MDCO-216 Does Not Induce Adverse Immunostimulation, in Contrast to Its Predecessor ETC-216.

PURPOSE: Aim of this study was to demonstrate that MDCO-216 (human recombinant Apolipoprotein A-I Milano) does not induce adverse immunostimulation, in contrast to its predecessor, ETC-216, which was thought to contain host cell proteins (HCPs) that elicited an inflammatory reaction. METHODS: Data were taken from a clinical trial in which 24 healthy volunteers (HV) and 24 patients with proven stable coronary artery disease (sCAD) received a single intravenous dose of MDCO-216, ranging 5-40 mg/kg. Additionally, whole blood from 35 HV, 35 sCAD patients and 35 patients requiring acute coronary intervention (aCAD group) was stimulated ex vivo with MDCO-216 and ETC-216. RESULTS: No inflammatory reaction was observed in HV and sCAD patients following MDCO-216 treatment, judging by body temperature, white cell counts, neutrophil counts, C-reactive protein, circulating cytokines (IL-6, TNF-α), and adverse events. In the ex vivo experiment, the geometric means (SD) of the ratio of MDCO-216 stimulated IL-6 over background levels were 0.8 (1.9), 0.7 (1.5), 1.0 (2.0) for respectively HV, sCAD, aCAD. The corresponding ETC-216 stimulated values were 15.8 (2.9), 9.5 (3.6), 3.8 (4.0). TNF-α results were comparable. Because many ETC-216 stimulated samples had cytokine concentrations >ULOQ, ratios were categorised and marginal homogeneity of the contingency table (MDCO-216 versus ETC-216) was assessed with the Stuart-Maxwell test. P-values were ≤0.0005 for all populations. CONCLUSIONS: MDCO-216 did not induce adverse immunostimulation in HV and sCAD patients, in contrast to ETC-216. Results from the ex vivo stimulation suggests the same holds true for aCAD patients.

A single infusion of MDCO-216 (ApoA-1 Milano/POPC) increases ABCA1-mediated cholesterol efflux and pre-beta 1 HDL in healthy volunteers and patients with stable coronary artery disease.

AIMS: Apolipoprotein A-1 (ApoA-1), based on epidemiology, is inversely associated with cardiovascular (CV) events. Human carriers of the ApoA-1 Milano variant have a reduced incidence of CV disease. Regression of atherosclerotic plaque burden was previously observed on intravascular ultrasound (IVUS) with ETC-216, a predecessor of MDCO-216. MDCO-216, a complex of dimeric ApoA-1 Milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, is being developed to reduce atherosclerotic plaque burden and CV events. We investigated the efficacy and safety of a single infusion of MDCO-216 in healthy volunteers and in patients with coronary artery disease (CAD). METHODS AND RESULTS: Twenty-four healthy volunteers and 24 patients with documented CAD received a 2-h infusion of MDCO-216 in a randomized, placebo controlled, single ascending dose study. Five cohorts of healthy volunteers and four cohorts of CAD patients received ApoA-1 Milano doses ranging from 5 to 40 mg/kg. Subjects were followed for 30 days. Dose-dependent increases in ApoA-1, phospholipid, and pre-beta 1 HDL and decreases in ApoE were observed. Prominent and sustained increases in triglyceride, and decreases in HDL-C, endogenous ApoA-1 and ApoA-II occurred at doses >20 mg/kg and profound increases in ABCA1-mediated cholesterol efflux were observed. Other lipid and lipoprotein parameters were generally unchanged. MDCO-216 was well tolerated. CONCLUSIONS: MDCO-216-modulated lipid parameters profoundly increased ABCA1-mediated cholesterol efflux and was well tolerated. These single-dose data support further development of this agent for reducing atherosclerotic disease and subsequent CV events.

Different inhibitory effects of immunosuppressive drugs on human and rat aortic smooth muscle and endothelial cell proliferation stimulated by platelet-derived growth factor or endothelial cell growth factor.

