bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular pathogenesis of human cytomegalovirus infection
Authors: Rasmussen L.
Published: March 2003

Despite progress in diagnosis and treatment of human cytomegalovirus (CMV) infection, we do not understand why, in hosts with comparable levels of immunosuppression, some CMV infections result in symptomatic CMV disease while others are limited to asymptomatic virus shedding with no discernible clinical consequences. CMV viral detection and quantification are useful for identifying those at highest risk, but do not consistently predict clinical outcome. Factors such as host genotype and immune response are active areas of research. However, the importance of CMV strain variability, recognized since 1976, is now receiving attention. Advances in technology that allow the rapid sequencing of viral DNA for purposes of strain characterization have fueled the renewed interest. The focus of this review will be to summarize our evolving knowledge of CMV strain variability and to document where possible a potential relationship to strain virulence. Studies with the UL55 (gB) envelope glycoprotein will be emphasized because of the ability to clearly identify naturally occurring variants, as well as the increasing number of reports that there are differences in biological activities that may contribute to virulence 1.
Mechanisms of human cytomegalovirus (HCMV) (re)activation and its impact on organ transplant patients
Authors: Reinke P. , Prösch S. , Kern F. , Volk H.‐D.
Published: March 2003

Human cytomegalovirus (HCMV) infection plays an important role in transplant patients. Its impact is both direct and indirect. This review focuses on new aspects of HCMV (re)activation and HCMV related pathology, particularly HCMV‐associated renal allograft injury. During the last two years we have learned that HCMV is more frequently (re)activated, even in healthy people, than previously expected. Inflammatory as well as stress mediators and some drugs may (re)activate the virus by using distinct intracellular pathways. Commonly, HCMV (re)activation is accompanied by HCMV antigenemia/DNAemia, suggesting that precursor cells in the bone marrow play an important role as a reservoir of latent virus. However, local HCMV (re)activation (colon, lung) without detection of active HCMV infection in the peripheral blood is possible. In healthy people a sufficient type 1 T‐cell response controls the active HCMV infection, while in patients with severe immune deficiency (AIDS, high‐dose immunosuppression) the virus can spread in an uncontrolled fashion and induce ‘classic’ HCMV disease. In patients with moderate immune deficiency (e.g. long‐term transplant patients on low‐dose immunosuppression) virus spreading is controlled but the elimination of cells harboring the active virus may be insufficient. The resulting persistent HCMV antigenemia may induce chronic inflammatory processes leading to tissue injury, particularly in the allograft. Therefore, antiviral therapy may be useful in patients suffering from graft deterioration with otherwise clinically symptomless HCMV infection. HCMV‐related immune deficiency with an increased risk of developing bacterial/fungal superinfections is frequently seen in patients with symptomatic HCMV disease but not in asymptomatic CMV antigenemia. The risk of developing superinfections can be predicted by flow‐cytometric monitoring of peripheral blood monocytes.
Toxoplasma gondii pneumonia in liver transplantation: survival after a severe case of reactivation
Authors: Barcán L.A. , Dallurzo M.L. , Clara L.O. , Valledor A. , Macías S. , Zorkin E. , Gerona S. , Livellara B. , Barcán L.A. , Dallurzo M.L. , Clara L.O. , Valledor A. , Macías S. , Zorkin E. , Gerona S. , Livellara B.
Published: March 2003

Abstract: Toxoplasmosis is an infrequent infection in solid organ transplantation, except in heart transplantation, where the grafting of a positive organ in a negative recipient transmits the infection in a high percentage of cases, in the absence of prophylaxis.

We report a case of pneumonia by Toxoplasma gondii in a woman who received a liver transplant and had pre‐transplant positive serology. Diagnosis was made by cytologic examination of bronchoalveolar lavage fluid, where the parasite was observed with hematoxylin–eosin and Giemsa staining. That finding was confirmed by direct immunofluorescence and positive polymerase chain reaction. The patient had a favorable outcome, although she had not initially received first‐choice drugs. This was a case of severe illness secondary to reactivation of Toxoplasma infection, diagnosed pre‐mortem and with a favorable outcome. Duration of treatment and need for secondary prophylaxis in these patients are discussed in the literature. Although infrequent, toxoplasmosis must be considered among the differential diagnoses of pulmonary infiltrates in solid organ transplantation.
Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts
Authors: Fiane Arnt E. , Mollnes Tom E. , Videm VIBEKE , Hovig TORSTEIN , Høgåsen Kolbjørn , Mellbye Ove J. , Spruce LYNN , Moore William T. , Sahu ARVIND , Lambris John D.
Published: March 2003

Fiane AE, Mollnes TE, Videm V, Hovig T, Høgåsen K, Mellbye OJ, Spruce L, Moore WT, Sahu A, Lambris JD. Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused xenografts. Xenotransplantation 1999; 6: 000‐000 ©Munksgaard, Copenhagen

