bluuebunny/crossref_metadata_2025 huggingface dataset:
Desert dogma revisited: coupling of stomatal conductance and photosynthesis in the desert shrub, Larrea tridentata
Authors: Ogle K. , Reynolds J. F.
Published: March 2003

The success of the desert shrub Larrea tridentata (creosotebush) has been largely attributed to temperature acclimation and stomatal control of photosynthesis (A) under drought stress. However, there is a paucity of field data on these relationships. To address this void, we conducted a joint field and modelling study that encompassed a diverse set of environmental conditions. At a Larrea‐dominated site in southern New Mexico we manipulated soil moisture during the growing season over a 2‐year period and measured plant pre‐dawn water potential (Ψpd), stomatal conductance (g) and A of individual shrubs. We used these data to develop a semi‐mechanistic photosynthesis model (A–Season) that explicitly couples internal CO2 (Ci) and g. Vapour pressure deficit (VPD) and Ψpd affect instantaneous g in a manner that is consistent with a biophysical model of stomatal regulation of leaf water potential. Ci is modelled as a function of g, derived from a simplification of a typical A–Ci curve. After incorporating the effects of growing temperature on stomatal behaviour, the model was able to capture the large diurnal fluctuations in A, g and Ci and the observed hysteresis in g versus Ci dynamics. Our field data and application of the A–Season model suggest that dogma attributed to Larrea's success is supported with regard to stomatal responses to VPD and Ψpd, but not for mechanisms of temperature acclimation and CO2 demand.
Variation in economically and ecologically important traits in onion plant organs during reproductive development
Authors: Briggs W. H. , Goldman I. L.
Published: March 2003

The spatial distribution of organosulphur compounds throughout the onion (Allium cepa L.) plant body during reproduction is of ecological and horticultural interest. These secondary metabolites are associated with both pest resistance and many of the vegetable's culinary and medicinal properties, including the ability to inhibit platelet aggregation. Inhibition of platelet aggregation can be of benefit to human cardiovascular health. Organosulphur compound concentrations are associated with elemental sulphur, pungency, soluble solids and effect on human platelet aggregation. These parameters were evaluated in extracts collected separately from bulb scales, leaf blades, scapes and umbels biweekly throughout the reproductive phase of the life cycle of the onion. Significant variation in pungency, platelet inhibition, total sulphur content and soluble solids existed among samples of organs and within organs over time during reproductive growth. Furthermore, some extracts from leaf, scape and bulb induced rather than inhibited platelet aggregation.
Impact of waterlogging on the N‐metabolism of flood tolerant and non‐tolerant tree species
Authors: Kreuzwieser J. , Fürniss S. , Rennenberg H.
Published: March 2003

The present study was conducted to characterize the N‐metabolism of important European tree species with different degrees of flooding tolerance. The roots of Fagus sylvatica (sensitive to flooding), Quercus robur (moderately flood tolerant) and Populus tremula × P. alba (flood tolerant) saplings were exposed to different flooding regimes and N uptake, amino acid, protein and chlorophyll concentrations as well as gas exchange were measured. The effects of these treatments on the tree species varied distinctly. In general, the N metabolism of beech was severely affected whereas less impacts were observed on oaks and almost no effects on poplars. The concentrations of amino compounds, particularly of Asp, Asn, Glu and Gln, were lower in the roots of flooded trees than in controls. By contrast, γ‐amino butyric acid concentrations increased. Root protein concentrations remained unaffected in oak and poplar but decreased in beech in response to flooding. The concentrations of pigments remained unaffected by flooding in all tree species investigated. However, photosynthesis and transpiration were severely affected in beech but much less in oak and poplar. The data obtained show a clear correlation between the different flooding tolerances of the trees investigated and the impacts of flooding on N uptake and N metabolism.
Nuclear magnetic resonanceimaging of membrane permeability changes in plants during osmoticstress
Authors: Van Der Weerd L. , Claessens M. M. A. E. , Efdé C. , Van As H.
Published: March 2003

The cell water balance of maize (Zea mays L., cv LG 11) andpearl millet (Pennisetum americanum L., cv MH 179) duringosmotic stress was studied non‐invasively using 1H nuclearmagnetic resonance (NMR) microscopy. Single NMR parameter imagesof (i) the water content (ii) the transverse relaxation time (T2)and (iii) the apparent diffusion coefficient (Dapp)were used to follow the water status of the stem apical region duringosmotic stress. During stress there are hardly any changes in watercontent or T2 of the stem region of maize. Incontrast, the apical tissue of pearl millet showed a ∼ 30% decreaseof T2 within 48 h of stress, whereasthe water content and Dapp did not change. Thesechanges can be explained by an increase of the membrane permeabilityfor water. This conclusion is supported by results from scanningelectron microscopy, relaxation measurements of sugar solutionsand numerical simulations of the relaxation and (apparent) diffusionbehaviour of water in a plant cell.
Long‐distance transport of gases in plants: a perspective on internal aeration and radial oxygen loss from roots
Authors: COLMER T. D.
Published: March 2003