BACKGROUND: Vascular smooth muscle cell hyperplasia with resulting luminal narrowing is the main histologic feature of accelerated arteriosclerosis seen after organ transplantation (transplant arteriosclerosis) and after balloon angioplasty (restenosis). It limits long-term allograft survival, as well as the success rate of angioplasty. At present, effective prophylactic and therapeutic strategies for these complications are still missing. Studies of in vivo models of accelerated arteriosclerosis induced by allogeneic or mechanical injury to the vasculature indicate that certain immunosuppressive drugs have inhibitory properties on smooth muscle cell hyperplasia. METHODS: This study summarizes the inhibitory effects of different immunosuppressive drugs in vitro on the growth factor-induced proliferation of vascular smooth muscle cells and endothelial cells isolated from human and rat thoracic aortas. RESULTS: The immunosuppressants rapamycin and mycophenolic acid were potent in inhibiting smooth muscle and endothelial cell proliferation. Cyclosporine demonstrated some inhibition of smooth muscle and endothelial cell proliferation, but the inhibitory concentration50 (IC50) values were just below toxicity levels. FK506 revealed a moderate inhibitory activity but, interestingly, only for human cells. High concentrations of leflunomide inhibited in our experiments only rat smooth muscle and endothelial cell proliferation. Methylprednisolone showed a gradual inhibition over a broad concentration interval of rat and human smooth muscle cells and of rat but not of human endothelial cells. CONCLUSIONS: These data indicate that all of the established and new immunosuppressants tested have antiproliferative properties on vascular cells. Rapamycin was by far the most potent one. Therefore immunosuppressants, especiallyrapamycin and mycophenolic acid, may be used for prevention of accelerated arteriosclerosis.

Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva.

Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5' and 3' regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3' region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

The drug resistance-related protein LRP is the human major vault protein.

Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein). LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma. We describe the cloning and chromosome localization of the gene coding for this novel protein. The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein. Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport. The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

A novel multi-gene family of sheep gamma delta T cells.

The WC1 protein is a cell surface constituent of bovine gamma delta T cells and is absent from most or all CD4+, CD8+ T cells and from B cells. It is a single polypeptide chain of 1413 amino acids consisting of 11 non-identical repeats of a 110 amino acid consensus sequence, homologous to the macrophage scavenger receptor cysteine rich (SRCR) domain. A 1059 nucleotide segment of the bovine WC1 cDNA sequence was used as a probe to molecularly clone homologous DNA segments from a sheep genomic library in which the presence of numerous positive plaques was documented. The high representation of such recombinants (1-2/1000 clones) within the library suggested the existence of multiple genes for WC1 (called T19 in sheep) and supported Southern blotting data which revealed an unexpectedly high number of WC1/T19 restriction fragments in sheep genomic DNA. Restriction digests of 27 samples of T19 genomic recombinants were examined by electrophoresis and Southern blotting. All but two pairs of recombinants exhibited non-overlapping restriction digest patterns. Four recombinant DNA samples were partially sequenced and in all cases putative exons were identified and exhibited high homology to appropriate segments of the WC1 cDNA at the levels of both nucleotide and amino acid sequence. Furthermore, multiple nucleotide and amino acid differences occurred between all sequences compared, establishing the existence of a repertoire of non-identical T19 genes, each with the potential to encode a different protein.

Soluble CD8 and CD25 in serum of patients after heart transplantation.

To evaluate the diagnostic value of serum cytokine levels and cytokine receptor levels in the diagnosis of acute rejection after heart transplantation, we measured soluble CD8 and soluble CD25 in the serum of heart transplant recipients. The results were compared with endomyocardial biopsy (EMB) histopathology, lymphocyte activation by morphologic inspection of peripheral blood cells (cytoimmunologic monitoring), clinically manifested infections, and the maintenance immunosuppressive therapy. Significantly increased levels were observed in cases of lymphocyte activation in cytoimmunologic monitoring indicative of either rejection or infection. In clinically documented cytomegalovirus (CMV), bacterial, and Pneumocystis carinii infections, increased levels of soluble CD25 were observed. Soluble CD8 was only increased in a single case of P. carinii infection. A statistically significant correlation was calculated between the levels of soluble CD8 and whole blood cyclosporin A level. Considering chemotherapy, the levels of soluble CD8 showed an inverse correlation with the daily dosage of azathioprine. In conclusion, the levels of soluble CD8 and CD25 are associated with lymphocyte activation in peripheral blood, but do not differentiate between lymphocyte activation indicative of rejection or infection. No relationship was observed between levels of soluble CD8 and CD25, and EMB histopathology. Therefore, the assessment of these two cell products has no diagnostic potential for monitoring acute rejection after heart transplantation.

Cytoimmunologic monitoring as an adjunct in monitoring rejection after heart transplantation: results of a 6-year follow-up in heart transplant recipients.