Compstatin, a newly described C3‐binding peptide, inhibits complement activation by blocking C3 convertase‐mediated cleavage of C3. As the complement activation is an essential part of the rejection reaction, we evaluated the ability of Compstatin to delay or prevent hyperacute rejection in an ex vivo xenograft model. Porcine kidneys were perfused with fresh human blood containing either Compstatin (n=6) or a control agent (n=6). Graft survival and activation of complement, leukocytes and platelets both in the fluid phase and in the tissue were examined. The survival of the Compstatin‐perfused kidneys (median, 380 min) was significantly (P=0.0036) longer than that of the controls (median, 90 min). The classical complement pathway (C1rs‐C1inhibitor and C4bc) was significantly and equally activated in both groups during the first 60 min. C3 activation products increased fivefold and terminal complement complex eightfold in the control group, but no increase occurred in the Compstatin group during this period. Immunohistochemistry showed less C3 and fibrin deposition and immuno‐electron microscopy showed less terminal SC5b‐9 complement complex deposition in the Compstatin group. A significant change in total white cells, neutrophils, myeloperoxidase, and expression of the surface activation markers CD11b (CR3) and CD35 (CR1) and CD62 L (
l‐selectin) was observed in both groups. Leukocyte activation was lower in the Compstatin group but the difference was not statistically significant. There were no differences in platelet counts, thrombospondin, soluble P‐selectin or β‐thromboglobulin between the groups. We conclude that Compstatin prolongs graft survival and suggest that it may be a useful agent for attenuating hyperacute rejection by inhibiting C3 and thus terminal complement pathway activation.
Immunohistologic evaluation of mechanisms mediating hyperacute lung rejection, and the effect of treatment with K76‐COOH, FUT‐175, and anti‐Gal column immunoadsorption
Authors: Zhang Jian‐ping , Blum Matthew G. , Chang Andrew C. , Shyr Yu , Blair Kelly S. A. , Awwad Michel , Pierson Richard N.
Published: March 2003

Abstract: Although most investigators agree that lung dysfunction occurs rapidly in various pig‐to‐primate hyperacute lung rejection (HALR) models, the basic mechanisms mediating this phenomenon remain in question. Here we describe an immunohistochemical method for assessment of mechanisms driving HALR. Using an established model wherein piglet lungs are perfused ex vivo with human blood, six experimental groups (K76 COOH; FUT‐175; K76 with FUT; anti‐α‐Gal column adsorption; column with FUT; and column with K76) and two control groups (unmodified human blood; autologous pig blood) were studied. Each lung was biopsied serially during perfusion, and assessed using an immunohistochemical technique, with vWF staining as an internal control to quantitate binding of human IgM, IgG, C3, C5b‐9, properdin, and C1q. The effect of each treatment and subsequent lung perfusion on IgG and IgM anti‐α‐Gal titers(by ELISA) and on pig endothelial cell cytotoxicity were correlated with histologic findings. We found that [1] the classical complement activation pathway was activated, as has been shown for other pig organs in primate or human blood environments [2]; alternative complement pathway activation is also seen, which has not been described for other organs in pig‐to‐primate models, but only in the context of classical pathway activation; and [3] anti‐Gal column absorption, pharmacologic inhibition of complement, or combination therapy each was associated with histologic evidence of partial protection, consistent with what would be predicted for each intervention. Further, immunohistologic differences correlated with physiologic outcomes [8] and with antibody assay results, and revealed that treatments used were incompletely effective. Our data suggest that more complete inhibition of antibody‐ and complement‐driven pathways than was achieved in these experiments will be necessary to prevent the antibody and complement‐mediated facets of hyperacute lung rejection. This immunohistologic technique may also help us identify additional pathogenic mechanisms important to eventual clinical application of pig‐to‐human lung xenografts.
Porcine endogenous retroviruses (PERVs): Generation of specific antibodies, development of an immunoperoxidase assay (IPA) and inhibition by AZT
Authors: Stephan Oliver , Schwendemann Jochen , Specke Volker , Tacke Stefan J. , Boller Klaus , Denner Joachim
Published: March 2003

Abstract: Xenotransplantation may be associated with the risk of transmission of microorganisms. In particular, the porcine endogenous retroviruses (PERV) have raised concerns as in vitro experiments show susceptibility of human cells for PERV infection. However, it remains unclear whether PERVs are able to infect transplant recipients in vivo and whether they are pathogenic. It is therefore essential that the risks are evaluated and for this purpose specific and sensitive screening methods for PERVs have to be developed. We generated specific antibodies against all major structural proteins of PERV and developed several assays which allow antibodies against PERV to be detected as indirect evidence of infection. For direct detection of PERV production, reverse transcriptase (RT) assays were used. PCR methods were used to detect provirus integration and the presence of viral mRNA. Here we present an immunoperoxidase assay (IPA), which would allow the detection of viral proteins in infected cells as well as antibodies against PERV in the serum of an infected host. The specificity of the sera used in the assay was determined by several methods, including immunoelectron microscopy, and the sensitivity of the assay was compared with other methods. This IPA was used to detect PERV infection in in vitro experiments for evaluation of the virus host range, for titrating the virus and for testing anti‐viral properties of AZT. Using this method it was shown that AZT inhibits replication of PERV. This IPA may be very useful for the surveillance of preclinical and clinical xenotransplantations.
Enzymatic removal of αGal antigen in pig kidneys by ex vivo and in vivo administration of endo‐β‐galactosidase C
Authors: Liu DaGe , Kobayashi Takaaki , Yokoyama Itsuo , Ogawa Haruko , Nagasaka Takaharu , Muramatsu Hisako , Kadomatsu Kenji , Oikawa Tadashi , Shimano Yasunobu , Morozumi Kunio , Uchida Kazuharu , Muramatsu Takashi , Nakao Akimasa
Published: March 2003