Internal transport of gases is crucial for vascular plants inhabiting aquatic, wetland or flood‐prone environments. Diffusivity of gases in water is approximately 10 000 times slower than in air; thus direct exchange of gases between submerged tissues and the environment is strongly impeded. Aerenchyma provides a low‐resistance internal pathway for gas transport between shoot and root extremities. By this pathway, O2is supplied to the roots and rhizosphere, while CO2, ethylene, and methane move from the soil to the shoots and atmosphere. Diffusion is the mechanism by which gases move within roots of all plant species, but significant pressurized through‐flow occurs in stems and rhizomes of several emergent and floating‐leaved wetland plants. Through‐flows can raise O2concentrations in the rhizomes close to ambient levels. In general, rates of flow are determined by plant characteristics such as capacity to generate positive pressures in shoot tissues, and resistance to flow in the aerenchyma, as well as environmental conditions affecting leaf‐to‐air gradients in humidity and temperature. O2diffusion in roots is influenced by anatomical, morphological and physiological characteristics, and environmental conditions. Roots of many (but not all) wetland species contain large volumes of aerenchyma (e.g. root porosity can reach 55%), while a barrier impermeable to radial O2loss (ROL) often occurs in basal zones. These traits act synergistically to enhance the amount of O2diffusing to the root apex and enable the development of an aerobic rhizosphere around the root tip, which enhances root penetration into anaerobic substrates. The barrier to ROL in roots of some species is induced by growth in stagnant conditions, whereas it is constitutive in others. An inducible change in the resistance to O2across the hypodermis/exodermis is hypothesized to be of adaptive significance to plants inhabiting transiently waterlogged soils. Knowledge on the anatomical basis of the barrier to ROL in various species is scant. Nevertheless, it has been suggested that the barrier may also impede influx of: (i) soil‐derived gases, such as CO2, methane, and ethylene; (ii) potentially toxic substances (e.g. reduced metal ions) often present in waterlogged soils; and (iii) nutrients and water. Lateral roots, that remain permeable to O2, may be the main surface for exchange of substances between the roots and rhizosphere in wetland species. Further work is required to determine whether diversity in structure and function in roots of wetland species can be related to various niche habitats.
Logistics of water and salt transport through the plant: structure and functioning of the xylem
Authors: DE BOER A. H. , VOLKOV V.
Published: March 2003

The xylem is a long‐distance transport system that is unique to higher plants. It evolved into a very sophisticated plumbing system ensuring controlled loading/unloading of ions and water and their effective translocation to the required sinks. The focus of this overview will be the intrinsic inter‐relations between structural and functional features of the xylem. Taken together the xylem is designed to prevent cavitation (entry of air bubbles), induced by negative pressures under transpiration and to repair the cavitated vessels. Half‐bordered pits between xylem parenchyma cells and xylem vessels are on the one hand the gates to the vessels but on the other hand a serious ‘bottle‐neck’ for transport. Hence it becomes evident that special transport systems exist at the interface between the cells and vessels, which allow intensive fluxes of ions and water to and out of the xylem. The molecular identification and biophysical/biochemical characterization of these transporters has just started. Paradigms for the sophisticated mechanism of controlled xylem transport under changing environmental conditions are SKOR, a Shaker‐like channel involved in K+‐loading and SOS1, a Na+/H+ antiporter with a proposed dual function in Na+ transport. In view of the importance of plant water relations it is not surprising to find that water channels dominate the gate of access to xylem. Future studies will focus on the mechanism(s) that regulate water channels and ion transporters and on their physiological role in, for example, the repair of embolism. Clearly, progress in this specific field of research will greatly benefit from an integration of molecular and biophysical techniques aimed to understand ‘whole‐plant’ behaviour under the ever‐changing environmental conditions in the daily life of all plants.
Cadmium loading into potato tubers: the roles of the periderm, xylem and phloem
Authors: REID R. J. , DUNBAR K. R. , MCLAUGHLIN M. J.
Published: March 2003

The mobility of Cd in potato plants (Solanum tuberosum) was examined using both short‐term radioisotopic labelling with 109Cd and long‐term growth experiments in soil supplemented with Cd, with an emphasis on the pathways through which Cd is taken up by tubers. Split‐pot experiments showed that tubers and their associated stolons and stolon roots contribute only a minor fraction to the overall Cd absorption by the plant. Most of the Cd was absorbed by the basal roots. 109Cd absorbed from the soil was rapidly exported to other parts of the plant, especially the stem, with significant amounts appearing in the tubers within 30 h. Application of 109Cd to leaves showed that Cd can be rapidly distributed via the phloem to all tissues. The results suggest that unlike Ca, Cd has high mobility in plants in both xylem and phloem, and that stems may have an important role in transfer between these two pathways.
Distribution of Zimbabwean Armillaria groups and their pathogenicity on cassava
Authors: Mwenje  , Ride  , Pearce
Published: March 2003

Disease surveys carried out between 1989 and 1992 showed Armillaria to be restricted to the northern and eastern parts of Zimbabwe and to be absent from the western and southern parts. Armillaria disease is of local economic significance especially in fruit and clonal pine seed orchards. Pathogenicity studies showed significant differences in infection of cassava (Manihot esculenta) variety Zanaga (Ccv1) between isolates belonging to the three Zimbabwean Armillaria groups (I, II and III), but there were no significant differences in the infection of another cassava variety, Zcv1, from Zimbabwe. The group II isolates failed to cause any infection on the cassava clones. Rhizomorphs were the main means of infection in the pathogenicity studies, although they are rare in nature in Zimbabwe. Results using detached cassava tubers to assess pathogenicity proved to be inconsistent. Periderm restoration responses, which have been observed in woody plants and are presumed to function by restricting pathogen ingress, were observed in cassava infected with Armillaria.
Relationship between cultivar height and severity of Fusarium ear blight in wheat
Authors: Hilton  , Jenkinson  , Hollins  , Parry
Published: March 2003

