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Biomedical subjects

M L Rose

Publications and source records attributed to M L Rose.

At least 19 recordsLinked to original sources
PubMed2006

Sensitization following LVAD implantation using leucodepleted blood is not due to HLA antibodies.

Implantation of left ventricular assist devices (LVAD) is associated with HLA antibody sensitization. The objective of this study was to determine the specificity of antibodies produced by LVAD recipients using a combination of ELISA, Luminex and microcytotoxicity assays. Fifty-one LVAD patients were studied, from 44 to 838 days post-implantation. No patient developed HLA antibodies, although 24 produced IgG antibodies detectable in both ELISA and Luminex assays. These antibodies manifest as positive reactions with class I and class II wells of the ELISA and also blank wells. In Luminex assays, they produce high MFI readings with the negative control beads. Antibodies were detected 18 to 228 days after implantation. This reactivity was found to be directed against bovine serum albumin (BSA), commonly used to block non-specific binding in ELISA and Luminex assays; absorption of sera with BSA-coated beads completely abrogated reactivity in all solid phase assays, but did not eliminate anti-HLA antibodies in control sera. Ten of the 24 patients have proceeded to transplantation, with a 1-year graft survival of 69%. In conclusion, it appears that implantation of LVADS disrupts immunoregulatory pathways leading to production of anti-albumin antibodies. These can be misinterpreted as anti-HLA antibodies in solid phase assays.

Adult↗
PubMed2005

Heat shock protein 27 is associated with freedom from graft vasculopathy after human cardiac transplantation.

Experimental studies have suggested that protective genes protect allografts from cardiac allograft vasculopathy (CAV), the major complication after cardiac transplantation. Here we have sought to confirm this hypothesis using long-term heart transplant recipients. Twenty-two patients that were 9 years or older after transplant were investigated; 11 of these were without angiographic evidence of CAV; 11 had developed early CAV at 1 to 3 years after transplant. To identify proteins that may act as protectors from CAV, a global proteomic approach was used comparing cardiac biopsies from 12 patients taken within the first 2 weeks after transplant and those taken after 9 years from the same patient. Proteins were separated by 2-D gel-electrophoresis, detected by silver staining, and analyzed using Progenesis software. A particular protein spot was found in 4/6 biopsies from patients without CAV, but absent from 5/6 biopsies from those with CAV (P=0.24); however, quantitative analysis of spot intensity showed a significant difference (0.061+/-0.05 versus 0.003+/-0.01, P=0.04). This spot was identified by mass spectrometry and a combination of techniques as a diphosphorylated form of HSP27. Immunohistochemistry of further biopsies not only validated that HSP27 was more abundantly expressed on biopsies without CAV but also showed it to be localized to blood vessels. In contrast, vessels from patients with CAV did not express HSP27 (P=0.028x10(-4)). Immunohistochemistry of 12 further early biopsies and nontransplanted heart showed HSP27 to be present in normal blood vessels. These findings suggest that expression of a specific diphosphorylated form of HSP27 is associated with healthy blood vessels; it appears to be lost from vessels of patients with graft vasculopathy.

Apoptosis↗
PubMed2005

Expression of human ecto 5' nucleotidase in pig endothelial cells and its implication for adenosine production and xenotransplantation.

Human endothelial activity of ecto-5'-nucleotidase (E5'N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and antiaggregatory adenosine. We have shown in this study that human E5'N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5'N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.

5'-Nucleotidase↗
PubMed2001

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Animals↗
PubMed2001

Antivimentin antibodies are an independent predictor of transplant-associated coronary artery disease after cardiac transplantation.

BACKGROUND: Transplant-associated coronary artery disease (TxCAD) is the most serious long-term complication after cardiac transplantation. Anti-endothelial antibodies are associated with disease, and one of the major endothelial antigens recognized in the sera of patients has been shown to be the protein filament vimentin. In this study, we investigated whether antivimentin antibodies are associated with TxCAD and whether their presence can be used to identify patients at high risk of developing angiographically detectable TxCAD. METHODS: Up to 5 years after transplantation, 880 sequential sera (7.07+/-1.8 samples/patient) were collected retrospectively from 109 patients; the majority were collected in the first 2 years. Sera were assessed for antivimentin antibodies using ELISA. TxCAD was assessed by annual angiography. RESULTS: Mean titres of antivimentin antibodies, calculated up to 1, 2, and 5 years, were significantly higher in patients who developed TxCAD than those who remained disease free (P<0.0001, P<0.0038, and P<0.0001, respectively). A predictive test based on the first-year mean vimentin titre alone (> or = 120) produced a test with 63% sensitivity and 76% specificity. Inclusion of persistent rejection or high 1-year mean titre (> or = 270) as a risk factor produced a test with 66% sensitivity and 82% specificity. Multivariate analysis of time to occurrence of transplant vasculopathy showed that mean titre at 1 or 2 years was an independent predictor of time until disease in the presence of all other variables. CONCLUSIONS: Antivimentin antibodies are an independent predictor of TxCAD and can be used to identify some of the patients who are at high risk of developing this complication.

