bluuebunny/crossref_metadata_2025 huggingface dataset:
Imazethapyr, an inhibitor of the branched‐chain amino acid biosynthesis, induces aerobic fermentation in pea plants
Authors: Gaston Susana , Zabalza Ana , González Esther M. , Arrese‐Igor Cesar , Aparicio‐Tejo Pedro M. , Royuela Mercedes
Published: March 2003

Acetolactate synthase (ALS; EC 4.1.3.18) inhibition is the primary mechanism of action of imazethapyr (IM). However, the precise mechanisms that links ALS inhibition with plant death have not been elucidated. Supply of IM to pea (Pisum sativum L) plants produced an immediate cessation of growth, caused a 50% inhibition of the in vivo ALS activity within 1 day of treatment, and a remarkable accumulation (2.7‐times) of free amino acids after 3 days. Carbohydrates (soluble and starch) were accumulated in both leaves and roots. Accumulation of soluble sugars in roots preceded that of starch in leaves, suggesting that the accumulation of carbohydrates in leaves is not the reason for the arrested root growth. A transient pyruvate accumulation was observed in roots, 1 day after the onset of IM supply. This was coincident with an increase in pyruvate decarboxylase (EC 4.1.1.1), and later increases in alcohol dehydrogenase (EC 1.1.1.1), lactate dehydrogenase (EC 1.1.1.27), and alanine amino transferase (EC 2.6.1.2) activities. This enhancement of fermentative activities was coincident with a slight decrease in aerobic respiration. The overall data suggest that the impairment of ALS activity may lead to a fermentative metabolism that may be involved in growth inhibition and plant death.
Age‐related loss of rooting capability inArabidopsis thalianaand its reversal by peptides containing the Arg‐Gly‐Asp (RGD) motif
Authors: Díaz‐Sala Carmen , Garrido Germán , Sabater Bartolomé
Published: March 2003

We describe here an experimental system to study the age‐related decline of adventitious root formation inArabidopsis thalianaL. (Heynh), ecotype Landsbergerecta(Ler). The system is based on the different rooting capacity of hypocotyls from de‐rooted juvenile (12‐day‐old) and adult (26‐day‐old) plants. Hypocotyls from de‐rooted juvenile plants rooted readily within a week of culture, and the rooting process was not dependent on exogenous auxin. In contrast, hypocotyls from de‐rooted adult plants rooted poorly and only after longer periods of time. Exogenously applied auxin had no effect on rooting of hypocotyls from de‐rooted adult plants. Rooting capacity, although correlated with the transition to flowering, did not depend on this transition. Root induction declined in a similar manner when the transition to flowering was delayed, either genetically with thefvemutant or physiologically with short days. The results showed that rooting of hypocotyls from de‐rooted adult plants depended on the effect of peptides containing the RGD motif. Both the percentage of rooting and the number of roots were largely increased when the hypocotyls were treated transiently with the RGD peptide. The effect of the RGD peptide was a necessary, but not sufficient, condition for rooting of hypocotyls from de‐rooted adult plants.
Cadmium accumulation in the chloroplast of Euglena gracilis
Authors: Mendoza‐Cozatl David , Devars Silvia , Loza‐Tavera Herminia , Moreno‐Sánchez Rafael
Published: March 2003

Intracellular distribution of Cd, cysteine, glutathione, and Cd‐induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 mM CdCl2, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll‐purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd2+, since uptake of added CdCl2 in isolated chloroplasts exhibited a hyperbolic type of kinetics with a Km of 57 µM and Vmax of 3.7 nmol (mg protein)−1 min−1. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd2+, although they were always higher in the cytosol. Thiol‐containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol‐peptides.
ABA and polyamines act independently in primary leaves of  cold‐stressed tomato (Lycopersicon esculentum)
Authors: Kim Tae Eun , Kim Seong‐Ki , Han Tae Jin , Lee June Seung , Chang Soo Chul
Published: March 2003

The effects of ABA and putrescine, a polyamine, on cold‐induced membrane leakage were investigated using primary leaves of wild‐type and an ABA‐deficient mutant, flacca, of tomato (Lycopersicon esculentum Mill.). The amount of chilling‐induced electrolyte leakage from flacca leaves was much higher than that from the wild‐type leaves. When applied exogenously ABA reduced cold‐induced electrolyte leakage from leaves of both wild‐type and the flacca mutant. However, the cold‐induced electrolyte leakage from flacca leaves was not as pronounced as in the wild‐type indicating that ABA is an important mediator in response to cold stress in the leaves. Putrescine reduced cold‐induced electrolyte leakage from both wild‐type and flacca leaves. Synthesis of putrescine in the leaves was increased by cold treatment. DFMO, a biosynthetic inhibitor of the polyamine, increased electrolyte leakage from cold‐treated leaves, and exogenously applied putrescine decreased the enhanced leakage to the control level. Therefore, this polyamine is thought also to be involved in the response to cold stress of tomato leaves. Both ABA and putrescine were protective against cold stress, but exogenously applied ABA decreased the endogenous level of putrescine in the leaves. Furthermore, the DMFO‐increased electrolyte leakage in cold‐stressed leaves was completely abolished by the application of ABA. These results suggest that ABA is a major regulator in the response to cold stress in tomato leaves and that it does not exert its role via putrescine in the response to cold stress.
Far red end‐of‐day treatment restores wild type‐like plant length in hybrid aspen overexpressing phytochrome A
Authors: Olsen Jorunn E. , Junttila Olavi
Published: March 2003

