in animals immunized intradermally, reflecting remobilization and recruitment of specific cell populations. Splenomegaly, due to granulocytosis, monocytosis and megakaryocytosis, was most prominent in the OMP vaccine group. Histological examination of spleen sections confirmed that these changes were due primarily to splenic haematopoiesis. Circulating numbers of granulocytes and monocytes increased significantly (P < 0.05) in the blood of the OMP vaccine group, as did granulocyte numbers in the lungs (P < 0.05). No changes in T‐ and B‐cell numbers were detected by flow cytometry in the spleens, lungs or blood over the 28‐day period in any treatment group. Thymocyte numbers (predominantly CD4+CD8+ cells) in the OMP vaccine group fell by 95% within 3 days of immunization. Identical cellular responses were obtained when an innocuous antigen, ovalbumin, was complexed with SAMA4 instead of OMP, thus demonstrating that the adjuvant effects of SAMA4 were due to synergistic interaction between antigen and adjuvant and not due to the presence of toxic components. The association of strong adaptive immune responses with such complex changes in leucocyte homeostasis induced by complexing adjuvant and antigen suggested that the changes were important for effective vaccination and were not purely circumstantial.
Allospecific Helper T‐Lymphocyte Repertoire in Monozygotic Twins
Authors: Reisæter  , Thorsby  , Brinchmann
Published: March 2003

In the course of maturation in the thymus there is a selection of T lymphocytes based on the avidity between their T‐cell receptors and HLA/peptide complexes expressed on stromal thymic cells. The repertoire of mature T lymphocytes is further modulated by encounters with foreign antigens. Thus, the antigen specific repertoire of the peripheral T‐lymphocyte pool is determined by genetic and environmental factors. We recently reported that pairs of monozygotic twins often display significant differences in their allospecific cytotoxic T‐cell repertoire, suggesting an important role of confrontation with foreign antigens on the CD8+ T‐cell repertoire. We have now performed similar studies on the repertoire of allospecific CD4+ T lymphocytes. Using positively selected CD4+ T cells in limiting dilution analyses we compared the differences in the allospecific helper T‐lymphocyte precursor frequencies (HTLpf) between pairs of genetically identical monozygotic twins and pairs of unrelated, HLA disparate individuals. We found that all monozygotic twin pairs and most unrelated pairs had similar HTLpf to the same stimulator, i.e. the 95% confidence intervals were overlapping. However, when studied in greater detail, the differences in HTLpf within monozygotic twin pairs were found to be significantly smaller than the differences within pairs of unrelated responders. Thus, we find evidence of an influence by environmental antigens also on the repertoire of allospecific CD4+ T cells, but polymorphic genetic factors seem to be more important here than for the repertoire of allospecific CD8+ T cells.
Expression and Function of c‐Met, a Receptor for Hepatocyte Growth Factor, During T‐Cell Development
Authors: Tamura  , Sugawara  , Tokoro  , Taniguchi  , Fukao  , Nakauchi  , Takahama
Published: March 2003

The c‐Met oncoprotein is a cell‐surface receptor for hepatocyte growth factor (HGF). Signals through HGF and c‐Met have been appreciated for their crucial roles in the development of many cell types, including liver cells. The present study examined whether c‐Met is expressed in the thymus and whether c‐Met/HGF signals can regulate T‐cell development in the thymus. We have found that mRNA transcripts encoding c‐Met are expressed in mouse thymus. The c‐Met transcripts were expressed at higher levels in fetal and neonatal thymus than in adult thymus, and were mostly expressed by lymphoid cells rather than by stromal cells. Interestingly, the addition of HGF to fetal thymus organ cultures increased the generation of mature T cells expressing high levels of T‐cell antigen receptors. These results indicate that c‐Met is expressed in the thymus during early ontogeny, and that c‐Met/HGF signals can promote T‐cell development.
Gene‐Encoded Peptide Antibiotics and the Concept of Innate Immunity: An Update Review
Authors: Boman
Published: March 2003