Adolescent↗
PubMed2001

Gadolinium chloride-induced hepatocyte proliferation is prevented by antibodies to tumor necrosis factor alpha.

Gadolinium chloride (GdCl(3)) destroys large Kupffer cells and has been used extensively in mechanistic studies in a number of disease and toxicity processes; however, it cannot be used to study hepatocyte turnover since it increases cell proliferation itself. The mechanism by which GdCl(3) activates cell turnover in liver is unknown, but several possibilities exist. Here it was demonstrated that a direct mitogenic action on hepatocytes is unlikely since GdCl(3) did not stimulate the growth of primary rat hepatocyte in vitro. Therefore, it was hypothesized that GdCl(3) acts indirectly through mitogenic cytokines of nonparenchymal cell origin. Antibodies to tumor necrosis factor alpha (TNFalpha) were used to evaluate if TNFalpha is causally responsible for GdCl(3)-induced cell proliferation. GdCl(3) treatment of rats in vivo increased hepatocyte replication 5-fold in 24 h and 3-fold in 48 h. Pretreatment with specific anti-TNFalpha antibodies completely prevented these effects. However, when antibody treatment was delayed until 24 h after GdCl(3), increased cell proliferation was not prevented, suggesting that TNFalpha production during the first 24 h after treatment is responsible for activation of a signaling cascade involving other mitogens that sustain hepatocyte replication at 48 h. Twenty-four hours after treatment with GdCl(3), TNFalpha mRNA transcripts were increased 2-fold over control, an effect that was prevented by pretreatment with anti-TNFalpha antibody. NFkappaB, which is known to be involved in TNFalpha transcription, was activated by GdCl(3) about 4.5-fold over control 8 h after treatment in vivo, an increase not observed when antibodies to TNFalpha were present. When GdCl(3) was added to macrophages in culture, TNFalpha was nearly doubled 4 h after treatment. Additionally, conditioned media harvested from macrophages treated with GdCl(3) for 2 to 8 h stimulated the growth of HepG2 cells in culture about 2-fold, while antibodies to TNFalpha completely prevented this effect. Taken together, these data are consistent with the hypothesis that TNFalpha released from Kupffer cells at early time points prior to their destruction is causally responsible for triggering a cascade of events responsible for GdCl(3)-induced cell proliferation.

Animals↗
PubMed2001

Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells: induction of VCAM-1 but no proliferation.

OBJECTIVE: Intercellular adhesion molecule (ICAM)-1 is an immunoglobulin-like cell adhesion molecule expressed by several cell types, including proliferating vascular smooth muscle cells (VSMC). Cross-linking ICAM-1 on the surface of different cell types has previously been shown to cause an increase in cellular activation within the cytoplasm. Here, our objective was to examine events following ligation of ICAM-1 on the surface of human VSMC. METHODS: VSMC were isolated by explant from human pulmonary arteries or aortic tissue from cardiac transplant donors. ICAM-1 was ligated with monoclonal antibodies, followed by cross-linking with a secondary antibody. Activation of signalling pathways, proliferation and expression of a second adhesion molecule, vascular cell adhesion molecule (VCAM)-1 were investigated. RESULTS: ICAM-1 cross-linking caused an increase in activation of extracellular regulated kinase (Erk)-1/-2 and Jun N-terminal kinase (JNK)-1/-2. mRNA and protein for VCAM-1 was observed after ICAM-1 cross-linking, and this was abrogated by addition of an upstream inhibitor of Erk-1/-2, PD98059. No increase in cell proliferation was observed. CONCLUSIONS: Ligation of ICAM-1 on the surface of vascular smooth muscle cells in vitro, leads to the expression of adhesion molecules associated with monocyte infiltration, but does not contribute to smooth muscle cell proliferation. In vivo, this might lead to prolongation of the inflammatory response within diseased blood vessels, by arresting monocytes within atherosclerotic plaques.