A significant negative relationship between tiller height and resistance to Fusarium ear blight (FEB), following inoculation, was observed in 17 cultivars of winter wheat in the 1995/96 growing season. Tall cultivars such as Kraka and Spark showed fewer symptoms of FEB (6 and 4%) than the shorter cultivars Brigadier and Virtue (35 and 51%). To determine if this relationship was caused by a genetic association or an effect of the microclimate, height and disease were measured in segregating populations derived from tall × short cultivars and humidity was measured in near‐isogenic lines with and without the Rht1 and Rht2 dwarfing genes. Among random F3 populations there was a clear tendency for tall strawed lines to show less severe disease symptoms than shorter strawed lines following inoculation. The effect of the individual dwarfing genes Rht1 and Rht2 on the severity of FEB was also studied in an inoculation trial using a number of near‐isogenic lines of Maris Huntsman and Maris Widgeon. Within isogenic lines of Maris Huntsman, there was a clear tendency for tall straw to be associated with fewer symptoms, but this was not apparent within lines of Maris Widgeon. Monitoring relative humidity at ear height in a short and tall isogenic line of Maris Huntsman revealed no significant differences between these genotypes from GS 65 to GS 85, suggesting that microclimate cannot explain differences in severity of FEB between these lines. It is suggested that there are independent genes affecting the severity of FEB that may allow plant breeders to select resistant cultivars of any height.
Production and immunodetection of ascospores of Mycosphaerella brassicicola: ringspot of vegetable crucifers
Authors: Kennedy  , Wakeham  , Cullington
Published: March 2003

Pseudothecia containing abundant ascospores of Mycosphaerella brassicicola were produced in vitro on Brussels sprout decoction agar at 15°C under a 16‐hour photoperiod of different light regimes. Spermogonia containing spermatia were also produced on the decoction agar. Ascospores were released when cultures were misted with SDW and placed under continuous light. Germination of ascospores was highest between 20°C and 25°C and spores remained viable at relative humidities above 93.5%. Exposure of ascospores to 55% relative humidity for 24 h reduced their germination to 75%. A polyclonal antiserum raised against whole ascospores was used to detect, by immunofluorescence, the ascospore and mycelial wall of M. brassicicola, following reaction with anti‐rabbit IgG FITC conjugate. Autofluorescence of spore and mycelial components of other fungal species could be eliminated using the counterstains Evan's blue and eriochrome black at 0.2% and 0.5%, respectively, in phosphate buffered saline (pH 7.2). A procedure was developed to detect, by immunofluorescence, ascospores of M. brassicicola on artificially inoculated Melinex spore tape. Coating of the spore tape with bovine serum albumin provided a suitable support medium and blocking agent for detection of ascospores in the field. The potential use of the system for selective detection of ascospores of M. brassicicola in infected crops of vegetable brassicas in the presence of other ascosporic fungi is discussed. Keywords: ascospores, immunofluorescence, Mycosphaerella brassicicola, spore production, spore trapping.
Dry‐dispersal and rain‐splash of brown (Puccinia recondita f.sp. tritici) and yellow (P. striiformis) rust spores from infected wheat leaves exposed to simulated raindrops
Authors: Geagea  , Huber  , Sache
Published: March 2003

The dispersal of spores from lesions of brown (Puccinia recondita f.sp. tritici) or yellow (P. striiformis) rusts of wheat by impacting drops was studied. Using a generator of uniform‐size drops, drops of 2.5, 3.4, 4.2 and 4.9 mm in diameter were released from rest at heights of 5, 50 and 100 cm above horizontal and primary leaves uniformly covered with sporulating lesions. Dry‐dispersal and rain‐splash occurred simultaneously in response to drop impaction. A coloration technique allowed separate counting of dry‐dispersed and rain‐splashed spores caught on slides. More spores were rain‐splashed than dry‐dispersed. Neither removal mechanism affected in‐vitro germination of spores, which was higher in brown than in yellow rust. For both rusts, the number of both dry‐dispersed and rain‐splashed spores, as well as their travel distance, increased with drop diameter and fall height. The fall speed of incident drops in relation to diameter and fall height was obtained by solving numerically the equation of vertical drop motion. The number of spores removed by a given impacting drop was found to be a power function of the calculated kinetic energy of the impacting drop. Based on this experimental relationship, a simulation study showed the relevance of rain type in the removal of spores.
Assessing the susceptibility of chrysanthemum cultivars to tomato spotted wilt virus
Authors: van de Wetering  , Posthuma  , Goldbach  , Peters
Published: March 2003

Three methods were compared to assess the susceptibility of vegetatively propagated chrysanthemum to tomato spotted wilt tospovirus (TSWV): mechanical and thrips‐mediated inoculation of whole plants, and a leaf‐disc assay. As symptom expression was often poor or even absent, TSWV infections and subsequent susceptibility to TSWV were determined by ELISA. All 15 chrysanthemum cultivars tested were susceptible to TSWV, irrespective of their degree of vector resistance (based on feeding‐scar damage rates). Thrips‐mediated inoculation using different numbers of thrips revealed that 100% infection was obtained when plants were challenged by six thrips per plant, whereas 80 and over 50%, respectively, of the plants became infected when inoculated by a single male or female thrips. However, false negatives were scored even after intensive sampling because of erratic, cultivar‐specific and time‐dependent virus distribution after inoculation in the plants. Labour‐intensive samplings and long incubation periods could be overcome by a readily applicable leaf‐disc assay. This assay was as reliable as thrips‐mediated inoculation of whole plants, and its use is therefore favoured to assess chrysanthemum cultivars for TSWV susceptibility.
Pathogenicity of Bipolaris sorokiniana isolates from wheat roots, leaves and grains in Mexico
Authors: Duveiller E. , García Altamirano I.
Published: March 2003