Antibacterial peptides were first considered rather species‐specific. However, the perspective began to change in 1987–89. Five years later there were two symposium volumes and several reviews on gene‐encoded peptide antibiotics which covered the known peptides irrespective of origin. The field is rapidly growing and a first update was published in this Journal in 1996. At that time a database was made with about 100 entries; now it has over 400, with some redundancy. Recently a methodological handbook was published and there are many specialized reviews covering only defensins or insect immunity. In the last 2 years, the larger perspective of innate immunity and the role of gene‐mediated peptide antibiotics have evolved in ways which justify a new update. Today insects and plants are known to have similar overall design of their defensins while insects and mammals have very similar control mechanisms. The signal pathways are beginning to appear and the future perspective may involve additional changes.
Differentiation of T Lymphocytes in the Human Adenoid as Measured by the Expression of CD45 Isoforms
Authors: Mattila  , Tarkkanen
Published: March 2003

Encounter of antigen by T lymphocyte on antigen‐presenting cells results in changes in the expression of several cell surface molecules, including the abundant cell surface glycoprotein CD45. We have characterized the expression of the CD45 isoforms CD45RA and CD45RO in CD4+ and CD8+ T lymphocytes in the adenoids and peripheral blood of young children. We found that the relative proportions of CD45RA−,CD45RO+ antigen‐experienced T cells was higher in the adenoids than in peripheral blood, and that the proportion of naive or resting CD45RA+,CD45RO− T cells was lower in the adenoids than in peripheral blood. The frequency of bright double‐positive CD45RA+,CD45RO+ T cells, which represent cells in transition from the CD45RA+ to CD45RO+ phenotype, was higher in the adenoids than in peripheral blood. The frequency of another double‐positive cell population, but with unknown ontogeny, expressing both CD45RA and CD45RO at a low level, was higher in peripheral blood than in adenoidal T cells. It was found that the frequency of adenoidal antigen‐experienced CD45RA−,CD45RO+ T lymphocytes increased with increasing age of the child. These results are consistent with the model that the adenoids serve as a site for conversion of CD45RA+ to CD45RO+ T lymphocytes, and that the maturation of the immune system in young children is associated with phenotypic changes in T lymphocytes residing in secondary lymphoid organs.
RANTES Production in HIV‐1 Antigen‐Stimulated Whole Blood Culture: Relationship with Type 1 Immune Response and Plasma Viral Load in Individuals Infected with HIV‐1
Authors: Benyoucef  , Hober  , De Groote  , Bocket  , De La Tribonnière  , Mouton  , Wattré
Published: March 2003

Host factors which control replication and clearance of human immunodeficiency virus (HIV) are poorly understood. RANTES (regulated on activation, normal T cell expressed and secreted) and other β‐chemokines may be HIV‐1‐suppressive factors but their role in the progression of HIV‐1 infection is a subject of controversy. We investigated the relationship between production of RANTES and correlates of disease progression in 15 patients infected with HIV‐1. We used whole blood culture to study the production of RANTES, interferon (IFN)‐γ, interleukin (IL)‐4 and IL‐13 in response to supernatant of T cells infected with HIV‐1. A defect of RANTES production was associated with a predominant type 2 and decreased type 1 cytokine profile (IL‐4 and/or IL‐13 > IFN‐γ). We obtained a positive correlation between RANTES and IFN‐γ (P = 0.004) and the ratio of type 1 and type 2 cytokines IFN‐γ/IL‐4 (P = 0.04) and IFN‐γ/IL‐13 (P = 0.003), and a negative correlation between RANTES production and HIV‐1 RNA copy number in plasma (P = 0.01). The same pattern of correlation was observed between HIV‐1 p24‐stimulated production of RANTES and the plasma viral load (P = 0.02, n = 15). The measurement of RANTES produced by heparinized whole blood in response to HIV‐1 antigens appears as a potentially valuable tool to assess the defect of type 1 immune response in individuals infected with HIV‐1 and to define whether the absence of a RANTES response may play a role in the increased rate of HIV‐1 replication.
High‐Dose Intravenous Immunoglobulin Treatment Activates Complement In Vivo
Authors: Mollnes  , Høgåsen  , De Carolis  , Vaquero  , Nielsen  , Fontana  , Perricone
Published: March 2003