Cell Communication↗
PubMed2001

Acute rejection and cardiac graft vasculopathy in the absence of donor-derived ICAM-1 or P-selectin.

BACKGROUND: ICAM-1 and P-selectin are molecules that facilitate adhesion of circulating leukocytes to vessel walls. We have investigated the role of donor-derived ICAM-1 and P-selectin in acute and chronic cardiac allograft rejection. METHODS: C57BL/6J (H-2(b)) mice were used as donors for heterotopic heart transplantation into CBA/Ca (H-2(k)) recipients. The donors were wild-type or homozygous for gene mutations of ICAM-1 or P-selectin. We measured acute rejection in non-immunosuppressed recipients by daily palpation and sacrificed mice at Days 2, 4, and 6 for immunohistochemical analysis. For chronic rejection, recipients received monoclonal antibody against CD4+ T cells. We removed hearts at Days 60 to 62 for histologic assessment of vasculopathy using quantitative morphometry to measure intimal thickening. RESULTS: Time (days) to rejection was 7.1 +/- 0.57 for wild-type (n = 10), 7.0 +/- 0.71 for ICAM-1 -/- (not significantly different, n = 7) and 6.1 +/- 0.33 (p = 0.001) for P-selectin -/- donors. ICAM-1 deficiency was associated with delayed infiltrate at Day 4 compared with wild-type. In the model of chronic rejection, elastin-positive vessels showed a mean occlusion of 34% +/- 3% in transplanted wild-type hearts; vessels were divided into those showing 0% to 20%, 20% to 50%, and 50% to 100% occlusion. We observed no difference in the number of affected vessels or the amount of vascular thickening in donors lacking ICAM-1 or P-selectin compared with wild-type controls. CONCLUSIONS: The absence of ICAM-1 or P-selectin in donor tissues neither lengthens the time of allograft survival nor inhibits the vascular lesions associated with chronic rejection. Indeed, the absence of P-selectin may exacerbate alloimmune injury.

Animals↗
PubMed2001

Screening of a HUVEC cDNA library with transplant-associated coronary artery disease sera identifies RPL7 as a candidate autoantigen associated with this disease.

A HUVEC cDNA library was screened with sera from two patients who had developed transplant-associated coronary artery disease (TxCAD) following cardiac transplantation. A total of six positive clones were isolated from a primary screen of 40 000 genes. Subsequent DNA sequence analysis identified these to be lysyl tRNA synthetase, ribosomal protein L7, ribosomal protein L9, beta transducin and TANK. Another gene whose product could not be identified showed homology to a human cDNA clone (DKFZp566M063) derived from fetal kidney. Full-length constructs of selected genes were expressed as his-tag recombinant fusion proteins and used to screen a wider patient base by ELISA to determine prevalence and association with TxCAD. Of these ribosomal protein L7 showed the highest prevalence (55.6%) with TxCAD sera compared to 10% non-CAD.

Adult↗
PubMed2001

Valve interstitial cells induce donor-specific T-cell anergy.

OBJECTIVES: Valve allografts produce an immune response, which can influence their performance. The exact role of the interaction between recipient T cells and the different cellular components of the donor valve in stimulating an immune response is not known. Therefore the T-cell response to valve endothelial and interstitial cells was investigated in vitro. METHODS: Valve endothelial and interstitial cells were characterized for cell-surface molecules before and after interferon gamma treatment by means of a panel of specific monoclonal antibodies and flow cytometry. The proliferative response of highly purified T lymphocytes was used to assess the immunogenicity of cultured valve endothelial and interstitial cells. This was further investigated by using a 2-step tolerance-induction protocol. RESULTS: Valve endothelial and interstitial cells express similar levels of human leukocyte antigens and adhesion and costimulatory molecules, which are either induced or upregulated after interferon gamma treatment. T-cell responses to endothelial cells were detected after interferon gamma treatment, but responses to interferon gamma-treated interstitial cells were not detected. This lack of response resulted in the induction of T-cell anergy, which was reversed by the presence of the costimulatory molecule B7-1. CONCLUSIONS: Although valve endothelial and interstitial cells express a similar range of cell-surface molecules, it is only the endothelial cells that are immunogenic. In addition, we have shown that these 2 cell types interact in a donor-specific manner to orchestrate the immune response and therefore may have clinical relevance in the allogeneic response of the heart valve recipients.