Pathogenicity of 27 Bipolarissorokiniana isolates from roots, leaves and grains of spring wheat, collected at a site in Mexico, showed no clear differences between groups of isolates, based on lesion density after seedling inoculation of the susceptible cultivar Ciano T‐79 under controlled conditions. Results were variable when tests were repeated over time or in experiments using a smaller number of isolates. The lesion number per leaf was significantly different depending on the isolate used for inoculation, irrespective of the plant part group. No difference was found between individual isolates when pathogenicity was assessed using a disease severity scale. No cultivar–isolate interaction was observed for lesion density when 12 isolates were tested on six cultivars, but there were some significant differences between cultivars and between isolates. Results obtained confirmed that infection by B. sorokiniana is highly variable, and very sensitive to environmental conditions. In this study no physiological specialization was observed. The fungus appeared as a continuum of isolates differing in aggressiveness. This work was conducted with isolates from a single site, and highlights the need to assess carefully the biological significance of any differences observed among B. sorokiniana isolates.
Resistance to powdery mildew in mutant lines of the susceptible wheat cultivar Hobbit ‘sib’
Authors: Duggal V. , Jellis G. J. , Hollins T. W. , Stratford R.
Published: March 2003

Mutant lines of the wheat (Triticum aestivum) cultivar Hobbit ‘sib’, produced by irradiation with fast neutrons, were evaluated for their response to powdery mildew (Erisyphe graminis f.sp. tritici) at various growth stages to identify and characterize resistance. Two mutants, I3‐54 and I3‐48, were found to be significantly more resistant than Hobbit ‘sib’, with the resistance being expressed in both seedlings and adult plants. Fungal hyphal development was slower in both the mutants than in Hobbit ‘sib’ between 18 and 24 h after inoculation, and fewer haustoria were formed in the mutants, particularly in I3‐54, 72 h after inoculation. Constitutive expression of a thaumatin‐like protein gene was detected in both I3‐48 and I3‐54 but not in Hobbit ‘sib’.
Detection, quantification and characterization of Erwinia carotovora ssp. carotovora contaminating pepper seeds
Authors: Hadas R. , Kritzman G. , Gefen T. , Manulis S.
Published: March 2003

Erwinia carotovora ssp. carotovora (Ecc) was isolated from commercial pepper (Capsicum annuum) seed lots and identified according to biochemical and pathogenicity tests, cellular fatty acid composition, and PCR reaction with primers based on the pel gene sequence. RAPD‐PCR analysis using nine arbitrary primers showed similarity between Ecc isolates obtained from pepper. Ecc isolates from pepper, tomato, potato and cabbage formed four distinct groups, according to the original host. A natural Ecc population higher than 100 CFU g−1 seed significantly affected seed germination. A significant correlation between the level of Ecc on the seeds and the percentage of diseased seedlings was also observed. Surface sterilization of the seeds with sodium hypochlorite eliminated the pathogen, indicating that the bacteria were located on the surface of the seeds. Contaminated pepper seeds can be a primary and important source of inoculum.
Effect of phosphite on in planta zoospore production of Phytophthora cinnamomi
Authors: Wilkinson C. J. , Holmes J. M. , Dell B. , Tynan K. M. , McComb J. A. , Shearer B. L. , Colquhoun I. J. , Hardy G. E. St. J.
Published: March 2003

The efficacy of phosphite to control the production of zoospores of Phytophthora cinnamomi on infected trees grown in a glasshouse and in a revegetated mined area was examined. Banksia grandis and Eucalyptus marginata seedlings in the glasshouse and E. marginata seedlings in the minepit were sprayed with 0, 5 and 10 g phosphite L−1. In both trials, zoospores were produced from infected tissue of plants treated with all concentrations of phosphite. In the glasshouse, spray application of 5 and 10 g phosphite L−1 significantly reduced the production of zoospores from both B. grandis and E. marginata seedlings. In the mined area there was a similar, though nonsignificant, reduction in the number of zoospores produced from phosphite‐treated and nontreated E. marginata seedlings. However, the average number of zoospores produced was greater in plants not treated with phosphite (1·75 zoospores mL−1) than from plants treated with 5 or 10 g phosphite L−1 (0·04 and 0·09 zoospores mL−1, respectively). Pimelea ferruginea leaves were used to bait the water surrounding the plants in the mined area to determine if zoospores produced from phosphite‐treated plants were able to infect plant material. Significantly more baits were infected by zoospores from plants not treated with phosphite compared with plants treated with 5 or 10 g phosphite L−1. These results suggest that phosphite reduces, but does not prevent, the production of viable zoospores on infected trees. Thus phosphite application may not remove the risk of P. cinnamomi spreading from infested, sprayed areas.
Relationship between brown foot rot and DNA ofMicrodochium nivale, determined by quantitative PCR, in stem bases of winter wheat
Authors: Turner A. S. , Nicholson P. , Edwards S. G. , Bateman G. L. , Morgan L. W. , Todd A. D. , Parry D. W. , Marshall J. , Nuttall M.
Published: March 2003