Xenotransplantation using the pig as a donor species is considered to be a promising solution to the serious shortage of organ donors. Both hyperacute and acute vascular rejection (AVR) are believed to be associated with xenoreactive antibody binding to αGal epitopes on the pig vascular endothelial cells. Thus, suppression of this antigen‐antibody reaction would appear essential for successful long‐term xenograft survival. The purpose of this study was to examine the efficacy of ex vivo and in vivo administration of recombinant endo‐β‐galactosidase C (EndoGalC which, in previous in vitro studies, has been proven to digest αGal antigens completely) on αGal epitopes expressed in pig kidneys. Excised pig kidneys were perfused with University of Wisconsin solution containing EndoGalC and preserved for 4 h. After cold storage, the pig kidney was transplanted into another pig. Ex vivo perfusion and cold storage with EndoGalC reduced αGal epitope expression on vascular endothelial cells to an undetectable level. However, αGal antigens began to be expressed again as early as 1 day after transplantation. The digestion of αGal epitopes by EndoGalC did not cause any damage to the kidney graft. EndoGalC was intravenously administered to two pigs (15 kg), without causing any serious adverse effect. Twelve hours later, >98% of αGal antigens on pig red blood cells (RBCs) had been digested. Immunohistochemical study revealed almost complete elimination of αGal expression on vascular endothelial cells of the kidney graft 4 and 8 h after in vivo administration, but reappearance within 24 h. EndoGalC was administered to a baboon after an interval of 2 months. The second administration did not result in any serious toxicity or reduction in efficacy. These results suggest that ex vivo and in vivo administration of EndoGalC is simple and useful in removing αGal epitopes from pig organs. As the effect of EndoGalC is temporary, multiple in vivo administrations of EndoGalC would be required to inhibit the reappearance of αGal epitopes. Alternatively, transgenic techniques of introducing the gene for EndoGalC into the donor organ might permanently prevent αGal expression.
Porcine mononuclear cells adhere to human fibronectin independently of very late antigen‐5: implications for donor‐specific tolerance induction in xenotransplantation
Authors: Theodore Pierre R. , Simon Andre R. , Warrens Anthony N. , Sackstein Robert , Sykes Megan
Published: March 2003

To combat the shortage of donor organs, transplantation across species barriers has been proposed. Induction of tolerance would overcome the substantial immunologic barriers to xenotransplantation and would avoid the chronic use of immunosuppressive agents. Successful transplantation of hematopoietic cells induces robust specific tolerance to donor antigens in allogeneic and xenogeneic models. The β1 integrin class of adhesion molecules and their interactions with extracellular matrix components are thought to be integral to the engraftment and maturation of hematopoietic stem cells. We therefore examined the efficacy of porcine very late antigen‐5 (VLA‐5) and VLA‐4 interactions with the human extracellular matrix (ECM) protein, fibronectin. Peripheral blood mononuclear cells (PBMCs) from humans and miniature swine were flourochrome labeled and adhesion to plates coated with whole human fibronectin (whFN) or its 120 KDa fragment containing the VLA‐5 binding region was determined. Flow cytometry and immuno‐ precipitation were used to identify a monoclonal antibody that cross‐reacted on porcine VLA‐5. Human and pig PBMC adhesion to human fibronectin (hFN) or 120 kDa fragment‐coated plates was assessed following incubation with control ab, anti‐VLA‐4, anti‐VLA‐5, or soluble fibronectin. Using rabbit complement, cells expressing VLA‐5 were purged from PBMC preparations before performing the adhesion assay. Porcine and human PBMC both adhered to hFN in a divalent cation‐dependent and activation‐dependent manner. Adhesion to hFN of human but not pig PBMC was blocked by anti‐VLA‐5 monoclonal antibody SAM‐1, although this mAb immunoprecipitated a heterodimeric cell surface molecule (155/135 kDa) resembling VLA‐5 from pig PBMC. Complement‐mediated depletion of VLA‐5‐expressing cells ablated specific binding of human but not porcine cells to hFN and its 120 kDa fragment. Addition of soluble fibronectin was capable of blocking adhesion of PBMC of both species to hFN. Anti‐VLA‐4 reduced the binding of PBMC from both species to hFN to a similar extent. Human and pig cells can specifically adhere to hFN and its 120 kDa fragment, suggesting that this critical cell–ECM interaction is preserved across species. While human cells exclusively use VLA‐5 for binding to the 120 kDa fragment, porcine cells could not be shown to adhere to whFN or its 120 kDa fragment via VLA‐5. However, porcine VLA‐4 is capable of mediating adhesion to human FN. We conclude that disparities in the adhesive interactions of β1 integrins may be a barrier to the use of porcine hematopoietic stem cell transplantation as a means of inducing donor‐specific tolerance in the pig to human species combination.
Pig endogenous retroviruses and xenotransplantation
Authors: Blusch Jürgen H. , Patience Clive , Martin Ulrich
Published: March 2003

Xenotransplantation of porcine organs might provide an unlimited source of donor organs to treat endstage organ failure diseases in humans. However, pigs harbour retroviruses with unknown pathogenic potential as an integral part of their genome. While until recently the risk of interspecies transmission of these porcine endogenous retroviruses (PERV) during xenotransplantation has been thought to be negligible, several reports on infection of human cells in vitro and spread of PERV from transplanted porcine islets in murine model systems have somewhat challenged this view. Here, we compile available data on PERV biology and diagnostics, and discuss the significance of the results with regard to the safety of clinical xenotransplantation.
Pre‐transplant analysis of accommodation in donor pigs
Authors: Beschorner William E. , Shearon C. Carson , Yang Tianyu , Langnas Alan N. , Thompson Scott C. , Zhao Yong , Franco Kenneth L. , Radio Stanley J. , Sudan Debra L.
Published: March 2003