Antibodies, Monoclonal↗
PubMed2000

Intragraft interleukin-4 mRNA expression after short-term CD154 blockade may trigger delayed development of transplant arteriosclerosis in the absence of CD8+ T cells.

BACKGROUND: It has recently been shown that, although anti-CD154 induces CD4+ T-cell tolerance, it is unable to prevent allograft rejection mediated by CD8+ T cells. We have also shown that anti-CD154 monotherapy does not protect the graft from the development of transplant arteriosclerosis even in the absence of CD8+ T cells. This study was designed to investigate and characterize possible mechanisms responsible for the development of transplant arteriosclerosis after CD154 blockade in the absence of CD8+ T cells. METHODS: C57BL/6 (H2b) recipients received a fully MHC-mismatched BALB/c donor aorta (H2d). Animals were either treated with anti-CD154 monoclonal antibody (mAb) in the presence or absence of CD8 T cells. Histology, morphometric measurements, immunohistochemistry, and the production of alloantibodies (IgM, IgG1, IgG2a) were analyzed on days 14, 30, and 50 after transplantation. Cytokine production within the graft was investigated by competitive reverse transcription-polymerase chain reaction on day 14. RESULTS: Combined treatment with anti-CD154 and a depleting CD8 mAb resulted in a delay in the development of transplant arteriosclerosis (intimal proliferation: 33+/-10% vs. 67+/-11% untreated control, day 30) but ultimately did not prevent its progression (intimal proliferation: 55+/-10% vs. 78+/-9% untreated control, day 50). Although there was a significant decrease in the number of CD4+, CD11b+, and CD40+ graft-infiltrating cells and a reduction in the formation of donor-specific IgG1 alloantibodies in recipients treated with anti-CD154 and anti-CD8 mAbs, mRNA for interleukin (IL)-4 was increased, suggesting a shift in the intragraft cytokine profile towards a Th2-like pattern. CONCLUSIONS: Our data provide evidence that short-term CD154 blockade is insufficient to prevent transplant arteriosclerosis, even in combination with CD8+ T-cell depletion. Moreover, the increased expression of the Th2 cytokine interleukin-4 within the graft may be responsible for the development of transplant arteriosclerosis in the long term.

Animals↗
PubMed2000

An epithelial cell line that can stimulate alloproliferation of resting CD4+ T cells, but not after IFN-gamma stimulation.

It has previously been shown that IFN-gamma-induced up-regulation of HLA class II on the surface of epithelial cells is not sufficient to induce proliferation of allospecific CD4+ T cells in vitro. To further investigate this phenomenon, a human epithelial bladder carcinoma, T24, was induced to constitutively express HLA class II without IFN-gamma stimulation, by permanent transfection with the full-length class II transactivator (CIITA) gene. Proliferation of allospecific T cells to transfected and wild-type cells with and without prior activation with saturating levels of IFN-gamma for 4 days was examined. IFN-gamma-activated T24 did not induce any response from CD4+ T cells. However, T24.CIITA induced significant levels of alloproliferation, which could be abrogated by pretreatment of T24.CIITA with a mAb to LFA-3. Prestimulation of T24. CIITA with saturating levels of IFN-gamma for 4 days also prevented allospecific CD4+ T cell proliferation. These findings suggest that epithelial cells may be intrinsically able to process and present alloantigen and provide adequate costimulation. We propose that IFN-gamma has a secondary, as yet unidentified, effect that acts to negatively regulate this response, at least in some epithelial cells.

CD4-Positive T-Lymphocytes↗
PubMed2000

CD8+ T cells contribute to the development of transplant arteriosclerosis despite CD154 blockade.

BACKGROUND: The CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4+ and CD8+ T cells are differentially affected by CD154 blockade. METHODS: BALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8+ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation. RESULTS: Only combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33 +/-10% vs. 67+/-14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups. CONCLUSIONS: Our data provide direct evidence that CD8+ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.

Animals↗
PubMed2000

Significant frequencies of T cells with indirect anti-donor specificity in heart graft recipients with chronic rejection.

BACKGROUND: The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection. METHOD AND RESULTS: Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I-derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection. DISCUSSION: iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

Animals↗