Relationships between the incidence and severity of brown foot rot and of pathogenic fungi, determined by diagnostic and quantitative PCR, were investigated during the growth of nine winter wheat crops in three cropping seasons.Microdochium nivalevarsnivaleandmajuswere the only brown foot rot pathogens present in significant amounts. Relationships between disease symptoms and amounts of pathogen DNA were often weak in early spring (when shoot‐base symptoms are usually most difficult to ascribe to particular pathogens by visual examination) because of indistinct symptoms and small amounts of pathogen. Relationships were strongest during stem elongation. The amount ofM. nivalein the tissues tended to decline in the summer as the plants matured, apparently disappearing partially from necrotic lesions to which it contributed, resulting in a weakened relationship between symptoms and pathogen DNA. Regression analyses of brown foot rot on amounts ofM. nivaleDNA for different wheat cultivars generally produced lines with similar slopes but were often most significant for the cultivar with most eyespot resistance (i.e. with least confounding eyespot) or most apparently genuine brown foot rot. DNA ofFusariumspp. was rarely present in amounts sufficient to quantify.
Evolution and distribution of virulence in the German population of Bremia lactucae
Authors: Lebeda A. , Zinkernagel V.
Published: March 2003

Changes in the genetic structure of the lettuce crop (Lactuca sativa) and populations of the lettuce downy mildew pathogen (Bremia lactucae) in Germany from 1974 to 1997 were studied and the genetic and population genetic results of race surveys of B. lactucae were reinterpreted. Data on 176 B. lactucae isolates collected from L. sativa cultivars at 48 sites in 11 German federal states were analysed. The host population contained 12 well‐defined race‐specific resistance genes (Dm1, Dm2, Dm3, Dm4, Dm5/8, Dm6, Dm7, Dm11, R12, Dm13, Dm16 and R18). However, only some of these genes (e.g. Dm3, Dm7, Dm11, Dm16 and R18) played an important role in management of resistance in the host crop. Substantial changes in the frequencies of these R‐genes were recorded over the period as a reaction to the occurrence of new virulent phenotypes in the pathogen population. Generally, the durability of resistance was very short. However, distinct regional patterns were observed for the frequencies of different virulence (v) factors. The data on changes in the virulence of B. lactucae population exemplified well the process of host–pathogen coevolution. During the period studied, there was a considerable increase in the frequency of some v‐factors (v2, v3, v5/8, v6, v7, v11 and v16) and in the complexity of v‐phenotypes, while the diversity in the pathogen population in general decreased. In 1996/97, 12 of the 14 v‐factors evaluated reached frequencies higher than 0·90 in the pathogen population. Relatively low frequencies were observed only for factors v14 (0·17) and v18 (0·24). The presence of 23 v‐factors was studied in six German isolates virulent on lettuce cultivar Titan, possessing the resistance gene R36 derived from Lactuca saligna and introduced in German lettuce crops during the mid‐1990s. Isolate DEG2 showed the highest complexity of v‐phenotype; it overcame the resistance in cv. Mariska (R18) and line CS‐RL (R18+?), but some sporulation occurred also on cv. Titan (Dm6 + R36). Conversely, the isolates SR2 and SAW1 overcame resistance based on R36, but were avirulent to R18. Future utilization of R‐genes in commercial lettuce cultivars is proposed; R‐genes R18 and R36 could be considered most efficient for this purpose. However, they may be effective only in certain regions.
Cow Milk Feeding Induces Antibodies to Insulin in Children – A Link Between Cow Milk and Insulin‐Dependent Diabetes Mellitus?
Authors: Vaarala  , Paronen  , Otonkoski  , Åkerblom
Published: March 2003

Exposure to cow milk (CM)‐based formulas in early infancy has been associated with an increased risk of insulin‐dependent diabetes mellitus (IDDM), but studies on the possible pathogenic mechanism(s) linking CM and IDDM are contradicting. We hypothesized that if CM formulas contained bovine insulin (BI), exposure to them could lead to immunization against insulin, which is the only known β‐cell‐specific autoantigen in IDDM. We measured immunoglobulin G (IgG) antibodies by enzyme immunoassay (EIA) to BI and human insulin (HI) in children who received, during the first 9 months of life, either a formula containing whole CM proteins or a formula containing hydrolyzed casein (HC) peptides. BI was detectable by radioimmunoassay (RIA) and immunoblotting in the CM‐based formula. At 6 months of age the children who received CM formula had higher levels of IgG antibodies to BI than children who received either HC formula or children who were exclusively breast‐fed (median levels 0.480 versus 0.185, P = 0.04; and 0.480 versus 0.160, P = 0.04; respectively). Also, at 9 months of age, children in the CM group differed from the HC group (0.403 versus 0.230; P = 0.02). Antibodies to BI and HI showed a positive correlation and cross‐reacted in inhibition studies. The high incidence of insulin‐binding antibodies in young children with IDDM may be explained by oral immunization to BI present in CM. Exposure to BI, which differs from HI only by three amino acids, may break the tolerance to insulin.
Generation of Dendritic Cells from Peripheral Blood Adherent Cells in Medium with Human Serum
Authors: Anton  , Dabadghao  , Palucka  , Holm  , Yi
Published: March 2003

Dendritic cells (DC) provide an effective pathway for presenting antigens to T cells, both self‐antigens during T‐cell development and foreign antigens during immunity. As such, these cells may be promising adjuvants for immunotherapy. Thus, it is important to establish simple and fast method(s) to generate sufficient numbers of human DC in medium free of calf serum so that the cells can be used for both experimental and clinical purposes. In this report, we used peripheral blood adherent cells, without laborious cell purification or depletion, as the starting population and cultured them in medium supplemented with granulocyte/macrophage colony‐stimulating factor and interleukin‐4. Substantial numbers of cells with the phenotypical and functional characteristics of immature DC were obtained in a 7‐day culture. We then compared DC cultured in medium supplemented with either fetal calf serum or pooled human ABRh+ serum and found no difference in cell yields and in their ability to stimulate alloreactive T cells or to present soluble antigens to T cells. Irradiated cells were less efficient than non‐irradiated cells in antigen presentation and stimulation of T cells. Finally, we have examined DC with or without additional tumour necrosis factor‐α treatment and found that antigen‐pulsed mature cells could as efficiently present antigen to T cells as did immature cells. This method is suitable for the generation of DC in studies of large clinical materials.
Opsonophagocytic and Bactericidal Activity Mediated by Purified IgG Subclass Antibodies After Vaccination with the Norwegian Group B Meningococcal Vaccine
Authors: Aase  , Høiby  , Michaelsen
Published: March 2003

