A. Heinold

Salinomycin induces apoptosis and overcomes apoptosis resistance in human cancer cells

Salinomycin is a polyether antibiotic isolated from Streptomyces albus that acts in different biological membranes as a ionophore with a preference for potassium. It is widely used as an anticoccidial drug in poultry and is fed to ruminants to improve nutrient absorption and feed efficiency. Salinomycin has recently been shown to selectively deplete human breast cancer stem cells from tumorspheres and to inhibit breast cancer growth and metastasis in mice. We show here that salinomycin induces massive apoptosis in human cancer cells of different origin, but not in normal cells such as human T lymphocytes. Moreover, salinomycin is able to induce apoptosis in cancer cells that exhibit resistance to apoptosis and anticancer agents by overexpression of Bcl-2, P-glycoprotein or 26S proteasomes with enhanced proteolytic activity. Salinomycin activates a distinct apoptotic pathway that is not accompanied by cell cycle arrest and that is independent of tumor suppressor protein p53, caspase activation, the CD95/CD95L system and the proteasome. Thus, salinomycin should be considered as a novel and effective anticancer agent that overcomes multiple mechanisms of apoptosis resistance in human cancer cells.

No Association of Kidney Graft Loss With Human Leukocyte Antigen Antibodies Detected Exclusively by Sensitive Luminex Single-Antigen Testing: A Collaborative Transplant Study Report

Background. It is unclear whether kidney transplant recipients with preformed donor-specific human leukocyte antigen (HLA) antibodies (DSA) detectable only in the highly sensitive Luminex single-antigen (LSA) assay are at an increased risk of graft failure. Methods. We studied 3148 patients who received a deceased donor kidney graft between 1996 and 2008 and were enrolled in the prospective serum project of the Collaborative Transplant Study. There were 118 patients with graft loss during the first 3 years after transplantation on whom recipient and donor DNA was available for complete HLA typing. We compared the incidence of LSA-detected DSA in these patients with graft failure and matched controls with functioning grafts. All patients were found negative in the less-sensitive complement-dependent lymphocytotoxicity and enzyme-linked immunosorbent assays. Results. When mean fluorescence intensity (MFI) of greater than or equal to 1000 was used as a cutoff for Luminex positivity, 118 patients with graft loss did not show a higher incidence of DSA against HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, or -DPB1 antigens than 118 matched controls without graft loss (for all loci P not significant). The incidence of strong DSA (MFI ≥2000 or MFI ≥3000) detected only by LSA was low (for all loci between 0% and 5%) and did not identify unacceptable antigens that were relevant for graft loss within the first 3 years after transplantation. Conclusion. We conclude that, given currently practiced crossmatch procedures and immunosuppressive regimens, exclusion of donor organs carrying “unacceptable” HLA based exclusively on sensitive LSA antibody testing is not justified.

Role of minor histocompatibility antigens in renal transplantation.

In hematopoietic stem cell transplantation (HSCT), disparities between recipients and donors for minor histocompatibility antigens (mHags) have been shown to be related to graft‐versus‐host disease (GVHD) and graft‐versus‐leukemia (GVL) effects. We investigated the effect of mHag mismatches on kidney allograft survival. Out of 33 785 kidney transplants on which DNA and clinical data were available to the Collaborative Transplant Study (CTS), 702 recipient/donor pairs could be identified as HLA‐A, ‐B and ‐DRB1 matched first transplants of Caucasian origin. These pairs were typed for genetic polymorphisms of the mHags HA‐1, HA‐2, HA‐3, HA‐8, HB‐1, ACC‐1 and UGT2B17. Because mHags are presented in an HLA‐restricted manner, only HLA‐A*02 positive pairs were included in the analysis of HA‐1, HA‐2 and HA‐8. Similarly, only HLA‐A*01, HLA‐B*44 and HLA‐A*24 positive pairs were considered for the evaluation of HA‐3, HB‐1 and ACC‐1, respectively, whereas UGT2B17 compatible transplants were assessed in HLA‐A*29 and HLA‐B*44 positive pairs. None of the mHag disparities showed a statistically significant effect on death‐censored 5‐year graft survival. This report represents the first large‐scale study on the relevance of mHags in kidney transplantation.

Deleterious impact of mismatching for human leukocyte antigen-C in presensitized recipients of kidney transplants.

Background. Allocation of deceased donor kidneys is commonly based on matching for human leukocyte antigen (HLA)-A, -B, and -DR, whereas HLA-C is currently disregarded. We investigated the influence of HLA-C compatibility on renal allograft survival. In addition, we tested an approach of matching for HLA-C epitopes. Methods. A cohort of 2260 deceased donor kidney transplants were typed for HLA-C using polymerase chain reaction-sequence specific primer method. Samples for DNA typing, serum results on presensitization (lymphocytotoxicity), and clinical data were provided by transplant centers participating in the Collaborative Transplant Study. Results. HLA-C mismatch was found to be associated with significantly decreased graft survival in presensitized (P<0.001) but not in non-presensitized (P=0.75) recipients. Mismatch of certain HLA-C epitopes seemed to be more influential than others, with mismatches showing a negative impact on graft survival in presensitized patients. Conclusions. HLA-C mismatch has a substantial deleterious effect on graft survival in presensitized kidney recipients. Our study points out the need for investigations directed at identifying donor-specific HLA-C antibodies and evaluating their relevance in transplantation. In view of the fact that current assays for determining HLA-C specific antibodies are hampered by additional detection of clinically irrelevant antibodies, matching for HLA-C may provide a reasonable approach to improve transplant outcomes in presensitized kidney transplant recipients.