Beschorner WE, Shearon CC, Yang T, Langnas AN, Thompson SC, Zhao Y, Franco KL, Radio SJ, Sudan DL. Pre‐transplant analysis of accommodation in donor pigs. Xenotransplantation 2003; 10: 66–71. © Blackwell Munksgaard, 2003

Accommodation could lead to xenograft acceptance without the need for severe immune suppression. Generally graft accommodation is appreciated in the sensitized recipient, after transplantation. By inducing accommodation in chimeric donors, however, the risk and cost of inducing accommodation in the recipient would be reduced. An indirect assay of accommodation in the donor pig is needed for screening donors prior to procurement of the xenograft. The resistance of peripheral blood lymphocytes to cytolysis by antibody and complement was assessed in chimeric pigs and compared with control pigs. Peripheral blood lymphocytes (PBL) from chimeric pigs demonstrated a wide range of cytolysis (0 to 85%, median 13%) whereas PBL from control pigs were consistently lysed with these conditions (86 to 99%, median 96.5%, P < 0.0001). Accommodation or reduction in cytolysis did not correlate with the amount of chimerism. A longitudinal study demonstrated persistent accommodation of the PBL for as long as 15 weeks, when the donors averaged 68 kg in weight. Accommodation has been induced by low levels of antibodies interacting with the target tissue. An ELISA for sheep IgG was developed and the serum from newborn pigs assessed. Sheep IgG (up to 4.6 μg/ml) was detected in four of seven piglets with chimerism detectable by flow cytometry and in one of four piglets with minimal chimerism, detectable only by PCR. Lymphocyte accommodation was observed in all pigs with detectable sheep IgG. Of four pigs without accommodation, none had sheep IgG. Three pigs without detectable sheep IgG also had accommodation, suggesting that factors other than sheep IgG may induce accommodation. Acute vascular rejection was not observed in the heterotopic heart transplants from six donors with PBL accommodation. Only one incident of moderate diffuse cellular rejection (grade 3) was observed.
Pig islet xenografts are resistant to autoimmune destruction by non‐obese diabetic recipients after anti‐CD4 treatment
Authors: Koulmanda Maria , Qipo Andi , Smith R. Neal , Auchincloss Hugh
Published: March 2003

Abstract: In addition to providing a large source of donor tissue, xenogeneic islet transplantation might avoid recurrent autoimmunity in patients with type 1 diabetes. To examine this possibility further, xenogeneic pig islets were transplanted into recipient mice in the presence or absence of autoimmunity. Spontaneously, non‐obese diabetic (NOD) recipients rejected isografts rapidly whether or not the recipients were depleted of CD4+ T‐cells. Young NOD mice made diabetic with streptozotocin accepted islet isografts without immunosuppression, indicating that destructive autoimmunity did not develop in these recipients. Pig xenografts were rejected equally quickly in the two types of NOD recipients in the absence of immunosuppression and survived for up to 9 weeks in both types of NOD recipients after CD4 depletion. BALB/c mice often accepted pig xenografts indefinitely after anti‐CD4 antibody treatment. These results suggest that pig islets are resistant to recurrent autoimmunity when CD4+ T‐cells are depleted. The difficulty in obtaining indefinite islet xenograft survival in NOD recipients occurs independently from the development of destructive autoimmunity.
B cell tolerance to xenoantigens
Authors: Ohdan Hideki , Sykes Megan
Published: March 2003

Abstract: Xenotransplantation of pig organs to humans is a possible solution to the shortage of donor organs for transplantation. Multiple immunologic barriers need to be overcome if pig‐to‐primate transplantation is to be successful. The presence, in humans, of natural antibodies (Abs) directed against Galα1–3Galβ1–4GlcNAc epitopes on pig vascular endothelium provides the major barrier, as antibody–antigen binding initiates the process of hyperacute rejection. Even if hyperacute rejection is prevented, acute vascular rejection develops. Acute vascular rejection is also mediated, in part, by xenoreactive Abs and may be complement‐independent. Efforts being made to overcome antibody‐mediated rejection include depletion of antibody by extracorporeal immunoadsorption, prevention of an induced Ab response by pharmacologic reagents, B‐cell and/or plasma cell depletion, depletion or inhibition of complement, and the use of organs from pigs transgenic for human complement regulatory proteins. The ultimate solution would be the induction of B‐cell tolerance to xenogeneic antigens, which is being explored by attempting to induce xenogeneic hematopoietic chimerism. Here, we review the properties of the B cell types responding to xenoantigens and the strategies for tolerizing those B cells.
Scavenging effect of melatonin on hydroxyl radicals generated by alloxan
Authors: Brömme H.J. , Mörke W. , Peschke E. , Ebelt H. , Peschke D.
Published: March 2003

