Assessment of TREC, KREC and telomere length in long-term survivors after allogeneic HSCT: the role of GvHD and graft source and evidence for telomere homeostasis in young recipients.

Reconstitution of the adaptive immune system following allogeneic hematopoietic stem cell transplantation is crucial for beneficial outcome and is affected by several factors, such as GvHD and graft source. The impact of these factors on immune reconstitution has been thoroughly investigated during the early phase after transplantation. However, little is known about their long-term effect. Similarly, leukocyte telomere length (TL) shortening has been reported shortly after transplantation. Nevertheless, whether TL shortening continues in long-term aspect is still unsettled. Here, we assessed T-cell receptor excision circle (TREC), kappa deleting recombination excision circle (KREC) and leukocyte TL in recipients and donors several years post transplantation (median 17 years). Our analysis showed that, recipients who received bone marrow (BM) as the graft source have higher levels of both TREC and KREC. Also, chronic GvHD affected TREC levels and TL but not KREC levels. Finally, we show that recipient's TL was longer than respective donors in a group of young age recipients with high KREC levels. Our results suggest that BM can be beneficial for long-term adaptive immune recovery. We also present supporting evidence for recipient telomere homeostasis, especially in young age recipients, rather than telomere shortening.

Approaches to Investigating Complex Genetic Traits in a Large-Scale Inbred Mouse Aging Study.

Inbred mice are a unique model system for studying aging because of the genetic homogeneity within inbred strains, the short life span of mice relative to humans, and the rich array of analytic tools that are available. A large-scale aging study was conducted on 28 inbred strains representing great genetic diversity to determine, via histopathology, the type and diversity of spontaneous diseases that aging mice develop. A total of 20 885 different diagnoses were made, with an average of 12 diagnoses per mouse in the study. Eighteen inbred strains have had their genomes sequenced, and many others have been partially sequenced to provide large repositories of data on genetic variation among the strains. This vast amount of genomic information can be utilized in genome-wide association studies to find candidate genes that are involved in the pathogenesis of spontaneous diseases. As an illustration, this article presents a genome-wide association study of the genetic associations of age-related intestinal amyloidosis, which implicated 3 candidate genes: translocating chain-associated membrane protein 1 (Tram1); splicing factor 3b, subunit 5 (Sf3b5); and syntaxin 11 (Stx11). Representative photomicrographs are available on the Mouse Tumor Biology Database and Pathbase to serve as a reference when evaluating inbred mice used in other genetic or experimental studies to rule out strain background lesions. Many of the age-related mouse diseases are similar, if not identical, to human diseases; therefore, the genetic discoveries have direct translational benefit.

Alpha/beta T-cell depleted grafts as an immunological booster to treat graft failure after hematopoietic stem cell transplantation with HLA-matched related and unrelated donors.

Allogeneic hematopoietic stem cell transplantation is associated with several complications and risk factors, for example, graft versus host disease (GVHD), viral infections, relapse, and graft rejection. While high levels of CD3+ cells in grafts can contribute to GVHD, they also promote the graft versus leukemia (GVL) effect. Infusions of extra lymphocytes from the original stem cell donor can be used as a treatment after transplantation for relapse or poor immune reconstitution but also they increase the risk for GVHD. In peripheral blood, 95% of T-cells express the αß T-cell receptor and the remaining T-cells express the γδ T-cell receptor. As αß T-cells are the primary mediators of GVHD, depleting them from the graft should reduce this risk. In this pilot study, five patients transplanted with HLA-matched related and unrelated donors were treated with αß T-cell depleted stem cell boosts. The majority of γδ T-cells in the grafts expressed Vδ2 and/or Vγ9. Most patients receiving αß-depleted stem cell boosts increased their levels of white blood cells, platelets, and/or granulocytes 30 days after infusion. No signs of GVHD or other side effects were detected. A larger pool of patients with longer follow-up time is needed to confirm the data in this study.

Phenotypic characterization of the KK/HlJ inbred mouse strain.