To study how the different immunoglobulin (Ig)G subclass antibodies may confer protection against systemic meningococcal disease, we isolated IgG1, IgG2 and IgG3 antibodies from plasma from vaccinees immunized with the Norwegian meningococcal outer membrane vesicle vaccine. Four IgG1, one IgG2 and four IgG3 preparations were purified. The IgG2 and IgG3 subclass preparations were free from contaminating subclasses, whereas the IgG1 preparations contained from 0 to 14% of IgG2 and/or IgG3. Immunoblotting against whole‐cell meningococcal antigens showed broad specificities of the various preparations, both within and between subclasses. These subclass preparations were tested for opsonophagocytic and bactericidal activity. As targets we used two different variants of the meningococcal vaccine strain, with low (44/76‐SL) and high (44/76‐1) expression of the outer membrane protein Opc. Using polymorphonuclear leucocytes as effector cells in the presence of human complement, all three IgG subclass preparations revealed high, and similar, opsonophagocytic activities against 44/76‐SL, whereas against 44/76‐1 the IgG2 preparation showed a reduced activity and most IgG3 preparations were slightly more active than the IgG1 preparations. Regarding bactericidal activity, all the three subclasses were highly active against 44/76‐SL. Against 44/76‐1 the bactericidal activities were somewhat more varied: all IgG1 and three IgG3 preparations exhibited higher activities than against 44/76‐SL. Due to the low concentration in the IgG2 preparations, only a weak activity was seen against 44/76‐1. One IgG3 preparation that was highly opsonophagocytic revealed no bactericidal activity against either of the two bacterial variants examined. In conclusion, we have shown that the IgG subclass effector functions differ from person to person, but that antibodies of IgG1, IgG2 and IgG3 subclasses, judged by their behaviour in the functional tests, may all contribute to protection against meningococcal disease.
Restricted and Individual Usage of T‐cell Receptor β‐Gene Variables in Nickel‐Induced CD4+ and CD8+ Cells
Authors: Silvennoinen‐Kassinen  , Karvonen  , Ikäheimo
Published: March 2003

Nickel allergy is manifested as contact allergic eczema elicited by delayed‐type hypersensitivity, the reaction being mediated by T lymphocytes. We examined the T‐cell receptor (TCR) β‐chain variable gene segment (Vβ) use of nickel‐induced CD4+ and CD8+ T cells in the peripheral blood of nickel‐sensitive and non‐sensitized subjects. The results show that each patient had an individual Vβ repertoire overexpressed, these being in CD4+ cells Vβ10 and Vβ13 (in subject A); Vβ1, Vβ2, Vβ13 and Vβ21 (subject B); Vβ1 and Vβ10 (subject C); Vβ9 and Vβ19 (subject D). Thus, no single Vβ gene dominated in a majority of the CD4+ samples. The Vβ genes overexpressed in patient CD8+ nickel‐induced T cells were Vβ1 (in subject A), Vβ1 (subject B), Vβ1 and Vβ2 (subject C) and Vβ7 (subject D), domination of Vβ1 being seen in most of the CD8+ samples (75%). No specific overexpression of any Vβ genes in the nickel‐allergic subjects was found in comparison with the non‐sensitized subjects. In conclusion, an individual pattern of restricted Vβ genes was induced with nickel in CD4+ and CD8+ T cells in each nickel allergy patient.
The Primary Antibody Repertoire of κ‐Deficient Mice is Characterized by Non‐Stochastic Vλ1 + VH Gene Family Pairings and a Higher Degree of Self‐Reactivity
Authors: Knott  , Bona  , Kaushik
Published: March 2003

We have investigated the primary antibody repertoire of genetically manipulated 129/Sv κ‐deficient (JCκD) mice, in order to understand the contributions of the λ‐light chain, in the absence of an otherwise predominant κ‐light chain, to the development of humoral immunity. The expression of Vλ1 gene (λ1 and λ3 subtypes) and the Vλ1 + VH (J558, 36–60, VH11 and S107) gene family associations were studied in 7.43 × 103 mitogen‐activated splenic B‐lymphocyte clones of JCκD origin. Furthermore, the functional significance of the exclusive expression of the λ‐light chain, in the peripheral B‐cell repertoire of JCκD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B‐lymphocyte clones in the peripheral B‐lymphocyte repertoire. These experiments revealed that: first, of the three available Vλ genes at the λ locus, the Vλ1 gene is the one that is expressed most frequently (59.9%); second, non‐random Vλ1 + VH (J558, 36–60) gene family pairings occur in κ‐deficient mice; and third, a higher degree of self‐reactivity is generated as a result of exclusive use of the λ‐light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B‐lymphocyte clones in κ‐deficient (129/Sv JCκD) mice. These observations suggest that the high murine κ/λ ratio in mice may, apart from high sequence diversity at the κ‐locus, be a result of endogenous selection against the λ‐light chain to restrict self‐reactivity within the homeostatic threshold.
Regulation of β‐Chemokine mRNA Expression in Adult Rat Astrocytes by Lipopolysaccharide, Proinflammatory and Immunoregulatory Cytokines
Authors: GUO  , JIN  , ISHIKAWA  , HUANG  , VAN DER MEIDE  , LINK  , XIAO
Published: March 2003