Alloxan can act as a generator of reactive oxygen species (ROS) as long as sufficient suitable reducing agents (e.g. reduced glutathione) and oxygen are available. Using electron spin resonance‐spectroscopy and the oxygen‐centered spin trap DEPMPO, we demonstrate that hydroxyl radicals (OHzrad;) are formed in vitro by alloxan in the presence of glutathione (GSH) and chelated divalent iron. Furthermore, peroxidation of polyunsaturated fatty acids from phosphatidylcholine‐containing liposomes with concomitant formation of malondialdehyde (MDA) was used as a further indicator for a preceding OHzrad; formation. Melatonin, the main secretory product of the pineal gland, is an effective scavenger of OHzrad;. The 50%‐inhibitor concentration (IC50‐value) for melatonin to scavenge OHzrad; generated from the alloxan/GSH‐reaction in the presence of ferrous ions was 23 μmol/L. In contrast to the ability to effectively scavenge OHzrad;, the potential of melatonin to prevent lipid peroxidation is considerably less pronounced.
A new melatonin receptor ligand with mt1‐agonist and MT2‐antagonist properties
Authors: Nonno Romolo , Lucini Valeria , Spadoni Gilberto , Pannacci Marilou , Croce Assunta , Esposti Daniele , Balsamini Cesarino , Tarzia Giorgio , Fraschini Franco , Stankov Bojidar M.
Published: March 2003

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt1 and MT2. In the present work, we report the synthesis and pharmacological characterization of a new compound N‐{2‐[5‐(2‐hydroxyethoxy)‐1H‐indol‐3‐yl)] ethyl} acetamide or 5‐hydroxyethoxy‐N‐acetyltryptamine (5‐HEAT). To assess the activity of the compound, the following tests were performed: affinity determination for the high‐ and low‐affinity receptor states (2‐[
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I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([
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S]GTPγS binding assay). 5‐HEAT showed little selectivity for the mt1 receptor, with pKi values of 7.77 for mt1 and 7.12 for the MT2 receptors, respectively. 5‐HEAT was able to differentiate between the high‐ and the low‐affinity receptor states in the mt1 but not in the MT2 receptor. 5‐HEAT induced a high level of G protein activation when acting through the mt1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT2 receptor‐mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin‐induced MT2 receptor‐mediated G protein activation, with a pKB value of 7.4. In conclusion, it appears that 5‐HEAT possesses very different efficacies at the two melatonin receptors, behaving as a full melatonin receptor agonist at the mt1 and as an antagonist/weak partial agonist at the MT2 receptor. Therefore, it is a promising ligand for use in functional studies aimed at distinguishing between the effects mediated by the different melatonin receptors in the human.
The ancestral complement system in sea urchins
Authors: Smith L. Courtney , Clow Lori A. , Terwilliger David P.
Published: March 2003

The origin of adaptive immunity in the vertebrates can be traced to the appearance of the ancestral RAG genes in the ancestral jawed vertebrate; however, the innate immune system is more ancient. A central subsystem within innate immunity is the complement system, which has been identified throughout and seems to be restricted to the deuterostomes. The evolutionary history of complement can be traced from the sea urchins (members of the echinoderm phylum), which have a simplified system homologous to the alternative pathway, through the agnathans (hagfish and lamprey) and the elasmobranchs (sharks and rays) to the teleosts (bony fish) and tetrapods, with increases in the numbers of complement components and duplications in complement pathways. Increasing complexity in the complement system parallels increasing complexity in the deuterostome animals. This review focuses on the simplest of the complement systems that is present in the sea urchin. Two components have been identified that show significant homology to vertebrate C3 and factor B (Bf), called SpC3 and SpBf, respectively. Sequence analysis from both molecules reveals their ancestral characteristics. Immune challenge of sea urchins indicates that SpC3 is inducible and is present in coelomic fluid (the body fluids) in relatively high concentrations, while SpBf expression is constitutive and is present in much lower concentrations. Opsonization of foreign cells and particles followed by augmented uptake by phagocytic coelomocytes appears to be a central function for this simpler complement system and important for host defense in the sea urchin. These activities are similar to some of the functions of the homologous proteins in the vertebrate complement system. The selective advantage for the ancestral deuterostome may have been the amplification feedback loop that is still of central importance in the alternative pathway of complement in higher vertebrates. Feedback loop functions would quickly coat pathogens with complement leading to phagocytosis and removal of foreign cells, a system that would be significantly more effective than an opsonin that binds upon contact as a result of simple diffusion. An understanding of the immune response of the sea urchin, an animal that is a good estimator of what the ancestral deuterostome immune system was like, will aid us in understanding how adaptive immunity might have been selected for during the early evolution of the vertebrates and how it might have been integrated into the pre‐existing innate immune system that was already in place in those animals.

The authors are grateful to Drs Sham Nair and Paul Gross for their critique of the manuscript and helpful suggestions. This work was supported by the National Science Foundation (MCB 9603086).
Genesis of the ILT/LIR/MIR clusters within the human leukocyte receptor complex
Authors: Volz Armin , Wende Hagen , Laun Katja , Ziegler Andreas
Published: March 2003

Summary: The human leukocyte receptor complex (LRC) contains at least 26 genes which belong to the immunoglobulin superfamily. The genes include two clusters of immunoglobulin‐like transcript (ILT)/leukocyte immunoglobulin‐like receptor (LIR)/monocyte‐macrophage inhibitory receptor (MIR) loci, a cluster of killer cell inhibitory receptor (KIR) genes, two leukocyte‐associated immunoglobulin‐like receptor genes, as well as the Fc receptor for IgA and the natural cytotoxicity receptor 1 loci. It has already been postulated that these genes have evolved by multiple duplications, while the two ILT clusters are likely to have been generated by the inverse duplication of an ancient ILT cluster. To shed more light on the possible origin of the loci within the LRC, we have now investigated the presence of KIR and ILT loci in a variety of vertebrates by hybridizations and compared the genomic sequences of all ILT genes. Our results lead to the following conclusions: 1) the origin of KIR genes dates back to about 100 million years, but only primate and human KIRs are closely related; 2) in contrast, genes which are detectable with human ILT cDNAs are already found in birds, suggesting their presence already about 300 million years ago. Using the sequence data produced by the human genome project, we have developed a hypothesis that reconstructs the genesis of the two human ILT clusters in detail which will help to understand the function of the LRC.