Detailed histopathological diagnoses of inbred mouse strains are important for interpreting research results and defining novel models of human diseases. The aim of this study was to histologically detect lesions affecting the KK/HlJ inbred strain. Mice were examined at 6, 12, and 20 months of age and near natural death (ie, moribund mice). Histopathological lesions were quantified by percentage of affected mice per age group and sex. Predominant lesions were mineralization, hyperplasia, and fibro-osseous lesions. Mineralization was most frequently found in the connective tissue dermal sheath of vibrissae, the heart, and the lung. Mineralization was also found in many other organs but to a lesser degree. Hyperplasia was found most commonly in the pancreatic islets, and fibro-osseous lesions were observed in several bones. The percentage of lesions increased with age until 20 months. This study shows that KK/HlJ mice demonstrate systemic aberrant mineralization, with greatest frequency in aged mice. The detailed information about histopathological lesions in the inbred strain KK/HlJ can help investigators to choose the right model and correctly interpret the experimental results.

Immunosuppressive CD14+HLA-DRlow/neg IDO+ myeloid cells in patients following allogeneic hematopoietic stem cell transplantation.

Myeloid-derived suppressor cells (MDSCs) have emerged as a heterogeneic immunoregulatory population that can expand in response to inflammatory signals. Predominantly studied in cancer, MDSCs suppress T cells utilizing various mechanisms. In allogeneic hematopoietic stem cell transplantation (allo-HSCT) therapy-related toxicity and alloreactivity increase inflammatory cytokines that might favor an MDSC accumulation. To address this question, circulating CD14(+)HLA-DR(low/neg) cells were studied retrospectively in 51 allo-HSCT patients. These cells represent one of the few well-described human MDSC subsets under physiological and pathological conditions. The frequency of CD14(+)HLA-DR(low/neg) cells was significantly increased after allo-HSCT, especially in patients with acute graft-versus-host disease. Compared to healthy donor cells they were pSTAT1(low) (phosphorylated signal transducer and activator of transcription) and indoleamine 2,3-dioxygenase (IDO)(high). Serum levels of granulocyte colony-stimulating factor and interleukin-6, which both have been linked to MDSC induction, correlated positively with the frequency of CD14(+)HLA-DR(low/neg) cells. In vitro dysfunction of patient T cells, such as reduced proliferative capacity or CD3ζ-chain expression, was rescued by blocking the IDO activity of CD14(+)HLA-DR(low/neg) cells. Overall, we identified a T-cell-suppressive monocytic population that expands after allo-HSCT. The mechanisms responsible for such accumulation remain to be elucidated. It will be of great interest to prospectively investigate the influence of these cells on the graft-versus-tumor and -host reaction.

Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation.

Mesenchymal stromal cells (MSCs) are explored as a novel treatment for a variety of medical conditions. Their fate after infusion is unclear, and long-term safety regarding malignant transformation and ectopic tissue formation has not been addressed in patients. We examined autopsy material from 18 patients who had received human leukocyte antigen (HLA)-mismatched MSCs, and 108 tissue samples from 15 patients were examined by PCR. No signs of ectopic tissue formation or malignant tumors of MSC-donor origin were found on macroscopic or histological examination. MSC donor DNA was detected in one or several tissues including lungs, lymph nodes, and intestine in eight patients at levels from 1/100 to <1/1,000. Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, as DNA was detected from nine of 13 MSC infusions given within 50 days before sampling but from only two of eight infusions given earlier. There was no correlation between MSC engraftment and treatment response. We conclude that MSCs appear to mediate their function through a "hit and run" mechanism. The lack of sustained engraftment limits the long-term risks of MSC therapy.

Primary follicular dystrophy with scarring dermatitis in C57BL/6 mouse substrains resembles central centrifugal cicatricial alopecia in humans.

A number of C57BL/6 (B6) substrains are commonly used by scientists for basic biomedical research. One of several B6 strain-specific background diseases is focal alopecia that may resolve or progress to severe, ulcerative dermatitis. Clinical and progressive histologic changes of skin disease commonly observed in C57BL/6J and preliminary studies in other closely related substrains are presented. Lesions develop due to a primary follicular dystrophy with rupture of severely affected follicles leading to formation of secondary foreign body granulomas (trichogranulomas) in affected B6 substrains of mice. Histologically, these changes resemble the human disease called central centrifugal cicatrical alopecia (CCCA). Four B6 substrains tested have a polymorphism in alcohol dehydrogenase 4 (Adh4) that reduces its activity and potentially affects removal of excess retinol. Using immunohistochemistry, differential expression of epithelial retinol dehydrogenase (DHRS9) was detected, which may partially explain anecdotal reports of frequency differences between B6 substrains. The combination of these 2 defects has the potential to make high dietary vitamin A levels toxic in some B6 substrains while not affecting most other commonly used inbred strains.