Astrocytes constitute a part of the blood–brain barrier. Chemokine expression by astrocytes may contribute to leucocyte infiltration within the central nervous system (CNS) during inflammation. To investigate factor(s) regulating chemokine expression by astrocytes, we studied the induction of β‐chemokine mRNA expression in adult rat astrocytes. Astrocyte‐derived monocyte chemoattractant protein‐1 (MCP‐1), RANTES, macrophage inflammatory protein (MIP)‐1α and MIP‐1β mRNA were induced by interferon‐γ (IFN‐γ). Tumour necrosis factor‐α (TNF‐α) induced MCP‐1, RANTES and MIP‐1β mRNA expression, and lipopolysaccharide (LPS) induced MCP‐1, MIP‐1α and MIP‐1β mRNA expression in astrocytes. LPS‐induced MCP‐1, MIP‐1α and MIP‐1β mRNA expression by astrocytes was antagonized by transforming growth factor (TGF)‐β1 and interleukin (IL)‐10. TGF‐β1 and IL‐10 also down‐regulated MCP‐1 and RANTES mRNA expression induced by TNF‐α. IL‐10, but not TGF‐β1, inhibited MIP‐1β mRNA expression induced by TNF‐α. The results of this in vitro study suggest that β‐chemokine mRNA expression by adult rat astrocytes can be induced by LPS or proinflammatory cytokines, while regulatory cytokines, such as TGF‐β1 and IL‐10, down‐regulate astrocyte‐derived β‐family chemokine mRNA expression induced by LPS, IFN‐γ and TNF‐α. Further study of CNS chemokines will enhance our understanding of leucocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.
Soluble Fas and Soluble Fas Ligand Proteins in Human Milk: Possible Significance in the Development of Immunological Tolerance
Authors: SRIVASTAVA  , SAHAI SRIVASTAVA
Published: March 2003

Human milk contains a complex uncharacterized, immune system able to exert actions both locally and systemically. This study reports the results of an ELISA‐based quantitation of soluble Fas and soluble Fas ligand in human milk which may modulate the Fas/FasL system that is critical for the expression of immune tolerance and apoptosis. Production of Fas/FasL mRNAs by milk cells was also examined using RT–PCR. Fas is ubiquitously expressed in various cells and when bound by its ligand FasL, present predominantly on activated T‐ and NK cells, Fas‐expressing cells are killed. A large amount of soluble Fas (1746–4320 pg/ml) is detected in colostrum, transitional milk and the mature milk of mothers delivering prematurely or at full‐term, whereas FasL is present only in the range 123–310 pg/ml. Milk cells are positive for Fas mRNA, but negative for FasL mRNA. An excess of soluble Fas in human milk may bind to FasL preventing apoptosis and preserving epithelial barriers, and may represent an additional new mechanism whereby human milk favours immune tolerance and normal gastrointestinal development.
Induction of Phagocyte‐Stimulating Cytokines by In Vitro Stimulation of Human Peripheral Blood Mononuclear Cells with Haemophilus influenzae
Authors: ARVÅ  , ANDERSSON
Published: March 2003

The aim of this study was to analyse the in vitro response of human peripheral blood mononuclear cells to stimulation with killed Haemophilus influenzae strains of different capsular types, isolation sites and from cases with different forms of infections. The mean stimulatory index using 106 bacteria/well was 10, and 80 when 108 bacteria/well were used for stimulation. The mean ± SD level was 13 ± 4 ng/ml for interleukin (IL)‐1β, 128 ± 73 ng/ml for IL‐6, 203 ± 122 ng/ml for IL‐8, 3160 ± 1220 pg/ml for IL‐10, 29 ± 40 pg/ml for IL‐12, 2800 ± 1790 pg/ml for tumour necrosis factor (TNF)‐α and 4 ± 7 ng/ml for interferon (IFN)‐γ, when stimulating cells with the lower dose of 106 bacteria/well. Using the higher bacterial dose, the levels of IL‐1β, TNF‐α and IL‐12 remained similar, whereas the IL‐6, IL‐8 and IL‐10 levels were significantly lower, and IFN‐γ levels were significantly higher. Strains isolated from the bronchial tree induced significantly higher levels of IFN‐γ and significantly lower levels of IL‐6, IL‐8 and IL‐10 than strains from other isolation sites. In conclusion, H. influenzae generated phagocyte‐activating cytokines and an IL‐10/IL‐12 ratio that was 1090 times that described previously for Streptococcus pneumoniae.
Paroxysmal Nocturnal Haemoglobinuria: the Disease and a Hypothesis for a New Treatment
Authors: JARVA  , MERI
Published: March 2003

Paroxysmal nocturnal haemoglobinuria (PNH) is a disease entity that presents with intravascular haemolysis and an increased tendency for venous thrombosis. In recent years there has been a major breakthrough in our understanding of the pathogenesis of PNH. Most of the different symptoms can be tracked down to the deficiency of glycophosphoinositol (GPI)‐anchored proteins in cell lines deriving from a single haematopoietic stem cell. This deficiency is caused by a mutation in the X‐chromosomal PIG‐A gene whose product, a glycosyltransferase, participates in the first step of the GPI‐anchor biosynthesis. Lack of GPI‐linked complement inhibitors CD55 and CD59 predisposes red blood cells to lysis. The main unresolved question is why the stem cells lacking GPI‐anchored surface proteins gain a growth advantage over their normal counterparts. So far, our progress in understanding the pathogenesis has not resulted in better treatment of PNH and new ideas are warranted. In this regard, we propose a new mode of treatment for PNH by exploiting the increased susceptibility of affected bone marrow precursor cells to complement and targeting complement attack against them by a specific complement‐activating monoclonal antibody.
How Pre‐existing, Germline‐Derived Antibodies and Complement May Help Induce a Primary Immune Response to Nonself
Authors: LUTZ
Published: March 2003