Shoot elongation in woody plants is modulated by a multitude of light signals, including irradiance, photoperiod and spectral composition, for which the phytochrome system is the probable photoreceptor. In hybrid aspen (Populus tremula × tremuloides) overexpression of the oat phytochrome A (PHYA) prevents growth cessation in response to short photoperiod, and plants exhibit dwarf growth that is related to reduced cell numbers and reduced gibberellin contents. End‐of‐day far‐red treatment significantly enhances internode elongation in PHYA overexpressors as well as in the wild type, and this was found here to be caused by stimulation of cell division and cell extension. In PHYA overexpressors the effects were substantially larger than in the wild type, and resulted in complete restoration of wild type‐like plant length as well as cell numbers, and gibberellin content was greatly increased. No clear effect of far‐red end‐of‐day treatment on gibberellin levels could be detected in the wild type. It thus appears that the far‐red end‐of‐day treatment might modify the responsiveness of the tissue to GA rather than the GA levels. The observed effects were completely reversed by a subsequent irradiation with red light. The present data show that dwarfism due to PHYA overexpression can be completely overcome by far red end‐of‐day treatment, and the observations indicate that effects of far red end‐of‐day treatments appear to be mediated by phytochrome(s) other than phytochrome A.
Induction of tolerance to fast desiccation in black spruce  (Picea mariana) somatic embryos: relationship between partial water loss, sugars, and dehydrins
Authors: Bomal Claude , Le Van Quy , Tremblay Francine M.
Published: March 2003

Events associated with the induction of tolerance to fast desiccation in black spruce (Picea mariana) somatic embryos were investigated. An experimental approach using an initial period of partial water loss was developed to induce either no, partial, or complete tolerance to fast desiccation. Tolerance to subsequent fast desiccation was not promoted by decreasing embryo water content from 1.5 to 1.1 g H2O g−1 DW (g g−1) throughout the first 24 h of slow desiccation. However, tolerance increased from 10 to 95% germination during the second 24‐h period of slow desiccation after partial water loss from 1 to 0.55 g g−1. Emphasis was also placed on the relationship between observed tolerance, and sugar and dehydrin contents. Compared to controls, sucrose content in embryos doubled after 24 h of slow desiccation and more than tripled after 48 h. Conversely, starch content was decreased by one half after 24 h and by three quarters after 48 h. Sucrose abundance and raffinose occurrence after 48 h of slow desiccation were congruent with complete tolerance to fast desiccation. The period of slow desiccation between 24 and 48 h also increased the content of a 24‐kDa dehydrin and the appearance of a 42‐kDa dehydrin. The relationship between partial water loss, sugars and dehydrins is discussed with respect to tolerance to fast desiccation in black spruce somatic embryos.
In vitro regeneration and genetic transformation of the berberine‐producing plant, Thalictrum flavum ssp. glaucum
Authors: Samanani Nailish , Park Sang‐Un , Facchini Peter J.
Published: March 2003

Protocols have been developed for the in vitro regeneration and Agrobacterium‐mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum. Ten‐day‐old seedlings were bisected along the embryonic axis and the cotyledons were co‐cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l−1 sucrose, 2 mg l−1 kinetin, and 3 g l−1 Gelrite) containing 25 mg l−1 hygromycin B as the selection agent and 250 mg l−1 timentin to facilitate the elimination of Agrobacterium. Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin‐resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin‐resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l−1 hygromycin B, 250 mg l−1 timentin, and 3 g l−1 Gelrite). Detection of the β‐glucuronidase (GUS) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.
Ascorbate‐glutathione cycle and H2O2 detoxification in elongating leaf bases of ryegrass: effect of inhibition of glutathione reductase activity on foliar regrowth
Authors: Piquery Laurent , Huault Claude , Billard Jean‐Pierre
Published: March 2003

The role of the ascorbate‐glutathione cycle and AOS detoxification was investigated during leaf growth of defoliated and undefoliated plants of ryegrass (Lolium perenne L. cv. Bravo). Antioxidants and related enzymatic activities were located in elongating leaf bases (ELBs) of undefoliated plants, following a decreasing gradient from basal (meristem) to distal segments, inverse to H2O2 levels. In the meristematic zone, the intense activity of the ascorbate‐glutathione cycle and the supply of reducing power by the oxidative pentose phosphate pathway allowed the maintenance of both antioxidant reduction and H2O2 detoxification. BCNU (1–3 bis(2‐chloroethyl)‐N‐nitrosourea), a glutathione reductase inhibitor, induced an increase in the meristematic zone in both H2O2 and antioxidant levels and a decrease in reduced/oxidized ratios of glutathione and ascorbate. These changes were associated with a reduced foliar regrowth activity. In the absence of BCNU, defoliation did not modify the ratios of reduced/oxidized antioxidants, although it triggered a temporary increase in H2O2 level. The results are discussed on the basis of a possible control of leaf growth by glutathione and ascorbate.
Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants
Authors: Suárez Ramón , Márquez Judith , Shishkova Svetlana , Hernández Georgina
Published: March 2003

Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2‐fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.
Contribution of explant carbohydrate reserves and sucrose in the medium to bulb growth of lily regenerated on scale segments in vitro
Authors: Langens‐Gerrits Merel , Kuijpers Anne‐Marie , De Klerk Geert‐Jan , Croes Anton
Published: March 2003

Bulb size is an important factor determining phase change in Lilium: phase change only occurs in bulblets over a certain threshold weight. After phase change has occurred, bulblets sprout with a stem with many leaves. Juvenile bulblets sprout with only a few leaves. The factors contributing to bulb size were studied during in vitro regeneration of bulblets on scale segments. The larger the explants, the larger the regenerated bulblets. Explant size influenced bulb growth during the complete culture period. Bulb growth was stimulated by a high sucrose concentration. The contribution of the medium and the explant reserves to bulb growth were studied in large and small explants using labelled sucrose. Sucrose was mainly taken up through the cut surfaces. In freshly cut explants, the rate of uptake was correlated with the size of the contact area, but at later stages, when regenerating organs were present, the difference in uptake rate of small and large explants almost disappeared. Small explants had a larger sink activity than large ones. Explants with regenerating organs took up more sucrose than freshly cut explants. Sucrose uptake and bulb growth were rather constant in the later phases and doubled when the sucrose concentration was doubled. Partitioning of label from the sucrose over the various organs, was also rather constant in time: approximately 25% was accumulated in the bulblets, 35–45% in the explant and 30–35% was converted to CO2. About half of the label in the explant was recovered at the proximal side where regeneration takes place. Sucrose, once incorporated in the storage pools in the explant, either remained in the explant or was converted to CO2. Redistribution to the growing bulblets hardly occurred. The percentage of bulb growth that could be attributed to uptake of medium components was constant over the regeneration period: 45–50% for large and 65–75% for small explants.
Lectin interactions with α‐galactosylated xenoantigens
Authors: Kirkeby Svend , Moe Dennis
Published: March 2003

α‐Galactosylated xenoantigens (Galα1‐3Galβ1‐4GlcNAcβ1 and Galα1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4Glc) are often detected with the α‐Gal specific lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4). However, this lectin exhibits a broad and variable specificity for carbohydrates terminating in α‐Gal. Thus, both false positive and false negative results may occur when GS1 B4 is used to determine natural antigens in xeno (pig‐to‐primate) transplantation research. To refine the tools for detecting α‐galactosylated antigens we have studied the binding of various α‐galactophilic lectins to α‐galactosylated neoglycoproteins. The lectins were: Euonymus europaeus agglutinin (EEA), Griffonia simplicifolia 1 isolectin B4 (GS1 B4), Maclura pomifera agglutinin (MPA) and Pseudomonas aeruginosa agglutinin (PA‐IL). Although both GS1 B4 and MPA strongly bound glycoconjugates terminating in Gal there seems to be some differentiation in their sugar binding preferences. MPA was the only lectin that showed high affinity for the pentasaccharide Galα1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4Glc and for the Galα‐glycans on non‐primate thyroglobulin. The length of the xenoantigenic carbohydrate chain may influence the nature of the inhibition when a simple sugar is used to inhibit GS1 B4 binding to the xenoantigen. Inhibition studies of MPA GS1 B4 interaction further suggest that both lectins attach to the same site of the carbohydrate antigen and that GS1 B4 in addition binds to at least one other site that has no affinity for MPA. When lectins are used for recognition and investigation of natural Galα‐antigens, we propose that GS1 B4 and MPA should accompany each other.
Human serum‐induced porcine endothelial cell E‐selectin expression is associated with IgG3 and IgM anti‐Gal antibodies
Authors: Sæthre Marit , Sølvik Una Ø. , Haraldsen Guttorm , Fiane Arnt E. , Boretti Eva , Thorsby Erik , Platt Jeffrey L. , Mollnes Tom E.
Published: March 2003

