Development and performance of a next generation sequencing (NGS) assay for monitoring of dd-cfDNA post solid organ transplantation.

The aim of this study was to evaluate the analytical performance of a novel NGS assay, intended for monitoring of donor-derived cell-free DNA (dd-cfDNA), and describe its validity in clinical plasma samples from kidney transplanted patients. Artificial and clinical samples with increasing amounts of patient DNA were evaluated using NGS analysis of indel markers. Monitoring of dd-cfDNA with the NGS assay presented herein demonstrated a sensitivity of ≥0.

Early relapse prediction after allogeneic hematopoietic stem cell transplantation for acute lymphoblastic leukemia (ALL) using lineage-specific chimerism analysis.

Relapse is a major cause of treatment failure after hematopoietic stem cell transplantation (HSCT) for acute leukemia. Here, we report a monocentric retrospective study of all HSCTs for B cell acute lymphoblastic leukemia (ALL) performed during the years 2005-2021 ( = 138, including 51 children), aiming to identify the optimal use of lineage-specific recipient-donor chimerism analysis for prediction of relapse. In adults, relapse was associated with increased recipient chimerism in CD3 bone marrow cells sampled at least 30 days before a relapse.

Lineage-specific early complete donor chimerism and risk of relapse after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia.

Recipient-donor chimerism is routinely analyzed after allogeneic hematopoietic stem cell transplantation (HSCT) to monitor engraftment and graft rejection. For malignancies, chimerism can also be used to screen for disease relapse post-HSCT but methodology and interpretation of results are not standardized and likely depend on underlying diagnosis. We have implemented highly sensitive and accurate methodologies for chimerism analysis for the purpose of improving relapse prediction.

Development and performance of a next generation sequencing (NGS) assay for monitoring of mixed chimerism.

The aim of this study was to evaluate the performance of a novel NGS-based assay to monitor mixed chimerism (MC) and compare its technical capacity to established techniques for chimerism analysis. Artificial and clinical samples with increasing amounts of patient DNA were compared using real-time PCR detection of indels and SNP, fragment analysis of short-tandem repeats (STR) and NGS analysis of indels. Real-time PCR displayed excellent sensitivity (>0,01%) but poor accuracy (>20 CV% at MC > 20%), while fragment analysis exhibited good accuracy (<5 CV% at MC > 20%) with limited sensitivity (>2,5%).

No effect of HLA-C mismatch after allogeneic hematopoietic stem cell transplantation with unrelated donors and T-cell depletion in patients with hematological malignancies.

HLA-C mismatch in unrelated donor's hematopoietic stem cell transplantation (HSCT) has been associated with poor patient outcome. However, the impact of HLA-C mismatch in the context of HSCT combined with in vivo T-cell depletion remains unclear. We therefore performed a single-center, retrospective analysis of the clinical outcome on patients with hematological malignancies treated with allo-HSCT, who underwent T-cell depletion.

An unusual cause of fatal rapid-onset ataxia plus syndrome.

Background: Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder of the central nervous system caused by reactivation of the JC-virus and is in most cases associated with underlying immunosuppression. Acquired immune deficiency syndrome (AIDS) and hematological malignancies are well-known predisposing factors for PML. However, in the past ten years, various pharmacological agents have been associated with increased risk of PML.

De Novo Donor-Specific HLA Antibody Formation in Two Patients With Crigler-Najjar Syndrome Type I Following Human Hepatocyte Transplantation With Partial Hepatectomy Preconditioning.

Clinical hepatocyte transplantation is hampered by low engraftment rates and gradual loss of function resulting in incomplete correction of the underlying disease. Preconditioning with partial hepatectomy improves engraftment in animal studies. Our aim was to study safety and efficacy of partial hepatectomy preconditioning in clinical hepatocyte transplantation.

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching.

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM).

Evaluation of a new flow cytometry crossmatch procedure for simultaneous detection of cytotoxicity and antibody binding.

In this study we have evaluated an alternative 96-well format flow cytometry based (FCtox) method which enable simultaneous detection of cytotoxicity and human leukocyte antigen (HLA) antibody binding. Comparable results were obtained in side-by-side comparisons with conventional complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM) in terms of sensitivity and specificity. There was 91 and 93% agreement between results obtained by FCtox and CDC for T and B cells, respectively.

Immune modulation to prevent antibody-mediated rejection after allogeneic hematopoietic stem cell transplantation.

It has been shown that antibodies to donor CD34+/VEGFR-2+ stem cells or antibodies against mismatched HLA are associated with graft rejection after hematopoietic stem cell transplantation (HSCT). CD34/VEGFR-2 positive stem cells have been implicated to play a major role in engraftment after HSCT. In this study we treated four patients with an imminent risk of antibody-mediated rejection with immune modulation, i.

Improved survival after allogeneic hematopoietic stem cell transplantation in recent years. A single-center study.

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009.

A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial precursor cells.

Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes.

Cytotoxic crossmatch analysis before allo-SCT is a poor diagnostic tool for prediction of rejection.

The predictive value of cytotoxic crossmatch analysis before allo-SCT remains unclear. We retrospectively analyzed the clinical impact of cytotoxic T- and B-cell crossmatch testing before allo-SCT between January 2000 and June 2005. Cytotoxic crossmatches were performed in 157 patients receiving stem cells from matched unrelated donors or an HLA-A, -B or -DRB1 allele mismatched graft.

Outcome of haematopoietic stem cell transplantation in patients transplanted with matched unrelated donors vs allele-mismatched donors: a single centre study.