This work was supported by the European Union through grant BMH4‐CT96–1105 (to A.Z.). We also thank the Sonnenfeld‐Stiftung (Berlin) and the Berliner Krebsgesellschaft for financial support.
On the ontogeny and physiology of regulatory T cells
Authors: Annacker Oliver , Pimenta‐Araujo Ricardo , Burlen‐Defranoux Odile , Bandeira Antonio
Published: March 2003

Summary: Lymphocytes can interfere with the activity of other lymphocytes in a thousand and one ways. A particular subset of so‐called regulatory CD4+ T cells is capable of controlling the activity of other lymphocytes in yet another way. Their function is primarily defined by the ability to protect the integrity of tissues and organs in vivo. This was demonstrated in experimental models of natural tolerance to peripheral tissues, transplantation tolerance and the regulation of immune responses promoted by exogenous antigens at the level of the intestinal mucosa. Moreover, regulatory T cells also play a major role in the systemic homeostatic mechanisms that control total lymphocyte numbers. There is good evidence to support the contention that a significant fraction of the naturally occurring regulatory T cells is generated in the thymus following selection mediated by high avidity T‐cell receptor/ligand interactions. Symbolically, self‐reactive regulatory T cells do represent the breakthrough of concepts challenging the long‐lasting Burnetian dogma that all autoreactive cells should be eliminated or inactivated. Although clonal deletion of self‐reactive cells is a fundamental process in T‐cell development, controlled autoreactivity is part of the physiology of the immune system. Thus, autoreactive regulatory T cells also protect immunologists from the desperate hunting for the evil of horror autotoxicus.
Virus replication and evolution drive the kinetics and specificity of SIV‐specific cytotoxic T lymphocytes
Authors: Geretti Anna Maria , Osterhaus Albert D. M. E.
Published: March 2003

Summary: SIV (simian immunodeficiency virus) infection of cynomolgus macaques provides an excellent model for investigating the basis of protective immunity against HIV (human immunodeficiency virus). We explored the protective role of cytotoxic T lymphocytes (CTL) against the pathogenic molecular clone SIVmac‐J5. Vaccine‐induced CTL precursors (CTLp) against Env, Gag or Nef did not protect macaques against intravenous challenge. However, detection of Rev‐specific CTLp in infected macaques was associated with effective virus containment. Furthermore, CTL against an immunodominant Gag/p26 epitope (amino acids 242–250) resulted in the emergence of a mutant virus that uniformly replaced wild‐type virus in the spleen and partially escaped recognition. During primary infection, CTLp detection in blood coincided with decreasing viremia. After 12 months, two outcomes emerged. In one group of macaques, persistent viremia was associated with high viral load in lymphoid organs and declining CD4+ T‐cell counts. CTLp were maintained in asymptomatic macaques, but declined in the symptomatic phase of infection. In a second group, loss of detectable viremia was associated with low‐level virus reservoirs in lymphoid organs, asymptomatic status and maintained CD4+ T‐cell counts. CTLp peaked in the first 4 months of infection and subsequently declined in this group. These studies provide insights into the complex interplay between virus replication and host immunity.
The athymic nude rat: an animal experimental model to reveal novel aspects of innate immune responses?
Authors: Rolstad Bent
Published: March 2003

Summary: Athymic nude rats resemble nude mice in their lack of a normal thymus and functionally mature T cells. They have been useful in the study of mechanisms of tumor growth or graft rejection in immunocompromised hosts since they can accept major histocompatibility complex (MHC) mismatched organ allografts or xenografts for several months and because a number of tumor cell lines of human and rodent origin grow well in these rats. Injection of a few helper T (Th) cells from euthymic littermate rats partly restores the pool of mature T cells as well as full immunocompetence to reject organ allografts and has helped to reveal some of the cell interactions necessary for rejection to occur. In contrast, immunologically naïve athymic nude rats of certain strains, acutely reject allografts consisting of lymphocytes or bone marrow cells, which is due to the presence of alloreactive natural killer cells. These cells can recognize and kill MHC incompatible hematopoietic cells through the recognition of both mismatches within the classical (RT1.A) and non‐classical (RT1.C/E) MHC class I regions with a repertoire of inhibitory and activating killer lectin‐like receptors (KLR) for MHC‐I molecules, encoded by the Ly‐49 portion of the rat natural killer cell gene complex (NKC). Some of these receptors have been identified and molecularly cloned and show similarities with NK receptors identified in the mouse. Other leukocytes in nude rats, such as dendritic cells, may also contribute to specific innate immune responses in the absence of mature T cells. Nude rats develop T‐like cells expressing CD3 and T‐cell receptor (TCR) with increasing age. Though their phenotype in peripheral lymphatic tissues resembles that of normal T cells, consisting mainly of CD4+ or CD8+ cells, they lack alloreactivity in vivo and their TCR repertoire is more of an oligoclonal nature. Their contribution to allograft rejection in T‐cell‐reconstituted rats is therefore questionable, and their role in innate immune response in these rats still enigmatic.
Organization of thymic medullary epithelial heterogeneity: implications for mechanisms of epithelial differentiation
Authors: Farr Andrew G. , Dooley James L. , Erickson Matt
Published: March 2003