Naturally occurring anti‐Galα1‐3Gal (anti‐Gal) antibodies and complement induce hyperacute rejection (HAR) of porcine organs transplanted to primates. If the hyperacute reaction is prevented, an acute vascular rejection (AVR) occurs within hours to few days. Antibodies are important for the development of AVR, whereas the role of complement is still not clarified. AVR is characterized by protein synthesis‐dependent endothelial cell (EC) activation. In the present study we investigated the relation between EC activation as measured by E‐selectin expression, and the concentrations of anti‐Gal antibodies of IgM, IgG and IgG subclasses in sera from 80 healthy blood donors selected on the basis of sex and age. There was a significant correlation between E‐selectin expression and the concentration of IgG3 anti‐Gal (r=0.39; P=0.019), which was not seen for the other IgG subclasses or for total IgG anti‐Gal. A modest, but significant correlation was found between the concentration of IgM anti‐Gal and E‐selectin expression (r=0.38; P=0.040), but not between IgM and IgG3 anti‐Gal. There was a large interindividual variation in anti‐Gal antibodies, 50‐fold for IgM and 70‐fold for IgG. Females had significantly higher concentrations of IgM anti‐Gal than males (P=0.0006), which was explained by a substantial increase in IgM anti‐Gal concentration in younger women. The concentration of IgG anti‐Gal and the degree of E‐selectin expression did not differ between sex or age groups. In conclusion, the close correlation between anti‐Gal antibodies of the potent complement activating IgG3 subclass and porcine EC activation, may imply that these antibodies play a role in EC activation characteristic of AVR.
Immune parameters relevant to neural xenograft survival in the primate brain
Authors: Cicchetti F. , Fodor W. , Deacon T. W. , Van Horne C. , Rollins S. , Burton W. , Costantini L. C. , Isacson O.
Published: March 2003

Cicchetti F, Fodor W, Deacon TW, van Horne C, Rollins S, Burton W, Costantini LC, Isacson O. Immune parameters relevant to neural xenograft survival in the primate brain. Xenotransplantation 2003: 10: 41–49. © Blackwell Munksgaard, 2003

The lack of supply and access to human tissue has prompted the development of xenotransplantation as a potential clinical modality for neural cell transplantation. The goal of the present study was to achieve a better understanding of the immune factors involved in neural xenograft rejection in primates. Initially, we quantified complement mediated cell lysis of porcine fetal neurons by primate serum and demonstrated that anti‐C5 antibody treatment inhibited cell death. We then developed an immunosuppression protocol that included in vivo anti‐C5 monoclonal antibody treatment, triple drug therapy (cyclosporine, methylprednisolone, azathioprine) and donor tissue derived from CD59 or H‐transferase transgenic pigs and applied it to pig‐to‐primate neural cell transplant models. Pre‐formed αGal, induced αGal and primate anti‐mouse antibody (PAMA) titers were monitored to assess the immune response. Four primates were transplanted. The three CD59 neural cell recipients showed an induced anti‐αGal response, whereas the H‐transferase neural cell recipient exhibited consistently low anti‐αGal titers. Two of these recipients contained surviving grafts as detected by immunohistochemistry using selected neural markers. Graft survival correlated with high dose cyclosporine treatment, complete complement blockade and the absence of an induced PAMA response to the murine anti‐C5 monoclonal antibodies.
Nest‐site selection by golden plover: why do shorebirds avoid nesting on slopes?
Authors: Whittingham Mark J. , Percival Stephen M. , Brown Andrew F.
Published: March 2003

It is widely known that many upland‐breeding shorebirds tend to nest on plateaus but to date no studies have put forward explanations for this phenomenon. We examined the effect of slope and habitat on the distribution of ground‐nesting golden plover Pluvialis apricaria at two study sites in County Durham, U.K. Golden plovers showed strong selection for nesting on flat ground. Habitat significantly affected nest‐site location on one study site (heather burnt within the past 2 years was favoured and older stands of heather were avoided) but not on the other. Fifty‐nine per cent of all nests failed. We attributed 95% of all losses to predation. Seventy‐five per cent of nests, in which the predator was identified, were taken by ground predators, mainly stoats Mustela erminea. Nests on flat ground had significantly higher rates of survival than those on slopes. Nest survival did not vary significantly with habitat type nor with vegetation height or density around the nest. Neither habitat type, vegetation height nor vegetation density around nests differed between nests on slopes and on flat ground. In addition, individuals that nested on flat ground tended to have less black on their underparts (a suggested indicator of dominance). We suggest that birds nesting on slopes are less efficient at avoiding nest predation than those nesting on the flat. The nature or degree of a bird's response to a predator may be related to the efficiency of individual anti‐predator responses and/or to visibility from the nest. This study cannot differentiate between these two explanations. We encourage further work to investigate differences in behaviour between individuals nesting on slopes and those on flat ground.
Innate immunity and graft rejection
Authors: Fox Annette , Harrison Leonard C.
Published: March 2003

Summary: Although innate immunity evolved to combat pathogens, increasing awareness of a pivotal role in driving and shaping adaptive immunity has prompted this review on the role of innate immunity in graft rejection. We present evidence that grafts, especially xenografts, elicit innate responses, required for adaptive immunity. Particular attention is paid to studies by ourselves and others demonstrating the important role of innate immunity in T‐cell trafficking. The mechanisms by which grafts elicit innate immunity are a fertile subject for further investigation and an important target for therapeutic intervention.
Human blood platelet gC1qR/p33
Authors: Peerschke Ellinor I. B. , Ghebrehiwet Berhane
Published: March 2003