Several complement modulating effects of high‐dose intravenous immunoglobulins (IVIG) have been proposed from in vitro studies and experimental animal models. However, the in vivo effects of IVIG on plasma complement in humans are yet not known. We have investigated the in vivo effects of IVIG on complement in seven women with unexplained recurrent spontaneous abortion who were without evidence of autoimmune disease. Samples were obtained before and after the very first infusion of IVIG. There was a marked increase in immunoglobulin G (IgG) from (median and range) 12.4 (9.4–15.9) to 26.8 (22.4–30.0) g/l but no change in immunoglobulin A (IgA) or immunoglobulin M (IgM). A significantly increased complement activation was demonstrated using neoepitope‐specific enzyme immunoassays to the activation products C3bc (median increased from 9.8 to 31.2 AU/ml), Bb (0.66–1.66 g/ml), C5a (10.5–12.7 ng/ml), and TCC (0.81–2.19 AU/ml) (P = 0.015 for all). There were no changes in antigenic concentrations of individual complement components or regulators (C1q, C4, C3, C1‐inhibitor, C4b‐binding protein) and no decrease in complement haemolytic activity (classical and alternative CH50), which were all within the normal range. The classical pathway activation products C1rs/C1‐inhibitor complexes, C4bc and C4d were elevated in all patients before IVIG treatment and did not change significantly during treatment. In conclusion, IVIG induced a significant activation of complement in vivo.
Variation in Gut‐Homing CD27‐Negative Lymphocytes in Inflammatory Colon Disease
Authors: Meenan  , Spaans  , Grool  , Lammers  , Pals  , Tytgat  , Van Deventer
Published: March 2003

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin‐4 (IL‐4). Co‐expression of α4β7 identifies those cells with gut‐homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non‐inflammatory bowel disease (non‐IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, α4β7 and intracellular cytokines was performed. Circulating CD4+CD27– populations were increased in patients with CD (8.8 ± 0.8%, P < 0.001), UC (12.2 ± 1.9%, P < 0.001) and non‐IBD colitis (10.5 ± 1.3%, P < 0.01) as compared with controls (6.1 ± 0.5%). CD4+CD27−α4β7+ cells were increased in CD (P < 0.01). Lamina propria CD4+CD27− populations were depressed significantly in CD (P < 0.05), UC (P < 0.02) and non‐IBD colitis (P < 0.03). Mucosal CD4+CD27− cells synthesized IL‐4 in preference to interferon‐γ. Thus, colonic inflammation is associated with alterations in gut‐tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL‐4‐synthesizing cells.
Characterization of Soluble Terminal Complement Complex Assembled in C8β‐Deficient Plasma and Serum
Authors: Høgåsen  , Mollnes  , Nürnberger  , Pausa  , Fukumori  , Tedesco
Published: March 2003

Sera genetically deficient in either the α−γ or the β‐subunit of complement component C8 virtually lack haemolytic activity. We have studied the formation and the structural organization of the soluble terminal complement complex (TCC) assembled in these sera following activation with cobra venom factor (CVF). The TCC concentration in the activated C8α−γ and C8β‐deficient samples was 0.2% and 4%, respectively, when compared with zymosan‐activated normal serum. TCC was purified from the activated C8β‐deficient samples by affinity chromatography and analysed by immunoblotting and enzyme immunoassay. No C8β was detected in one TCC preparation, while 7% of the normal level was present in the other. The level of the other terminal components, including that of C8α−γ, was normal. The ability of C8α−γ to promote the assembly of TCC in the presence of a limited amount of C8β or in the apparent absence of this subunit was confirmed using purified components, by mixing C5b6 and either of the purified C8 subunits together with C7 and C9. These data show that soluble TCC can be formed in C8β‐deficient sera that contain little or no C8β.
Immunophenotypic Characteristics of Monocytes in Elderly Subjects
Authors: Stohlawetz  , Hahn  , Köller  , Hauer  , Resch  , Smolen  , Pietschmann
Published: March 2003