Summary: There are accumulating data to show that thymic epithelium expresses a remarkable array of molecules previously considered to be tissue‐specific antigens, such as parathyroid hormone, thyroglobulin, insulin, and C‐reactive protein. From an immunological perspective, this property of thymic epithelium would provide an ideal mechanism to effect central tolerance of epithelial‐restricted antigens. However, from a mechanistic perspective, this phenomenon remains mysterious. Two explanations have been proposed. One invokes promiscuous gene expression by medullary thymic epithelial cells that would allow transient derepression of selected gene expression. The other proposes that the expression of tissue‐restricted genes by thymic epithelium reflects alternate pathways of epithelial development by small numbers of cells to form a mosaic of different epithelial types within the thymus. Here we show thymic expression of lung‐associated gene products by an organized epithelial ‘organoid’ with ultrastructural features of respiratory epithelium and present data suggesting that the thymus also contains structures that ultrastructurally and phenotypically resemble solitary thyroid follicles. Based on these data, it is proposed that some thymic epithelial progenitor cells resemble pharyngeal endoderm in terms of their developmental potential and that alternative differentiation fates taken by these cells serve to maintain the spectrum of epithelial ‘self’ in the thymus.
Regulation of microtubule‐organizing center orientation and actomyosin cytoskeleton rearrangement during immune interactions
Authors: Sancho David , Vicente‐Manzanares Miguel , Mittelbrunn María , Montoya María C. , Gordón‐Alonso Mónica , Serrador Juan M. , Sánchez‐Madrid Francisco
Published: March 2003

Summary: The reorganization of membrane, cytoskeletal and signaling molecules during immune interactions is critical for the generation of immune response. At the initiation of the T cell–antigen presenting cell (APC) interaction, antigen‐independent weak adhesion forces allow the scanning of the APC surface by the T cell receptor for specific antigens. The stabilization of T cell–APC conjugates involves the segregation of membrane and intracellular signaling proteins, driven by reorganization of membrane microdomains and cytoskeletal changes. In early T cell–APC cognate interactions, the microtubular cytoskeleton undergoes drastic changes that lead to microtubule‐organizing center (MTOC) reorientation to the vicinity of the cell–cell contact area. Recent data on the dynamics of MTOC redistribution and its influence in T cell–APC conjugate stabilization, together with the description of an increasing number of signaling molecules associated to this complex, underscore the key role of MTOC translocation in the T cell response. We focus on the mechanisms that control the early MTOC reorientation during T cell–APC interaction and the relevance of this process to T cell activation.
Role of Shc in T‐cell development and function
Authors: Zhang Li , Lorenz Ulrike , Ravichandran Kodi S.
Published: March 2003

Shc is a prototype adapter protein that is expressed from the earliest stages of T‐cell development. Shc becomes rapidly tyrosine phosphorylated after T‐cell receptor (TCR) engagement. Expression of dominant negative forms of Shc in T‐cell lines had also suggested a role for this adapter downstream of the TCR. However, until recently, the relative significance of Shc compared to several other adapters in T cells was unclear. Mice lacking Shc expression specifically in the T‐cell lineage together with inducible expression of dominant negative Shc in transgenic mice have revealed an essential and nonredundant role for Shc in thymic T‐cell development. Functional defects in a Jurkat T‐cell line lacking Shc expression also suggest a role for Shc in mature T‐cell functions. While the requirement of Shc in T‐cell signaling is now established, precisely what signaling pathways downstream of Shc make this adapter unique are less clear. Although the Shc‐mediated activation of the extracellular signal regulated kinase (Erk)/mitogen‐activated protein kinase (MAPK) pathway could be one component, Shc likely signals to other pathways in T cells that are not yet discovered. A better molecular understanding of Shc function in the future could provide insights into how multiple adapters coordinate the various outcomes downstream of the TCR.
Telomerase activity correlates with cell cycle regulators in human hepatocellular carcinoma
Authors: Hsieh Huan‐Fa , Harn Horng‐Jyh , Chiu Shao‐Chih , Liu Yao‐Chi , Lui Wing‐Yiu , Ho Li‐Ing
Published: March 2003

Abstract: Aims/background: Mutation in cell cycle genes is the most common genetic change in malignant tumor cells. Telomerase activation, considered as essential in the immortality of cancer cells, is found in most cancers, where there may be an association with an active cell cycle. Methods: In this study study we used the TRAP assay to determine telomerase activity in liver tumor specimens from 25 cases of hepatocellular carcinoma (HCCs) as well as in corresponding non‐cancerous liver tissue in each patient. The expression of cyclin D1, cdk2, and cdk4 protein was also examined by Western blot. Results: Twenty‐one of the 25 cases of HCC were found to have increased telomerase activity, whereas only five out of the 25 non‐cancerous liver samples were found to have weak telomerase activity. Telomerase activity was not found to be related to tumor size, HBsAg, HBeAg, anti‐HCV, transaminase, or alpha‐fetoprotein serum titer. Furthermore, three out of the 25 cases of HCC showed cyclin D1 overexpression, whereas 15 of the 23 cases of HCC showed decreased cyclin D1 expression. Down regulation of cyclin D1, cdk2, cdk4 protein correlated with telomerase activity (p<0.004, p<0.013, and p<0.001 respectively). Conclusion: The results indicate that genetic defects in HCC facilitate the reactivation of telomerase activity, a process which may be dependent on cyclin D1 with its cyclin dependent kinase (cdk) partner defect.
Poor association of TT virus viremia with hepatocellular carcinoma
Authors: Yoshida Hideo , Kato Naoya , Shiratori Yasushi , Lan Keng‐Hsin , Ono‐Nita Suzane Kioko , Feng Zhu , Shiina Shuichiro , Omata Masao
Published: March 2003