Platelets are involved in the development of many types of vascular lesions. In addition to their role in primary hemostasis, they participate in inflammatory processes that may contribute to the development of thrombosis, atherosclerosis and vasculitis. In this regard, we have been interested in platelet interactions with the complement subcomponent C1q. C1q has been shown to modulate platelet interactions with collagen and immune complexes, and has been identified at sites of vascular injury and inflammation, as well as in atherosclerotic lesions. Platelets express a variety of C1q binding sites, including gC1qR/p33 (gC1qR), a multifunctional, multicompartment cellular protein. Here we focus on the structure and function of platelet gC1qR and its emerging role in modulating platelet function at sites of vascular injury and inflammation.

The authors are grateful to Wei‐Xing Guo M.D. for construction of the Exon 2 expression vector and GST‐E2 fusion protein expression, and to Yan Fang and Tara K. Murphy for technical assistance. This research was supported in part by grant HL50291 from the National Heart Lung and Blood Institute (EIBP, BG) and grant IM771 (BG) from the American Cancer Society.
The RT6 system of the rat: developmental, molecular and functional aspects
Authors: Thiele Heinz‐Günter , Haag Friedrich
Published: March 2003

Summary: RT6 is a developmentally regulated cell‐surface membrane adenosine 5′‐diphosphate‐ribosyltransferase/nicotinamide adenine dinucleotide‐glycohydrolase inserted within the membrane by a glycophosphatidylinositol anchor. In the rat it is restricted to mature T lymphocytes and a subpopulation of natural killer cells. With respect to the data now available, three aspects concerning the function of RT6 are discussed: first, the meaning of the marked polymorphisms; second, its enzymatic activity; third, its possible role concerning T‐cell survival. The observation that the rat RT6 gene contains two transcription start sites suggests their different use by distinct subpopulations of T cells. The fact that the expression of RT6 is defective in lymphopenic diabetes prone (DP‐BB) rats, although the RT6 gene is structurally not grossly altered in these animals, makes this rat strain a promising model to study the biological meaning of RT6. While it mostly is believed that the RT6 expression defect of the DP‐BB rat is a consequence of the lymphopenia, the present paper discusses the possibility that the RT6 expression defect is causally involved in the lymphopenia, and that a normal expression of RT6 may protect the recent thymic emigrants from apoptosis.
Rat models as tool to develop new immunotherapies
Authors: Link Hans , Xiao Bao‐Guo
Published: March 2003

Summary: Rat models (experimental autoimmune encephalomyelitis, EAE; myasthenia gravis, EAMG) have been developed that mimic certain aspects of the pathophysiology of multiple sclerosis (MS) and myasthenia gravis (MG) in humans. Rat EAE and EAMG are, therefore, widely used to evaluate immunotherapeutic strategies. Mucosal tolerance induced by oral or nasal administration of autoantigen effectively suppresses rat EAE and EAMG. Nasal administration of the cytokines interleukin (IL)‐4, IL‐10 or transforming growth factor (TGF)‐β1 also ameliorates clinical signs of rat EAE. However, neither mucosal tolerance nor cytokines affect clinical disease if administered during ongoing rat EAE or EAMG. Dendritic cells (DC) pulsed in vitro with autoantigen can mediate peripheral tolerance against rat EAE and EAMG, but do not affect ongoing EAE or EAMG. DC from healthy rats, modified in vitro by exposure to IFN‐γ or TGF‐β1, effectively suppress ongoing EAE and EAMG when given by the subcutaneous route. Most importantly, DC from EAMG rats, after being modified in vitro with IL‐10, also inhibit ongoing rat EAMG. These results demonstrate that different strategies influencing T‐ and/or B‐cell responses in rat EAE or EAMG are available, but that DC‐based immunotherapies are the most promising, in order to ameliorate ongoing organ‐specific autoaggressive immunity. Based on these observations, autologous DC, after being modified in vitro with cytokines, constitute a basis for new immunotherapeutic strategies in MS, MG and other diseases with autoimmune background.
Cellular and genetic factors involved in the difference between Brown Norway and Lewis rats to develop respectively type‐2 and type‐1 immune‐mediated diseases
Authors: Fournié Gilbert J. , Cautain Bastien , Xystrakis Emmanuel , Damoiseaux Jan , Lagrange Magali Mas Dominique , Bernard Isabelle , Subra Jean‐François , Pelletier Lucette , Druet Philippe , Saoudi Abdelhadi
Published: March 2003

Summary: The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune‐mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1‐mediated autoimmune diseases while BN rats are highly susceptible to Th2‐mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC‐dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC‐independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL‐4, IL‐13, IL‐10 and TGF‐β in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1‐mediated experimental autoimmune encephalomyelitis, and to Th2‐mediated disorders triggered by gold salts as well as the difference in the CD45RChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.
Regulation of eosinophil and neutrophil apoptosis – similarities and differences
Authors: Simon Hans‐Uwe
Published: March 2003