We studied the importance of human leucocyte antigen (HLA)-A, -B and -DRB1 high-resolution matching on the outcome of haematopoietic stem cell transplantation (HSCT) with matched unrelated donors (MUDs) vs single allele-mismatched unrelated donors. Fifty consecutive HSCT patients receiving an HLA-A, -B or -DR allele-level-mismatched unrelated graft (mmUD) were compared with a matched cohort of 100 patients with an HLA-A, -B and -DR-MUD. Rejection occurred in seven patients (14%) in the mmUD group and in four patients (4%) in the MUD group (P = 0.

Expression of hyaluronan synthases and hyaluronan in malignant mesothelioma cells.

Background: Hyaluronan is one of the main components of the extracellular matrix. It is synthesized at the cell plasma membrane by specific hyaluronan synthases (HAS). Although a large number of studies have described hyaluronan in pleural effusion from malignant mesothelioma, the source of hyaluronan in malignant mesothelioma has been subject to controversy.

Which donor should be chosen for hematopoietic stem cell transplantation among unrelated HLA-A, -B, and -DRB1 genomically identical volunteers?

The aim of this study was to identify significant prognostic factors by using unrelated genomically HLA-A, -B and -DRB1-identical donors. Such data could help to choose the best donor. We studied 136 consecutive patients with hematologic malignancies and a median age of 32 years (range, 0-55 years) who received hematopoietic stem cell transplantation.

An association between human leucocyte antigen alleles and acute and chronic graft-versus-host disease after allogeneic haematopoietic stem cell transplantation.

The association between various human leucocyte antigen (HLA) alleles and the occurrence of acute and chronic graft-versus-host disease (GVHD) was evaluated in 493 haematopoietic stem-cell transplant (HSCT) patients with HLA identical sibling donors. There were 307 men and 186 women with a median age of 30 years (0.2-77).

T lymphocyte expression of thrombospondin-1 and adhesion to extracellular matrix components.

The mechanisms controlling the formation of pseudopodia and other active cell edges in T lymphocytes are not understood. We show here that T lymphocytes express thrombospondin-1 (TSP-1). TSP-1 in T lymphocytes has a high turnover as shown by the fact that brefeldin and monensin rapidly increase while cycloheximide tend to decrease the cellular TSP-1 content.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours.

Porcine endothelium supports transendothelial migration of human leukocyte subpopulations: anti-porcine vascular cell adhesion molecule antibodies as species-specific blockers of transendothelial monocyte and natural killer cell migration.

Background: In cases where hyperacute rejection has been prevented, pig to primate organ transplantation results in a delayed rejection mediated by graft-infiltrating leukocytes. The migration of human leukocytes across porcine endothelium is poorly characterized, but may offer targets for species-specific antirejection therapy.

Methods: Transwell tissue culture inserts with endothelial cells growing on polycarbonate filters were used to characterize the migration of peripheral blood monocuclear cells and purified leukocyte subpopulations across pig and human endothelial cells and cell lines.

Urokinase-type plasminogen activator inhibits alpha 4 beta 1 integrin-mediated T lymphocyte adhesion to fibronectin independently of its catalytic activity.

The urokinase-type plasminogen activator (u-PA)/plasmin system plays an important role in promoting cell migration and invasion, an effect which is largely ascribed to the proteolytic activity of these enzymes. We investigated whether u-PA modulates integrin-dependent T lymphocyte migration and adhesion on fibronectin independently of its plasminogen activator function. Here we report that u-PA reduced the spontaneous and phorbol 12-myristate 13-acetate-induced migration of peripheral blood T lymphocytes on fibronectin by 20-50%, decreased the T lymphocyte and alpha4beta1(+)/alpha5beta1(+) K562 cell adhesion on fibronectin by 30-40%, and completely suppressed integrin alpha4beta1-dependent T lymphocyte and alpha4beta1(+)/alpha5beta1(+) K562 cell adhesion to the LDV-containing 40-kDa fibronectin fragment.

Spectrum of extracellular matrix degrading enzymes in normal and malignant T lymphocytes.

Human T cells produce and release fibronectin degrading neutral serine proteases with a molecular weight of 50 kD, 70-80 kD (doublet) and 95 kD and have a cell associated 400 kD fibronectin degrading enzyme. In addition, human T cells produce proteases with m.w.

Growth-factor-dependent migration of human lung-cancer cells.

Human lung tumors express different types of growth-factor receptors and corresponding ligands that might modulate several biological functions such as proliferation, differentiation, adhesion, and chemotaxis. In the present study, we have investigated the expression of different growth-factor receptors and their ligands in 5 established human lung-cancer cell lines. Using RT-PCR, we found that IGF-II/mannose-6-phosphate (M6P), c-met, EGF and c-kit receptors are expressed in 5/5 human lung-cancer cell lines.

Tenascin-C inhibits beta1 integrin-dependent T lymphocyte adhesion to fibronectin through the binding of its fnIII 1-5 repeats to fibronectin.

The extracellular matrix consists of different proteins interacting to form a meshwork-like structure. T lymphocyte adhesion to individual matrix proteins is mainly regulated at the adhesion receptor level, but it is conceivable that the composition of the matrix itself may affect T lymphocyte adhesion to individual proteins. We have addressed the latter point by studying the effect of the matrix protein tenascin-C (TN-C) on T lymphocyte adhesion to fibronectin.

Platelet-derived growth factor (PDGF) BB acts as a chemoattractant for human malignant mesothelioma cells via PDGF receptor beta-integrin alpha3beta1 interaction.

Platelet-derived growth factor BB (PDGF BB) and the PDGF receptor beta are expressed on mesothelioma cells, but their biological function has not yet been defined. In the present study we used Boyden chambers fitted with filters coated with the adhesive matrix proteins fibronectin, laminin, collagen type IV or the nonmatrix adhesive molecule poly-L-lysine (PLL). Mesothelioma cells migrated towards PDGF BB at concentrations ranging from 0.