Since ageing is accompanied by various alterations of the immune system, the aim of the present study was to investigate the effect of age on the expression of surface markers in peripheral blood monocytes. We studied 28 healthy young subjects and 28 elderly subjects fulfilling the criteria of the SENIEUR protocol. Peripheral blood mononuclear cells were isolated and the expression of various surface markers was analysed by double colour flow cytometry. The mean fluorescence intensity of the intercellular adhesion molecule‐1 (CD54), CD29, CD45RO and CD32 was increased significantly in CD14+ cells of elderly people when compared with the young subjects. No significant differences were found in the expression of CD11a, CD11b, CD15, CD26, CD27, CD33, CD45RA, CD45RB, CD49d, HLA‐DR and CD65. In summary, we demonstrated significant increase in four monocyte surface markers in subjects of old age; this finding may be related to certain immune phenomena observed in ageing subjects such as auto‐immunity.
Prostaglandin‐Independent Stimulation of Interleukin‐6 Production by Fibrinogen Degradation Product D in Perfused Murine Liver
Authors: Csala  , Léránt  , Bánhegyi  , Kardon  , Puskás  , Mucha  , Machovich  , Falus  , Mandl
Published: March 2003

Bacterial endotoxin (LPS) and fibrinogen degradation product D (FDP‐D) are both potent stimulators of interleukin‐6 (IL‐6) production in liver, however, there are differences in their metabolic effects. The aim of the present study was to compare the role of prostaglandins in the enhancement of IL‐6 production by LPS or FDP‐D in perfused mouse livers. Indomethacin inhibited the effect of LPS significantly but was ineffective in the case of FDP‐D. Accordingly, production of prostaglandins D2 and E2 was not elevated following the addition of FDP‐D, while their formation was increased several fold by LPS. At the same time interleukin‐1 (IL‐1) production in perfused liver rose markedly upon the addition of FDP‐D. It is suggested that prostaglandins are not involved in the effects of FDP‐D on the liver. The stimulatory effect of FDP‐P on IL‐6 production might be the consequence of elevated IL‐1 levels.
Experimental Infections of Rana esculenta with Aeromonas hydrophila: A Molecular Mechanism for the Control of the Normal Flora
Authors: Simmaco  , Mangoni  , Boman  , Barra  , Boman
Published: March 2003

Frogs can be useful models for studying the mechanisms that may regulate their natural microbial flora. Their skin glands produce a secretion containing 20–30 different peptides, some antimicrobial some neurotrophic. As they often live in soil or silt that is rich in microbes, they can be expected to be able to prevent or eliminate infections in very short peroids of time. The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans. From an alternative frog strain of A. hydrophila, Bo‐3, we isolated a spontaneous and stable mutant (Bo‐3N), resistant to nalidixic acid, here used to follow the host–microbe interactions in experimental infection of mouth and skin of Rana esculenta. The skin peptides had been previously isolated, sequenced and cloned. We showed that skin treatment with a glucocorticoid (GC) cream blocked de novo synthesis of these peptides and, simultaneously, prepropeptide mRNAs disappeared while IκBα was up‐regulated. Experimental mouth infections with 20 million cells of A. hydrophila Bo‐3N showed that a normal wild frog can eliminate the bacteria from the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo‐3N below 3500 colony‐forming units (CFU)/5 μl ‘saliva’. An in vitro comparison showed that frog blood or serum allowed bacteria to grow, while the skin secretion killed the bacteria within 10 min. Using different enzyme‐linked immunosorbent assays (ELISAs) with rabbit anti‐Bo‐3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A. hydrophila. An assay for immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A. hydrophila only corresponding to a few per cent of the positive control. For skin infections we bathed the frogs for 10 min in an overnight culture of Bo‐3N diluted to about 107 CFU/ml. Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pretreatment with GC cream reduced the activity to about one‐third of that of the non‐bathed control frog. HPLC analysis of the peptide pattern confirmed these findings. The survival value of antimicrobial peptides have earlier been demonstrated in vivo and in vitro only in Drosophila. The present experiments are the first combined in vivo and in vitro demonstrations of the function of peptide antibiotics in a vertebrate. One such function is involved in the control of the natural flora.
Tyrosine Phosphorylation of p72syk Induced by Anti‐9‐O‐Acetyl GD3 Antibodies in Human Peripheral Blood Mononuclear Cells
Authors: Reivinen  , HolthÖfer  , Miettinen
Published: March 2003