Summary: Apoptosis is the most common form of physiologic cell death and a necessary process to maintain cell numbers in multicellular organisms. In many chronic inflammatory diseases, reduced cell death of different types of granulocytes is one important mechanism for cell accumulation. Granulocytes are constantly produced in large amounts in the bone marrow and the same numbers die, under normal circumstances, within a defined time period. Changing the rate of apoptosis rapidly changes cell numbers in such systems. Overexpression of IL‐5 appears to be crucial for delaying eosinophil apoptosis in many allergic disorders, whereas overexpression of GM‐CSF and G‐CSF is associated with suppression of neutrophil apoptosis in bacterial and non‐bacterial inflammations. Cytokine withdrawal leads to the induction of apoptosis both in vitro and in vivo. In contrast to the role of survival cytokines, little is known about the role of death factors and their receptors in the regulation of granulocyte apoptosis. Recent observations suggest a role for mitochondria in both eosinophil and neutrophil apoptosis, although the mechanisms that trigger mitochondria to release pro‐apoptotic factors remain to be determined. Besides similarities, there are differences in the regulation of apoptosis between these granulocyte subtypes that include both expression and function of Bcl‐2 and caspase family members. The identification of differences in the apoptosis regulation may help to define new molecular targets that allow specific induction of either eosinophil or neutrophil apoptosis by pharmacological means.

Work in the author's laboratory is supported by the Swiss National Science Foundation (32‐58916.99), Helmut Horten Foundation (Madonna del Piano), Foundation Novartis (Basel), EMDO Foundation (Zurich), and Stiftung zur Krebsbekämpfung (Zurich).
TCR triggering on the move: diversity of T‐cell interactions with antigen‐presenting cells
Authors: Friedl Peter , Bröcker Eva‐B.
Published: March 2003

Summary: Polarized T cells are mobile cells optimized for migration, receptor scanning, and signaling. When in contact with antigen‐presenting cells (APCs), polarized T cells can develop a spectrum of biophysical interaction modes ranging from adhesive sticking to dynamic crawling. Both static and dynamic contacts support sustained triggering of the T‐cell receptor (TCR), leading to signal induction, T blast formation, and proliferation. In dynamic interactions, T cells crawl across the surface of the APC at speeds of 2–6 µm/min and simultaneously establish an asymmetric tight yet mobile junction plane, representing a dynamic immunological synapse. In dynamic synapses three functional compartments of the polarized T cell are in close contact with the APC surface, i.e. leading edge, cell body and uropod. Through its mobility, the asymmetric junction is topographically suited for receptor scanning and engagement at the leading edge, retrograde receptor movement along the junction, and exit from the uropod. Herein we develop a model on scanning encounters between T cells and APCs that includes the simultaneous engagement of T‐cell leading edge and uropod and implicates a serial receptor triggering mode in cell‐cell recognition.
Integrin structure: new twists and turns in dynamic cell adhesion
Authors: Arnaout M. Amin
Published: March 2003

Summary: The divalent‐cation‐dependent binding of αβ heterodimeric integrins to their ligands regulates most cellular processes. Integrin–ligand interactions are tightly controlled by inside‐out activation signals. Ligand‐bound integrins transduce outside‐in signals typical of other receptors. Precise information of how ligands bind to integrins is restricted to that of a small vWF A‐type domain present in some α‐subunits (αA). Both inside‐out and outside‐in signals elicit tertiary and quaternary changes in integrins, but the precise nature and scope and of these changes are unknown. The recently solved structures of the extracellular segment of integrin αVβ3 in its unliganded and liganded states are generating exciting new insights into the design, wiring, function and regulation of this protein family. The structures reveal a surprising degree of flexibility at defined regions in the structure that is potentially controlled by cations. The quaternary structure of the ligand‐binding region bears a striking resemblance to the nucleotide‐binding pocket of G‐proteins, implying analogous signaling mechanisms. Structural links exist through which ligand‐induced tertiary changes may be translated into quaternary changes and vice versa. The structures also raise the tantalizing hypothesis that αA is a regulated endogenous integrin ligand, so that no special regulatory features are needed in this integrin. These findings provide the framework for new investigations of structure–activity relationships in integrins, with important implications for targeting these receptors therapeutically.
Analysis of the B‐cell repertoire against antigens expressed by human neoplasms
Authors: Preuss Klaus‐Dieter , Zwick Carsten , Bormann Claudia , Neumann Frank , Pfreundschuh Michael
Published: March 2003