The cytoimmunologic monitoring assay has been proposed as a useful noninvasive technique in the diagnosis of rejection and infection after heart transplantation. In this study, we have analyzed the diagnostic usefulness of cytoimmunologic monitoring in 73 patients after heart transplantation. For individual patients, the follow-up varied between 2 and 78 months. Data were related to histopathologic characteristics of the endomyocardial biopsy. Significantly different cytoimmunologic monitoring results were not observed between groups according to endomyocardial biopsy histopathologic evaluation. The diagnostic usefulness of cytoimmunologic monitoring depended on the cutoff value applied. With higher cutoff values, the sensitivity decreased and the specificity and predictive value increased. For the previously reported cutoff value of 5%, the sensitivity was 0.29, the specificity was 0.73, and the predictive value was 0.66. Values of sensitivity, specificity, and predictive value were similar when only the first acute rejection was taken into account, or when only data on the first 4 weeks and the first 6 months after transplantation were considered. In calculating the diagnostic usefulness of the sensitivity, specificity, and predictive values were observed. We concluded that cytoimmunologic monitoring has a limited value for diagnosing acute rejection after heart transplantation.

Members of the novel WC1 gene family are differentially expressed on subsets of bovine CD4-CD8- gamma delta T lymphocytes.

CD4-CD8- gamma delta T cells of ruminants uniquely express a 220-kDa surface Ag recognized by several mAbs clustered as WC1. We recently reported the isolation of a cDNA clone encoding a WC1 Ag. Southern blotting suggested that the bovine genome contains multiple sequences highly related to the isolated WC1 cDNA. Here, we demonstrate that some of the clustered WC1 mAbs stain predominantly nonoverlapping subsets of bovine CD4-CD8- gamma delta T cells. By the isolation of two additional cDNA clones encoding molecules highly related to the original WC1 Ag, we provide a molecular basis for this phenomenon. Cells transfected with cDNAs encoding individual WC1 Ags are differentially recognized by various WC1 mAbs. Thus, expression of members of the WC1 gene family divides bovine CD4-CD8- gamma delta T cells into phenotypical subsets. Field inversion gel electrophoresis revealed that all WC1 genes map to a single, large (> 1 Mbp) Notl fragment. Although the function of WC1 remains unknown, it likely involves interaction with ligands that originate from a similarly complex genetic system.

Clustering of monoclonal antibodies recognizing different members of the WC1 gene family.

Mouse L cell lines expressing two different bovine WC1 glycoproteins were produced by transfection of the cells with the corresponding cDNAs. The cell lines were used to analyze the reactivities of 67 monoclonal antibodies (mAbs) which recognize bovine gamma/delta T cells. The results indicated that preliminary clustering of mAbs can be achieved based on their recognition of epitopes expressed on all gene products, or of epitopes encoded by individual members of the gene family. The studies also showed that at least three members of the WC1 gene family are expressed, although it is not yet known how many can be expressed by individual bovine gamma/delta T cells. Final clustering of the WC1 mAbs will not be possible until the exact number of expressed gene products is known, and the reactivities of the mAbs with these products have been analyzed.

Innervation of the endomyocardium in the first period after heart transplantation.

Serial endomyocardial biopsies from 5 patients during the first 3 months after heart transplantation were studied by immunohistochemistry for the neural markers neurofilament 200 kD, neuron-specific protein 9.5 (PGP9.5), S100 (Schwann cell marker), and tyrosine hydroxylase (TH). In normal endomyocardium, nerves immunoreactive for neurofilament 200 kD and PGP9.5 occurred in the interstitium around blood vessels, in close contact with myocyte fibrils. Immunoreactive fibers identified for S100 and TH were also present. In biopsies taken after transplantation, the basic nerve structure in neurofilament labeling was intact. There was a disappearance of immunolabeling for PGP9.5, S100, and TH during the first month after transplantation. Immunoreactivity reappeared during the second month, at first in the interstitium around blood vessels. This was observed for PGP9.5 and TH between 4 and 6 weeks after transplantation, and for S100 (in two of five patients) starting after 6 weeks. There was no apparent relation between reappearance and occurrence of rejection.

Endomyocardial biopsies after heart transplantation. The presence of markers indicative of activation.