A data-capture tool for mouse pathology phenotyping.

The Mouse Disease Information System is a free Microsoft Access database (http://research.jax.org/faculty/sundberg/index.html) designed by veterinary pathologists to aid veterinary pathologists in data acquisition, analysis, and coordination of tissue-sample archives. Linking the system to the Mouse Anatomy and Mouse Pathology Ontologies provides controlled vocabulary (and spelling) for organ, tissue, and diagnosis. Severity scores provide a quantitative assessment of all lesions to enable quantitative trait locus analysis for large-scale studies. Individual diagnoses can be verified for their definition by online linkage to Pathbase.net. Histologic images can be accessed from Pathbase by using the Mouse Pathology Ontology directly for comparison with slides being viewed at the time of data entry and providing the user with a reference and a "virtual second opinion."

No increased trapping of multipotent mesenchymal stromal cells in bone marrow filters compared with other bone marrow cells.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are candidates for cellular therapy in regenerative medicine and as treatment of graft-versus-host-disease (GvHD) after hematopoietic stem cell (HSC) transplantation. It has been suggested that MSC may be trapped in bone marrow (BM) filters during the stem cell procurement and lost from the HSC graft. METHODS: We investigated filtered BM and filters from six HSC donors. MSC were expanded from the two sources and investigated by flow cytometry, doubling capacity, differentiation ability and suppression in mixed lymphocyte cultures. RESULTS: A range of 0.3-3.4% cells was trapped in the filters. By flow cytometry, there was no difference in the proportions of different cell types between the filter-retrieved and filtered BM cells. The phenotype, immunosuppressive capacity, differentiation and growth were equal in MSC expanded from the two cell sources. DISCUSSION: Given the low number of trapped cells, filters do not appear to be a good source of MSC. When intended for clinical transplantation, MSC need to be expanded ex vivo to achieve sufficient doses for a clinical effect.

Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon.

Mesenchymal stem cells (MSC) possess anti-inflammatory properties and participate in tissue repair. We used MSC to heal therapy-induced tissue toxicity. Ten consecutive patients, treated with MSC due to tissue toxicity following allogeneic hematopoietic stem cell transplantation, (ASCT) were included. Their median age was 48 (13-64) years. Seven had hemorrhagic cystitis grades 2-5, two had pneumomediastinum and one had perforated colon and peritonitis. MSC donors were mainly third-party, HLA-mismatched (n=11), HLA-haploidentical (n=3) and, in two cases, the HLA-identical ASCT sibling donors. MSC were given intravenously, the median cell dose was 1.0 (range 0.7-2)x10(6)/kg. In five patients, the severe hemorrhagic cystitis cleared after MSC infusion. Gross hematuria disappeared after median 3 (1-14) days. Two patients had reduced transfusion requirements after MSC infusion, but died of multiorgan failure. In one of them, MSC donor DNA was demonstrated in the urinary bladder. In two patients, pneumomediastinum disappeared after MSC infusions. A patient with steroid-resistant graft-versus-host disease of the gut experienced perforated diverticulitis and peritonitis that was reversed twice by MSC. MSC is a novel treatment for therapy-induced tissue toxicity.

Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells.