We have previously shown that a subpopulation of peripheral blood CD4+ T‐cells are strongly positive for the ganglioside 9‐O‐acetyl GD3. Treatment of peripheral blood mononuclear cells (PBMC) with monoclonal anti‐9‐O‐acetyl GD3 antibodies (MoAb 27A) induced a dose‐dependent proliferative response in the cells. Here we show that binding of 27A IgG to PBMC induces rapid tyrosine phosphorylation of p72syk and mobilization of phosphoinositides. Equal phosphorylation was not seen after stimulation of PBMC with anti‐GD3 antibodies (MoAb R24) despite the apparent similar cell surface distribution of the two gangliosides. This suggests that the O‐acetyl modification enhances the signal transduction mediated by GD3 ganglioside.
Immunological Adjuvance of Metabolic Origin: Oxidative Stress, Postulated Impaired Function of Thiol Proteases and Immunogenicity
Authors: STARK
Published: March 2003

Autoimmune disease may sometimes arise because of dysfunction of thiol proteases, which are vulnerable to oxidation of their sulphydryl groups. This may be initially signalled by hyperinsulinaemia, regarded here as a telltale phenomenon of oxidative stress and indicating difficulty in protein catabolism. Initial immunogenic sensitization may take place when antigen processing is altered by a metabolic process, which has locally overwhelmed the antioxidant systems and led to diminished thiol‐protease digestion and to the repeated survival in critical cells of immunogenic peptide fragments. From this it follows that an immunological host may be either prejudiced towards or against tolerance by agents, respectively, which stabilize or destabilize antioxidant homeostasis.
Effect of Allergen‐Specific Immunotherapy on Interleukin‐4, Interleukin‐5 and Interferon‐γ mRNA Expression in the Nasal Mucosa of Rats with Allergic Rhinitis
Authors: EL‐Naggar  , Ukai  , Takeuchi  , Sakakura
Published: March 2003

To elucidate the mechanism of immunotherapy, we tested the effect of ovalbumin and ovalbumin–pullulan conjugate immunotherapy on the expression of interleukin (IL)‐4, IL‐5 and interferon‐γ (IFN‐γ) mRNA in the nasal mucosa of sensitized rats. Forty‐five rats were injected with ovalbumin intraperitoneally on three consecutive days and later were exposed to ovalbumin aerosol. The rats were injected intradermally, on six consecutive days, with saline, ovalbumin or ovalbumin–pullulan conjugate. Later, nasal mucosa was obtained and reverse transcription–polymerase chain reaction (RT–PCR) was performed. Nasal responses and specific immunoglobulin E (IgE) were measured. Although the immunotherapy significantly decreased nasal airway resistance, dye leakage and histamine content in nasal irrigation after allergen challenge, no significant difference was found in IL‐4 and IL‐5 mRNA expression or in specific IgE level among the three groups. We conclude that in this allergic model, the improvement of nasal responses after immunotherapy was the result of a mechanism other than decrease of T‐helper 2 (Th2) cytokines.
Cyclosporin A Suppresses the Induction of Nitric Oxide Synthesis in Interferon‐γ‐Treated L929 Fibroblasts
Authors: TRAJKOVIĆ  , JANKOVIĆ  , BADOVINAC  , SAMARDŽIĆ  , MAKSIMOVIĆ  , POPADIĆ
Published: March 2003