Abstract: Aim: The aim of this study was to clarify the relationship between TT virus (TTV) infection and the development of hepatocellular carcinoma. Methods: TTV from serum was examined in 224 patients with hepatocellular carcinoma (HCC) and 106 patients with chronic liver disease (CLD) but without HCC who were admitted to our hospital between 1995–1997. As controls, 48 patients without liver disease were also examined. TTV DNA was detected using nested PCR method after extraction of DNA from serum. Results: TTV DNA was detected in 29/224 (13%) of patients with HCC; in 14% (4/28) of HCC patients negative for both hepatitis B virus surface antigen (HBsAg) and anti‐hepatitis C virus antibody (anti‐HCV), in 9% (2/22) of HCC patients positive for HBsAg, and in 12% (21/170) of HCC patients positive for anti‐HCV. The prevalence of TTV DNA in HCC patients (13%) was not significantly higher than in CLD patients (22%). There were no significant differences in age, gender, liver function, tumor biology (size, TNM classification), other viral markers, or amount of alcohol intake between TTV‐positive and ‐negative HCC patients. Only a history of blood transfusion was significantly more frequent in TTV‐positive HCC patients than in TTV‐negative cases (p=0.02). Coinfection with TTV did not correlate with the severity of HCV‐positive liver disease. There was no significant difference in prognosis between TTV‐positive and ‐negative HCC patients. Conclusions: TTV does not seem to contribute to the development of HCC from chronic liver disease and is not correlated with severity of liver disease.
Seventh Day Syndrome – acute hepatocyte apoptosis associated with a unique syndrome of graft loss following liver transplantationNote
Authors: Memon Muhammed Ashraf , Karademir Sedat , Shen Jikun , Koukoulis George , Fabrega Francesc , Williams James W. , Foster Preston
Published: March 2003

Abstract: Aim: The aim of this study is to describe a unique 7th day syndrome (7DS), quite different from other causes of post‐transplantation allograft dysfunction in a group of orthotopic liver transplant (OLT) patients who needed retransplantation. Methods: A retrospective analysis of 594 consecutive OLT over an 8‐year period revealed that 10 patients developed allograft dysfunction approximately 7 days following an initially normal graft function. Results: The features included: (a) severe liver failure; (b) sudden peak of extremely high liver enzymes at approximately day 7; (c) serial liver biopsy findings of central lobular hemorrhage with minimal inflammatory cell infiltrate and (d) an explant with no evidence of vascular thrombosis. The biochemical and morphometric pathological data of these patients were compared with data of patitents who had early acute rejection (AR), hepatic artery thrombosis (HAT), primary non‐function (PNF), severe sepsis and no dysfunction. Lastly, serial liver core biopsies and explants were tested for evidence of apoptosis, which revealed a significantly higher number of apoptotic hepatocytes in 7DS compared to all control groups. Conclusions: Seventh Day Syndrome is a distinct entity associated with early graft dysfunction characterized by a marked apoptosis of hepatocytes. Fas receptor activation or other pathways of program cell death may be implicated in occurrence of 7DS.
Characteristics of the hepatic stellate cell‐selective carrier mannose 6‐phosphate modified albumin (M6P28‐HSA)
Authors: Beljaars Leonie , Olinga Peter , Molema Grietje , De Bleser Pieter , Geerts Albert , Groothuis Geny M. M. , Meijer Dirk K. F. , Poelstra Klaas
Published: March 2003

Abstract: Background/Aims: Drug targeting to hepatic stellate cells (HSC) may improve the pharmacological effects of antifibrotic drugs. Recently, albumin substituted with 28 mannose 6‐phosphate moieties (M6P28‐HSA) was found to distribute selectively to HSC in fibrotic rat livers. To assess whether this albumin can be used as a carrier for intracellular drug delivery, we explored the cellular handling of M6P28‐HSA in HSC. Methods/Results: Application of competitive substrates for the M6P/IGFII receptor or other receptors showed that the binding of M6P‐HSA to the M6P/IGFII receptor is specific. Binding was strong to activated HSC, but not to quiescent HSC. Furthermore, M6P28‐HSA was extensively internalized by these cells. Using monensin, a specific inhibitor of the lysosomal pathway, proof was obtained that M6P‐HSA is endocytosed via this route. The experiments performed with tissue slices, prepared from rat and human livers, revealed a specific binding and uptake of M6P28‐HSA in both normal and cirrhotic livers. In livers from cirrhotic patients, HSC contributed predominantly to the uptake of this neoglycoprotein. Conclusions: Based on our in vivo data demonstrating the HSC‐selectivity and on our in vitro data demonstrating binding and rapid internalization in activated HSC, we conclude that M6P28‐HSA is applicable as a stellate cell‐selective carrier for antifibrotic drugs that act intracellularly. This may have implications for the design of new strategies for the treatment of liver fibrosis.