A series of 104 endomyocardial biopsies (EMB) from patients after heart transplantation was evaluated for the presence of immunological markers on graft component and infiltrating cells. This included markers for cells expressing alpha beta-T-cell receptors and gamma delta-T-cell receptors, and cytotoxic T cells with granules bearing the serine esterase Granzyme B; the presence of activation markers identified by CD25 (interleukin 2 receptor), CD30, CD69 (activation inducer molecule), CDw70; macrophages using antibody CD14 (WT14), and cells with Fc gamma-receptors type III (CD16). Almost all cells in T-cell infiltrates expressed the alpha beta-T cell receptor. Cells bearing the gamma delta-T cell receptor were scarcely found. The analysis with respect to the histopathologic diagnosis for rejection showed an absence of significance for T cell subsets, Granzyme B-positive cells, and activation markers except CD25. The numbers of macrophages labeled by CD14 and cells expressing Fc gamma RIII showed a significant relation to histopathology of rejection. Apart from leukocytes, also endothelium in EMB with rejection was labeled by the two anti-Fc gamma RIII antibodies used. In addition, in a small series of biopsies investigated, Fc gamma RI- and Fc gamma RII-positive cells were increased in EMB with rejection, and endothelium was labeled by Fc gamma RII antibodies. A cluster analysis on the basis of scores for CD25, CD14, and anti-Fc gamma RIII revealed three main clusters, one cluster comprising biopsies without abnormalities, one cluster containing EMB with the histopathology of rejection and high scores in immunophenotyping for lymphocytes and macrophages, and one cluster in between. The present data emphasize the importance of macrophage assessment in evaluating pathologic processes during rejection of heart allografts and diagnosing rejection.

Molecular characterization of the WC1 antigen expressed specifically on bovine CD4-CD8- gamma delta T lymphocytes.

Although gamma delta T lymphocytes were identified several years ago, the functional importance of these cells remains to be established. gamma delta T cells of ruminants are unique in two respects. First, they are present at much higher levels compared to man and rodents. Second, ruminant CD4-CD8- gamma delta T cells uniquely express a 220 kD surface Ag recognized by a panel of mAb, recently clustered as WC1. WC1 has been most extensively studied in sheep with the use of the mAb T19. Here, we report on the isolation of a full length cDNA clone, encoding the WC1 Ag, from a COS cell cDNA expression library prepared from a bovine gamma delta T cell line. The protein encoded by the pWC1 cDNA clone was reactive with the bovine mAb CC15 and IL.A29, and with T19. The cDNA clone consisted of 4475 bp and contained a single long open reading frame of 1436 amino acids. The pWC1 cDNA clone encoded a type 1 integral membrane protein with an extracellular domain consisting of 11 scavenger receptor cysteine-rich-repeats with homology to CD5 and CD6. Southern blotting suggested that the bovine genome contained multiple sequences highly related to the isolated WC1 cDNA. Furthermore, WC1-like sequences were present in the genomes of all mammals tested including mouse and man. The molecular characterization of the WC1 Ag as reported here provides a starting point for the definition of its role in gamma delta T cell biology.

Transplantation tolerance in heart transplant recipients as demonstrated by unresponsiveness in cell-mediated lympholysis.

Transplantation tolerance or adaptation to an allograft is associated with unresponsiveness to donor-specific transplantation antigens measured in in vitro cell-mediated lympholysis (CML). We here demonstrate in a longitudinal follow-up that CML nonreactivity develops in seven of ten patients following heart transplantation. The first manifestation of this nonreactivity manifested between 3 and 27 months after transplantation. CML nonreactivity correlated with time after transplantation and the percentage of activated lymphocytes in peripheral blood. CML nonreactivity was also associated with good graft function, i.e., in condition of nonresponsiveness patients did not manifest acute rejection. The only exception was seen in one patient in whom the immunosuppressive therapy was strongly reduced. A more detailed evaluation of this patient indicated that the underlying mechanism for CML nonreactivity is clonal anergy or active suppression of the alloreactive cells.

Presence of CD16 on endothelial cells in heart transplant rejection: an immunohistochemical study.

We studied the distribution of Fc gamma RIII (CD16) in human lymphoid and non-lymphoid tissue by immunohistochemistry and two-colour immunofluorescence. In all tissues except stomach, skin, muscle and heart, infiltrating cells were stained. In tonsil and thymus, the CD16 antibody CLBgran 1 labelled some cells with high endothelial morphology in T-cell areas. Two-colour fluorescence combination of CD16 and antibody to Von Willebrand factor confirmed the endothelial nature of the CLB gran 1-positive cells. Fc gamma RIII expression was also demonstrated by antibody CLBgran11, which detects a product of the Fc gamma RIII-B gene of only the NA1 haplotype. A series of 95 endomyocardial biopsies (EMB) taken after heart transplantation was evaluated for Fc gamma RIII expression. In 18 cases Fc gamma RIII was unambiguously detected on endothelial cells by CD16 antibody CLBgran1. There was a significant correlation between capillary CD16 expression and transplant rejection. We conclude that Fc gamma R expression on endothelium represents endothelial activation found in immunopathological and inflammatory conditions.