Summary: The screening of tumor‐derived expression libraries for antigens detected by high‐titer immunoglobulin G antibodies from the sera of patients with cancer by SEREX (serological identification of antigens by recombinant expression cloning) allows a systematic search for antigens of human cancers. SEREX has led to the identification of a multitude of new tumor antigens in many different tumor entities. According to their specificities, the antigens can be grouped into different classes, of which the cancer testis antigens appear to be the most attractive candidates for vaccine development. Serologically defined human tumor antigens facilitate the identification of antigenic peptides recognized by tumor‐specific T lymphocytes, thus providing a molecular basis for polyvalent peptide‐based and gene‐therapeutic vaccine strategies in a wide variety of human neoplasms. Moreover, many of the SEREX‐identified antigens seem to play a functional role in the pathogenesis of malignant disease. With more than 2000 antigens listed in the SEREX database, it appears that tumor antigens that have resisted discovery to date are expressed in only a small minority of tumors, thus limiting their clinical usefulness. Novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of non‐Hodgkin's lymphoma patients.
Mapping genes for autoimmunity in humans: type 1 diabetes as a model
Authors: Onengut‐Gumuscu Suna , Concannon Patrick
Published: March 2003

Summary: Type 1 diabetes (T1D) arises from autoimmune destruction of the β cells of the pancreas leading to a complete dependence on exogenous insulin for survival. Like many autoimmune disorders, the etiology of T1D is complex, resulting from the action of multiple genes and environmental factors. Identification of genes that contribute to T1D susceptibility should improve disease prediction and contribute to the understanding of the underlying pathology of the disorder. Two regions of the human genome, the human leukocyte antigen (HLA) region and the insulin gene region are generally thought to contain susceptibility loci for T1D. Although additional putative T1D loci have been reported, the supporting evidence has often been of modest significance and findings have displayed poor reproducibility across multiple studies. This review summarizes the current state of genetic linkage and association studies in T1D and suggests future directions. We argue that much of the difficulty in mapping T1D susceptibility loci has resulted from inadequate sample sizes and we illustrate the substantial gains in power that can be achieved by pooling data across studies. These findings suggest that substantial progress toward the identification of susceptibility genes in T1D and other genetically complex disorders may be achieved through increased collaboration and consortium mapping efforts.
All roads lead to actin: the intimate relationship between TCR signaling and the cytoskeleton
Authors: Fuller Claudette L. , Braciale Vivian L. , Samelson Lawrence E.
Published: March 2003

Regardless of cell type, the regulation of the actin cytoskeleton is tightly linked to vital biological properties such as polarity, motility, cell–cell contact, exocytosis and proliferation. In the immune system, where rapid and efficient response to antigen‐provoked stimuli is crucial, an overwhelming amount of data implicate the actin cytoskeleton and its regulators as central to immune function. Increasingly, the cytoskeleton is considered an essential amplification step in T cell receptor (TCR)‐, costimulatory‐, and integrin‐mediated signaling. Advances in genetic manipulation and confocal imaging have led to a keener appreciation of the importance of TCR signal integration by the actin cytoskeleton. This review outlines recent advances in elucidating the regulation of T cell function through the actin cytoskeleton. We also examine intriguing parallels between the immune system and other models of cytoskeletal regulation.
Cholestatic syndrome with bile duct damage and loss in renal transplant recipients with HCV infection
Authors: Delladetsima Johanna K. , Makris Fotis , Psichogiou Mina , Kostakis Alkiviadis , Hatzakis Angelos , Boletis John N.
Published: March 2003

Abstract: Background/Aims: Bile duct cells are known to be susceptible to hepatitis B and C virus, while it has been recently suggested that hepatitis B virus (HBV) and hepatitis C virus (HCV) infection may have a direct role in the pathogenesis of vanishing bile duct syndrome (VBDS) after liver transplantation. We report the development of a cholestatic syndrome associated with bile duct damage and loss in four HCV‐infected renal transplant recipients. Methods: All four patients were followed up biochemically, serologically and with consecutive liver biopsies. Serum HCV RNA was quantitatively assessed and genotyping was performed. Results: Three patients were anti‐HCV negative and one was anti‐HCV/HBsAg positive at the time of transplantation and received the combination of methylprednisolone, azathioprine and cyclosporine A. Two patients became anti‐HCV positive 1 year and one patient 3 years post‐transplantation. Elevation of the cholestatic enzymes appeared simultaneously with seroconversion, or 2–4 years later, and was related to lesions of the small‐sized interlobular bile ducts. Early bile duct lesions were characterized by degenerative changes of the epithelium. Late and more severe bile duct damage was associated with bile duct loss. The progression of the cholestatic syndrome coincided with high HCV RNA serum levels, while HCV genotype was 1a and 1b. Two patients (one with HBV co‐infection) developed progressive VBDS and died of liver failure 2 and 3 years after biochemical onset. One patient, despite developing VBDS within a 10‐month period, showed marked improvement of liver function after cessation of immunosuppression because of graft loss. The fourth patient, who had mild biochemical and histological bile duct changes, almost normalized liver function tests after withdrawal of azathioprine. Conclusion:Á progressive cholestatic syndrome due to bile duct damage and loss may develop in renal transplant patients with HCV infection. The occurrence of the lesions after the appearance of anti‐HCV antibodies and the high HCV RNA levels are indicative of viral involvement in the pathogenesis. Withdrawal of immunosuppressive therapy may have a beneficial effect on the outcome of the disease.
Hepatic graft‐versus‐host disease resembling acute hepatitis: additional treatment with ursodeoxycholic acid
Authors: Chiba Tetsuhiro , Yokosuka Osamu , Kanda Tatsuo , Fukai Kenichi , Imazeki Fumio , Saisho Hiromitsu , Nishimura Miki , Saito Yasushi
Published: March 2003

