The significance of pretransplant donor-specific antibodies reactive with intact or denatured human leucocyte antigen in kidney transplantation.

Antibodies recognizing denatured human leucocyte antigen (HLA) can co-react with epitopes on intact HLA or recognize cryptic epitopes which are normally unaccessible to HLA antibodies. Their specificity cannot be distinguished by single antigen beads (SAB) alone, as they carry a mixture of intact and denatured HLA. In this study, we selected pretransplant sera containing donor-specific HLA class I antibodies (DSA) according to regular SAB analysis from 156 kidney transplant recipients. These sera were analysed using a SAB preparation (iBeads) which is largely devoid of denatured HLA class I, and SAB coated with denatured HLA class I antigens. A total of 241 class I DSA were found by regular SAB analysis, of which 152 (63%) were also found by iBeads, whereas 28 (11%) were caused by reactivity with denatured DNA. Patients with DSA defined either by regular SAB or iBeads showed a significantly lower graft survival rate (P = 0·007) compared to those without HLA class I DSA, whereas reactivity to exclusively denatured HLA was not associated with decreased graft survival. In addition, DSA defined by reactivity to class I SAB or class I iBeads occurred more frequently in female patients and in patients with historic HLA sensitization, whereas reactivity to denatured HLA class I was not associated with any of these parameters. Our data suggest that pretransplant donor-specific antibodies against denatured HLA are clinically irrelevant in patients already sensitized against intact HLA.

Pretransplant donor-specific HLA class-I and -II antibodies are associated with an increased risk for kidney graft failure.

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q-fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG-DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q-fixing DSA. Patients with both class-I plus -II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q-fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan-IgG SAB assay are generally related to C1q-fixation. We conclude that the presence of class-I plus -II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q-fixing IgG-DSA could not be assessed due to their low prevalence.

NK-KIR ligand identification: a quick Q-PCR approach for HLA-C epitope typing.

Interaction of donor natural killer (NK)-cell-associated killer cell immunoglobulin-like receptors (KIRs) with the patient's human leukocyte antigen-C (HLA-C) ligands can result in an alloreactive NK response after haematopoietic stem cell transplantation. In many retrospective studies, additional HLA-C-typing data are required to predict NK-cell alloreactivity. We developed a Taqman assay using the quantitative polymerase chain reaction (Q-PCR) technique that facilitates HLA-C epitope typing, allowing the assignment of HLA-C group 1 or 2 alleles based on the dimorphism at residues 77 and 80 rather than based on the sequence specific priming (SSP) and sequence-based typing allele types. Q-PCR analysis for HLA-C epitope detection showed three clusters reflecting homozygous group 1 or 2 and heterozygous samples. This new approach introduces a quick HLA-C epitope screening method to define the presence of the ligand for the KIR-HLA-C interaction.

Human primary T lymphocytes have a low capacity to amplify MLV-based amphotropic RCR and the virions produced are largely noninfectious.

The presence of replication-competent retrovirus (RCR) in retroviral-based gene therapy products poses a potential safety risk for patients. Therefore, RCR testing of clinical gene therapy products and monitoring of patients enrolled in gene therapy trials is required to assure viral safety. The requirement to test ex vivo-transduced cells originates from the presumed amplification of adventitious RCR during the transduction procedure. However, data on the capacity of different cell types to do so are lacking. In this study, we sought to analyze the amplification potential of primary human T lymphocytes after infection with amphotropic MLV-based RCR. The total number of viral particles produced after 1 or 2 weeks was measured by a quantitative 4070A env-specific RT-PCR assay. The fraction of infectious replication-competent viral particles was analyzed in the PG-4 S+L- assay. From this study, we conclude that the total number of viral particles RCR produced by T lymphocytes is 2-4 logs lower than the number produced by NIH-3T3 cells. Surprisingly, less than 1% of the viral particles produced by primary T lymphocytes appeared to be infectious, while nearly all virions produced by NIH-3T3 were. We conclude that primary human T lymphocytes are low producers of MLV-based amphotropic RCR.

Chemosensitization of myeloma plasma cells by an antisense-mediated downregulation of Bcl-2 protein.

An antisense oligodeoxynucleotide (ODN) complementary to the first six codons of the Bcl-2 mRNA, G3139 (oblimersen sodium; Genasense), has been shown to downregulate Bcl-2 and produce responses in a variety of malignancies including drug-resistant lymphoma. Incubation of ex vivo purified plasma cells from patients with multiple myeloma (MM) with carboxyfluorescein (FAM)-labeled antisense ODNs resulted in a time- and dose-dependent uptake in the cytoplasm and nucleus. No major differences in uptake of Bcl-2 antisense ODNs were observed among patients' samples. Incubation of purified myeloma plasma cells with G3139, but not solvent or reverse polarity control ODNs, resulted in a reduction (>75%) of Bcl-2 mRNA levels after 2 and 4 days, as measured by Real-Time PCR. Treatment with G3139 led to a sequence-specific reduction of Bcl-2 protein levels within 4 days of exposure in 10 out of 11 clinical samples from patients with chemosensitive and multidrug-resistant disease, without significant reduction of alpha-Actin, Bax, Bcl-XL, or Mcl-1 proteins. This resulted in a significantly enhanced sensitivity of the myeloma tumor cells to dexamethasone or doxorubicin-induced apoptosis. G3139 can consistently enter myeloma cells, downregulate the expression of Bcl-2, and enhance the efficacy of myeloma therapy. These data support further clinical evaluation of G3139 therapy in multiple myeloma.