Seven patients underwent treatment with mesenchymal stem cells (MSCs), together with allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were given to three patients for graft failure and four patients were included in a pilot study. HSCT donors were three human leukocyte antigen (HLA)-identical siblings, three unrelated donors and one cord blood unit. The conditioning was myeloablative in four patients and reduced in three patients. MSC donors were HLA-identical siblings in three cases and haploidentical in four cases. Neutrophil counts >0.5 x 10(9)/l was reached at a median of 12 (range 10-28) days. Platelet counts >30 x 10(9)/l was achieved at a median of 12 (8-36) days. Acute graft-versus-host disease (GVHD) grade 0-I was seen in five patients. Two patients developed grade II, which in one patient evolved into chronic GVHD. One severe combined immunodeficiency (SCID) patient died of aspergillosis, the others are alive and well. One patient, diagnosed with aplastic anemia had graft failure after her first transplantation and severe Henoch-Schönlein Purpura (HSP). After retransplantation of MSCs and HSCs, she recovered from both the HSP and aplasia. Thus, co-transplantation of MSC resulted in fast engraftment of absolute neutrophil count (ANC) and platelets and 100% donor chimerism, even in three patients regrafted for graft failure/rejection.

Mesenchymal stem cells stimulate antibody secretion in human B cells.

Mesenchymal stem cells (MSC) have immunomodulatory effects and inhibit T-cell responses to alloantigens and mitogens in vitro and in vivo. We wanted to examine the effect of MSC on human B cells. MSC stimulated IgG production, measured in an enzyme-linked immunospot (ELIspot) assay in blood and spleen lymphocytes. MSC only induced a low proliferation. When a semipermeable membrane separated MSC and mononuclear cells, the IgG production was stimulated in unfractionated lymphocytes. In contrast, enriched B cells required cell contact with MSC to produce IgG. Co-cultures of MSC and lymphocytes increased IFN-gamma production. MSC produce IL-6, and addition of MSC to spleen cells dramatically increased IL-6 levels. After lymphocyte stimulation with lipopolysaccharide (LPS), cytomegalovirus or varicella zoster virus, MSC either stimulated or inhibited IgG response, depending on the level of stimulation by LPS or the viral antigens. Similar results were obtained for enriched B cells. To conclude, MSC stimulate B-cell antibody secretion. The IgG secretion by activated B cells may be stimulated or inhibited by the addition of MSC, depending on the level of stimulation.

Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual.

Allogeneic stem cell transplantation is often complicated by reactivation of herpesviruses. Mesenchymal stem cells (MSC) are immunomodulatory and may be used to treat graft-versus-host disease. We investigated if herpesviruses infect and can be transmitted by MSC, and if MSC suppress immune responses to various infectious agents. Mesenchymal stem cells from healthy seropositive donors were evaluated with polymerase chain reaction for the most common herpesviruses: cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, Epstein-Barr virus (EBV) and varicella zoster virus. The cytopathological effect (CPE) was investigated and viral antigens analyzed by immunofluorescence after in vitro exposure to CMV, HSV-1 and EBV. We also studied MSC effect on lymphocyte stimulation induced by various infectious agents. No viral DNA could be detected in MSC isolated from healthy seropositive individuals. However, a CPE was noted and intracellular viral antigens detected after infection in vitro by CMV and HSV-1, but not by EBV. The CMV and HSV-1 infections were productive. Lymphocyte proliferation by herpesviruses, candida mannan and protein A from Staphylococcus aureus was suppressed by MSC. The data indicate that the risk of herpesvirus transmission by transplantation of MSC from healthy seropositive donors is low. However, MSC may be susceptible to infection if infused in a patient with CMV or HSV-1 viremia. MSC transplantation may compromise the host's defense against infectious agents.

Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes.

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.

Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.

We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.

Importance of the Gal alpha1-3 Gal antigen in discordant islet xenotransplantation: immunosuppression, which inhibits porcine islet xenograft rejection in ordinary mice, is equally effective in Gal-knockout mice.

BACKGROUND: Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. RESULTS: No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. CONCLUSIONS: After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.

Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.