Abstract: Hepatic graft‐versus‐host disease (GVHD) is a frequent complication after bone‐marrow transplantation (BMT). The disease is often refractory to immunosuppressive therapy. We present a 30‐year‐old Japanese male, who developed an abrupt elevation of aminotransferases, on day 135 after allogeneic BMT. A liver biopsy specimen revealed degeneration of the small bile ducts and portal fibrosis, and the diagnosis of chronic hepatic GVHD was confirmed. No manifestation of chronic GVHD was observed except liver dysfunction. The administration of prednisolone (PSL) and cyclosporin (CsA) ameliorated laboratory data to a degree, but they did not return to normal. Treatment with ursodeoxycholic acid (UDCA), subsequently added to the immunosuppressive therapy, apparently normalized the levels of biliary tract enzyme and total bilirubin. His liver function test completely returned to normal on day 260. We believe that it is worthwhile to administer UDCA as an additional treatment for not only common hepatic GVHD but also atypical cases presenting as acute hepatitis.
Impact of oestrogens on the progression of liver disease
Authors: Shimizu Ichiro
Published: March 2003

Abstract: As of this writing, the most common cause of hepatic fibrosis is chronic hepatitis C virus infection (HCV), the characteristic feature of which is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes, which in turn activates hepatic stellate cells (HSCs). HSCs are also thought to be the primary target cells for inflammatory stimuli, and produce extracellular matrix components. Based on available clinical information, chronic hepatitis C appears to progress more rapidly in men than in women, and cirrhosis is predominately a disease of men and postmenopausal women. It should be noted that estradiol (E2) is a potent endogenous antioxidant. A recent study has shown that hepatic steatosis became evident in an aromatase‐deficient mouse and was diminished in animals, after treatment with E2. Our studies showed that E2 suppressed hepatic fibrosis in hepatic fibrosis models, inhibited the activation of activator protein 1 and nuclear factor‐κB in cultured hepatocytes undergoing oxidative stress, and attenuated HSC activation in primary culture. Recently, variant oestrogen receptors (ERs) were found to be expressed to a greater extent in male patients with chronic liver disease than in female subjects. We also demonstrated decreased levels of ERs in postmenopausal women and cirrhotic patients of both genders. The actions of E2 are mediated through ER α and β. HSCs have also been found to possess functional ER β but not ER α. A better understanding the basic mechanisms underlying the gender‐associated differences observed in the development of hepatic fibrosis would provide valuable information relative to the search for effective antifibrogenic therapies.
Tumor necrosis factor‐α and interferon‐γ directly impair epithelial barrier function in cultured mouse cholangiocytes
Authors: Shinichiro Hanada , Masaru Harada , Hironori Koga , Takumi Kawaguchi , Eitaro Taniguchi , Ryukichi Kumashiro , Takato Ueno , Yoshiyuki Ueno , Motoyasu Ishii , Shotaro Sakisaka , Michio Sata
Published: March 2003

Abstract:Background/Aims:In primary biliary cirrhosis (PBC), cytokines fromCD4 + Tlymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity byCD8 + Tlymphocytes. Recently, we reported that immunolocalization of 7H6 – a tight junction (TJ)‐associated protein – was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines – tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), interleukin‐2 and 4 (IL‐2 and 4) – on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid(UDCA)on cytokine‐induced changes in paracellular permeability.Methods:Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and3H‐inulin flux. We also performed immunostaining and immunoblotting for TJ‐associated proteins – claudin‐1 and ‐3, occludin, zonula occluden‐1 (ZO‐1) and 7H6.Results:TNF‐α and IFN‐γ, but neither IL‐2 nor IL‐4, significantly decreased TER (P < 0.005).3H‐inulin flux studies confirmed IFN‐α‐induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ‐associated proteins were well preserved in TNF‐α‐ or IFN‐γ‐treated cells. Ursodeoxycholic acidhas been found to have no inhibitory effect on increased paracellular permeability induced by TNF‐α or IFN‐γ.Conclusion:These findings show that TNF‐α and IFN‐γ disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.
Hepatocellular adenoma and polycystic ovary syndrome
Authors: Toso Christian , Rubbia‐Brandt Laura , Negro Francesco , Morel Philippe , Mentha Gilles
Published: March 2003

Abstract: Various identified risk factors predispose to hepatocellular adenomas. We present the case of a young woman with liver adenoma in a context of polycystic ovary syndrome, associated with high levels of androgens and following a high dose hormonal therapy. In view of this complication, we recommend a close screening of patients with such hormonal imbalance, especially those who are treated with high doses of hormones, with repeated liver tests and ultrasonographies.