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) activity in murine L929 fibroblasts were investigated. IFN‐γ‐induced NO production in L929 cells was mediated through an iNOS‐dependent l‐arginine‐NO pathway, since it was abrogated by a selective inhibitor of iNOS, aminoguanidine. CsA applied simultaneously with IFN‐γ caused a dose‐dependent reduction of NO synthesis in L929 cells. However, CsA did not influence the enzymatic activity of iNOS, since it failed to affect NO production in cells in which iNOS had already been induced with IFN‐γ and any further induction was blocked by the protein‐synthesis inhibitor cycloheximide. IFN‐γ‐triggered expression of mRNA for interferon regulatory factor‐1 was not reduced by CsA‐treatment, suggesting that this iNOS transcription factor is not a target in CsA‐mediated inhibition of NO synthesis. Finally, FK506 was not able to mimic the inhibitory effect of CsA on NO production in L929 cells, indicating the calcineurin‐independent mechanism of CsA action. These results indicate that CsA suppresses NO synthesis in L929 cells independent of calcineurin inhibition, and interfering with intracellular pathways involved in the iNOS induction, rather than inhibiting its enzymatic activity.
Bruton's Tyrosine‐Kinase‐Deficient Murine B Lymphocytes Fail to Enter S Phase when Stimulated with Anti‐Immunoglobulin Plus Interleukin‐4
Authors: FORSSELL  , NILSSON  , SIDERAS
Published: March 2003

One of the earliest recognized defects of B cells carrying the xid mutation in the gene encoding for Bruton's tyrosine kinase (Btk) was their inability to proliferate in response to anti‐immunoglobulin plus interleukin (IL)‐4 stimulation. Previous attempts to define the stage at which this proliferative block occurred using xid B cells provided dissimilar results. We decided to reinvestigate this question using B cells from C57BL/6‐Btk‐protein‐deficient (BtkM) mice. Upon stimulation with anti‐IgM and IL‐4, BtkM cells increase in size and up‐regulate early activation markers such as CD69 and B7‐2, however, they do not progress into the cell cycle further than a very early G1 stage. They down‐regulate the cyclin‐dependent kinase (cdk) inhibitor p27 to some extent but fail to up‐regulate the G1‐phase cyclins D2 and E and the retinoblastoma protein (pRb) remains hypo‐phosphorylated. While ≈ 25% of the wild‐type cells enter S phase after 36 h stimulation, only ≈ 1% of the BtkM cells do so. The proliferative responsiveness of the BtkM cells is restored when the phorbol ester phorbol 12,13‐di‐butyrate (PDBu) is added to the anti‐IgM plus IL‐4 cultures. Collectively, our data demonstrate that a dramatically reduced frequency of responsive cells underlies the low proliferation of anti‐IgM plus IL‐4‐stimulated Btk‐deficient B cells and point towards an early block in the G1 phase due to inadequate activation of a pathway that regulates PKC activation.
Phenotypic and Functional Properties of Dendritic Cells Isolated from Human Peripheral Blood in Comparison with Mononuclear Cells and T Cells
Authors: HUANG  , XIAO  , WESTERLUND  , LINK
Published: March 2003