We aimed to study the effects of mesenchymal stem cells (MSCs) on alloreactivity and effects of T-cell activation on human peripheral blood lymphocytes (PBLs) in vitro. MSCs were expanded from the bone marrow of healthy subjects. MSCs isolated from second to third passage were positive for CD166, CD105, CD44, CD29, SH-3 and SH-4, but negative for CD34 and CD45. MSCs cultured in osteogenic, adipogenic or chondrogenic media differentiated, respectively, into osteocytes, adipocytes or chondrocytes. MSC added to PBL cultures had various effects, ranging from slight inhibition to stimulation of DNA synthesis. The stimulation index (SI = (PBL + MSC)/PBL) varied between 0.2 and 7.3. The SI was not affected by the MSC dose or by the addition of allogeneic or autologous MSCs to the lymphocytes. Suppression of proliferative activity was observed in all experiments after the addition of 10,000-40,000 MSCs to mixed lymphocyte cultures (MLCs). Lymphocyte proliferation was 10-90%, compared with a control MLC run in parallel without MSCs. In contrast, the addition of fewer MSCs (10-1000 cells) led to a less consistent suppression or a marked lymphocyte proliferation in several experiments, ranging from 40 to 190% of the maximal lymphocyte proliferation in control MLCs. The ability to inhibit or stimulate T-cell alloresponses appeared to be independent of the major histocompatibility complex, as results were similar using 'third party' MSCs or MSCs that were autologous to the responder or stimulating PBLs. The strongest inhibitory effect was seen if MSCs were added at the beginning of the 6 day culture, and the effect declined if MSCs were added on day 3 or 5. Marked inhibitory effects of allogeneic and autologous MSCs (15,000) were also noted after mitogenic lymphocyte stimulation by phytohaemagglutinin (median lymphocyte proliferation of 30% of controls), Concanavalin A (56%) and protein A (65%). Little, if any, inhibition occurred after stimulation with pokeweed mitogen. Low numbers of MSCs (150 cells) were unable to inhibit mitogen-induced T-cell responses. MSCs have significant immune modulatory effects on MLCs and after mitogenic stimulation of PBL. High numbers of MSCs suppress alloreactive T cells, whereas very low numbers clearly stimulated lymphocyte proliferation in some experiments. The effect of a larger number of MSCs on MLCs seems more dependent on cell dose than histocompatibility and could result from an 'overload' of a stimulatory mechanism.

A transcriptional roadmap to wood formation.

The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

Unravelling cell wall formation in the woody dicot stem.

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.

A new in vitro model for the study of pig-to-human vascular hyperacute rejection.

Studies on vascular hyperacute xenograft rejection (HAR) are usually conducted in vitro on cultured endothelial cells (EC) exposed to human serum, in complex whole organ perfusion models using heparinized blood or in vivo models. Here we describe a new model allowing perfusion of pig vessels with human whole blood without anticoagulants. Segments of the porcine iliac artery were connected to circular polyvinyl chloride (PVC) tubing, whose inner surface was conjugated with immobilized heparin. The vessels were perfused with 7 to 8 ml of fresh, non-anticoagulated human blood by rocking of the tubing device for 5, 15 or 60 min in an incubator at 37 degrees C. Human iliac arteries (n = 4) were perfused with fresh human ABO-compatible blood as controls. Perfusion of human vessels resulted in changes in the blood and plasma parameters similar to those in the PVC control loop. Overall, perfusion of the porcine vessels generated high levels of C3a, sC5b-9 and thrombin-anti-thrombin (TAT). Platelet consumption was near total (97.2 +/- 1.2%; "high" responders) in six of 13 vessels perfused and only moderate (55.8 +/- 9.9%; "low" responders) in the remaining seven vessels. The "high" responder vessel group showed a significantly higher platelet reduction, neutrophil loss and monocyte consumption and higher C3a and TAT factor at 60 min compared with the human vessels. The "low" responder porcine vessel group also generated significantly higher TAT levels at 60 min compared with the human vessels, but lower levels compared with the "high" responder porcine vessel group. Immunohistochemical examination of perfused porcine vessels revealed binding of human IgM, IgG, IgA, C1q, C3, fibrin and platelets at 5 min. The binding of these proteins was even stronger at 15 and 60 min, and at 60 min C9 could also be detected. Addition of soluble complement receptor 1 (sCR1) to the blood resulted in a significant reduction in C3a and sC5b-9 (P = 0.046 and P = 0.046, respectively). However, sCR1 did not reduce C1q, C3c or C5 staining, but did abolish C9 binding to the endothelium. In conclusion, in vitro perfusion of porcine vessel segments with non-heparinized, fresh human blood triggered events characterizing HAR. The small quantity of blood and xenogenic tissue that is needed makes this model ideal for investigations of the mechanisms and treatments of rejections associated with xenogeneic pig-to-human xenotransplantation.