In the hypothesis that pre‐existing, germline‐encoded antibodies (naturally occurring antibodies, NAb) bind to conserved epitopes on invading nonself antigens, bound NAbs may initiate complement deposition and become targets of nascent C3b, which generates C3b–C3b–NAb complexes that remain associated with the nonself antigen (C3b–C3b–NAb…antigen). The inactivated form of these complexes (C3dg–C3dg–NAb…nonself antigen) may bind bivalently and thus firmly to B cells via CR2, a process stimulating antigen presentation. In some cases, CR2‐bound ‘C3dg–C3dg–NAb…antigen complexes’ may further be recognized by immunoglobulin (Ig) determinants on B cells, whereby an immune response is elicited. As conserved epitopes on the nonself antigen are already complexed to NAbs, only B cells carrying Ig determinants specific for nonself epitopes may be stimulated. This hypothesis can explain directed affinity maturation towards nonself, protection from a strong immune response to conserved epitopes, down‐regulation of antibody formation and unresponsiveness to high‐dose antigen.
Characterization of the Three Immunoglobulin G Subclasses of Macaques
Authors: CALVAS  , APOIL  , FORTENFANT  , ROUBINET  , ANDRIS  , CAPRA  , BLANCHER
Published: March 2003

Southern blot experiments with genomic DNA samples of rhesus monkeys and crab‐eating macaques and human C gamma‐specific probes indicated that the two macaque species studied here possessed three C gamma genes per haploid genome. By amplifying the cDNA from macaque–mouse hybridomas, the coding sequences of two different rhesus monkey immunoglobulin (Ig)G subclasses, IgG1rh (Cγ1rh) and IgG2rh (Cγ2rh), and one crab‐eating macaque IgG subclass IgG1mafa (Cγ1mafa), were characterized. None of the 16 rhesus monkey–mouse hybridomas studied here secreted IgG of the third subclass IgG3rh (Cγ3rh). The Cγ3rh gene was partly characterized at the genomic level. The cDNA of the Cγ3rh gene was amplified from mRNA of rhesus monkey peripheral blood mononuclear cells (PBMC). The results are analysed in terms of phylogenesis of the C gamma genes. The cDNA sequences coding for the Cμ and the Cκ domains of rhesus monkey Ig were established and compared to their human and non‐human primate counterparts.
Inflammation and Severe Demyelination in the Peripheral Nervous System Induced by the Intraneural Injection of Recombinant Mouse Interleukin‐12
Authors: Pelidou  , Deretzi  , Zou  , Quiding  , Zhu
Published: March 2003

Inflammatory cytokines appear to be involved in damage to Schwann cells, myelin and axons, although the exact role of the different cytokines is uncertain. The direct injection model offers a new and simplified way of examining the mechanisms of early inflammation in the peripheral nervous system. The present study was performed to assess the direct effects of interleukin (IL)‐12 on rat sciatic nerves injected with recombinant mouse IL‐12. Histological and immunohistochemical examination 24 h after injection showed early inflammation as well as demyelination within the injected nerve fibres. By 4 days the inflammatory and demyelinating changes were significantly increased. Seven days after injection, the endoneurium still contained significant numbers of inflammatory cells and the demyelination was even more severe. Control rats injected with sterile phosphate‐buffered saline exhibited no such inflammatory and demyelinating response. These changes are similar to those seen in inflammatory and demyelinating disorders of the peripheral nervous system and suggest that IL‐12 could be relevant to the pathogenesis of demyelinating diseases such as Guillain–Barré syndrome.
Recognition of Auto‐ and Exoantigens by V4‐34 Gene Encoded Antibodies
Authors: Bhat  , Bieber  , Spellerberg  , Stevenson  , Teng
Published: March 2003

The antigenic specificities of 24 V4‐34‐encoded monoclonal antibodies were compared with the amino acid sequence. The specificities were divided into three categories, red blood cells, B lymphocytes and auto/exoantigens. Six anti‐I monoclonal antibodies, with multiple substitutions in their VH region, did not bind B lymphocytes or auto/exoantigens. Reactivity to these two antigens segregated with the 16 anti‐i monoclonal antibodies, which were derived from the near germline V4‐34 gene. All anti‐i monoclonal antodies bound B lymphocytes, albeit with varying intensities. B‐cell binding correlated with basic amino acids in the VH‐CDR3. Reactivity to auto/exoantigens was demonstrated only by a subset anti‐i monoclonal antibodies and did not correlate with B‐lymphocyte or i‐antigen binding. These anti‐ssDNA reactive monoclonal antibodies had basic amino acids in the VH‐CDR3, strongly supporting the suggested role of arginine in DNA binding. However, an arginine‐rich CDR3 was not enough to ensure DNA reactivity, since six other anti‐i monoclonal antibodies that fulfilled this criteria did not bind ssDNA. Thus it is possible that the anti‐DNA reactivity of V4‐34‐encoded monoclonal antibodies is mediated by the classic antigen‐binding groove generated by the CDRs of the heavy/light chains. In contrast, anti‐B‐cell/i‐antigen reactivity is mediated, unconventionally, by the V4‐34 protein with a dominant influence of the VH‐CDR3.