Dendritic cells (DCs) are pivotal for antigen presentation, T‐cell priming and B‐cell functions. Few studies have been carried out on DCs in human diseases, partly because the current procedures used for DC preparation include elaborate negative selection with monoclonal antibodies (MoAb) and prolonged culture in cytokine‐enriched milieu, which may influence DC functions. Using physical density and their adherent properties, DCs were prepared from the blood of healthy subjects. Approximately 2% of human blood mononuclear cells (MNC) were shown to consist of DCs, yielding DCs of 80–90% purity. They expressed markers related to DCs (CD1a, CD11c, CD32 and CD83), costimulatory molecules (CD40, CD80, CD86), human leucocyte antigen (HLA) class I and II molecules and inducible nitric oxide (NO) synthase (NOS2), and lacked lymphocyte and monocyte markers (CD3, CD19, CD20, CD56 and CD14). Compared with blood MNC and T cells, DCs showed a high level of spontaneous proliferation and nitric oxide production, as well as strong proliferative responses in mixed leucocyte reactions. Enzyme‐linked immunospot (ELISPOT) assays revealed higher levels of interleukin (IL)‐4‐, IL‐10‐ and interferon‐γ (IFN‐γ)‐secreting cells among DCs than among MNC or T cells obtained from the same blood specimens, while levels of tumour necrosis factor‐α (TNF‐α)‐ and IL‐6‐secreting cells did not differ. The results demonstrate that the method used is fast, effective and competitively priced, and should be useful for studies of DCs in disease states.
cDNA Sequence Coding for the α’‐Chain of the Third Complement Component in the African Lungfish
Authors: SATO  , SÜLTMANN  , MAYER  , FIGUEROA  , TICHY  , KLEIN
Published: March 2003

cDNA clones coding for almost the entire C3 α‐chain of the African lungfish (Protopterus aethiopicus), a representative of the Sarcopterygii (lobe‐finned fishes), were sequenced and characterized. From the sequence it is deduced that the lungfish C3 molecule is probably a disulphide‐bonded α:β dimer similar to that of the C3 components of other jawed vertebrates. The deduced sequence contains conserved sites presumably recognized by proteolytic enzymes (e.g. factor I) involved in the activation and inactivation of the component. It also contains the conserved thioester region and the putative site for binding properdin. However, the site for the interaction with complement receptor 2 and factor H are poorly conserved. Either complement receptor 2 and factor H are not present in the lungfish or they bind to different residues at the same or a different site than mammalian complement receptor 2 and factor H. The C3 α‐chain sequences faithfully reflect the phylogenetic relationships among vertebrate classes and can therefore be used to help to resolve the long‐standing controversy concerning the origin of the tetrapods.
Protein Kinase C Expression Links Natural Antibody Binding with Surveillance of Activated and Preneoplastic Cells
Authors: WANG  , CHOW
Published: March 2003

Extensive evidence supports a role for natural antibody (NAb) acting against small tumour foci in vivo. Ras‐transformation of murine C3H 10T½ fibroblasts, known to partially activate and down‐regulate endogenous PKC‐α, increased their serum NAb‐binding capacity consistent with the requirements for natural immune surveillance. Now a rat PKC‐β1‐overexpressing 10T½ clone, PKC‐4, with an 11‐fold increase in PKC activity and an activated, partially transformed phenotype, links higher susceptibility to transformation through v‐Ha‐ras infection with an 80% increase in NAb binding assayed by flow cytometry. H7 and E‐64d inhibition and phorbol ester depletion of PKC reduced NAb binding. PKC‐β1 expression and NAb binding exhibited a similar temporal recovery from TPA treatment. Thus, expression of NAb‐binding structures appears to be elevated by constitutive increases in the basal activation of PKC in both the ras‐transformation and the PKC‐β1‐preneoplasia models. This, coupled with corresponding decreases in membrane PKC‐α and NAb binding in confluent 10T½ cells raises the possibility that in general, cells activated through PKC are NAb sensitive. Together with the increased in vivo elimination of the high NAb‐binding PKC‐4 cells, the data extend the support for a role for NAb in immune surveillance, to resistance against preneoplastic cells, and argue for NAb contributing to homeostasis of the organism.