Deleterious impact of HLA-DRB1 allele mismatch in sensitized recipients of kidney retransplants.

Background Whether mismatches between donor and recipient of a kidney transplant at the HLA allele level can elicit an immune response strong enough to impact graft survival is not known. Methods We examined the influence of HLA-DRB1 allele level mismatch on graft survival based on high-resolution typing, utilizing blood samples and clinical data provided by the Collaborative Transplant Study. HLA-DRB1*04 was selected as a model for this investigation because it is the most common HLA-DRB1 allele group and consists of several alleles with relatively high frequencies, allowing for analysis of transplants matched at the antigen level but mismatched at the allele level. Nine hundred and ninety-six recipient/donor pairs were typed for HLA-DRB1 at high resolution. Results No effect of HLA-DRB1*04 allele mismatch was observed in first transplants. However, in retransplants, HLA-DRB1*04 allele mismatch was associated with significantly decreased graft survival, albeit only in sensitized (PRA>5%) patients (hazard ratio 3.98, P=0.014). Conclusion Our finding reinforces the concept that HLA compatibility significantly influences the outcome of kidney transplants, in sensitized retransplant recipients even at the allele level.

Characterization of mannose-binding lectin (MBL) variants by allele-specific sequencing of MBL2 and determination of serum MBL protein levels.

Mannose-binding lectin (MBL) is a major component of the lectin pathway of complement activation. High and low MBL levels have been associated with susceptibility and severity of a variety of infectious and autoimmune diseases. Several single-nucleotide polymorphisms (SNPs) in the promoter region and exon 1 of the MBL2 gene are responsible for variations in serum MBL levels. We developed a sequence-based typing method for allele-specific MBL2 genotyping and measured serum MBL protein levels in 24 German blood donors. We identified the common MBL2 haplotypes including five promoter polymorphisms in linkage with the Q allele and correlated serum MBL levels with the respective genotypes. The genotyping method presented here could provide a basis for confirmatory studies in larger cohorts.

Pitfalls of “hyper”-IgM syndrome: a new CD40 ligand mutation in the presence of low IgM levels. A case report and a critical review of the literature

Here, we report on a male infant with low serum IgG, IgA and IgM levels who suffered from Pneumocystis jirovecii and cytomegalovirus (CMV) pneumonia. The patient was tested to be HIV-negative. Absolute and relative numbers of lymphocyte subsets were normal, excluding the diagnosis of an X-linked agammaglobulinaemia (Bruton’s disease). Despite the decreased serum IgM level, an X-linked hyper-IgM syndrome (X-HIGM) was considered. X-HIGM is a rare immunodeficiency usually characterised by recurrent severe opportunistic infections, low serum IgG and IgA, but normal or increased serum IgM. The syndrome is caused by mutations of the CD40 ligand (CD40L) gene. In our patient, CD40L mutation analysis proved a novel mutation at codon 257 associated with non-detectable expression of CD40L on the surface of activated T cells. A literature search revealed that approximately 6.4% of X-HIGM patients had been found to have low serum IgM levels. Our statistical analysis of the IgM levels as reported by different studies arouses suspicion that many patients with low IgM levels may not have undergone diagnostic procedures for X-HIGM. In summary, in this report and critical review of the literature, we described a new mutation of CD40L and highlighted the pitfalls of the diagnosis of X-HIGM.

Characterization of four new HLA alleles: HLA-B*15:01:18, HLA-B*44:110, HLA-C*04:01:22 and HLA-DQB 1*05:14.

We describe four novel HLA alleles, HLA-B*15:01:18, HLA-B*44:110, HLA-C*04:01:22 and HLA-DQB1*05:14.

Genetic polymorphisms of adhesion molecules and kidney transplant survival.

Background. Adhesion molecules play a key role in the recruitment of leukocytes to sites of inflammation. Genetic polymorphisms of adhesion molecules may alter their expression or function and may thereby influence the process of leukocyte infiltration in the transplanted organ. It has also been suggested that polymorphic adhesion molecules may act as minor histocompatibility antigens. Methods. In two randomly selected cohorts (954 and 1002 kidney transplants), the effect of L-selectin/CD62L (codon 206 and 213), platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31; codon 125, 563, and 670), and activated leukocyte cell adhesion molecule (ALCAM/CD166; codon 258) single nucleotide polymorphisms on 5-yr allograft survival was investigated. DNA samples and clinical data were provided by the Collaborative Transplant Study. Recipients and donors were genotyped by polymerase chain reaction sequence-specific primer. A multivariate analysis was performed using a Cox regression model. Results. Incompatibility for L-selectin at codon 213 was significantly associated with better graft survival in the first cohort, but the effect could not be replicated in the second cohort. Polymorphisms of PECAM-1 and ALCAM had no impact on graft outcome. Conclusions. This is the first comprehensive and large-scale study on the relevance of L-selectin, PECAM-1, and ALCAM genetic polymorphisms in kidney transplantation, showing no significant associations of recipient or donor genotypes with allograft survival. Because the effect of L-selectin mismatch was not reproducible, a putative role of adhesion molecules as minor histocompatibility antigens cannot be confirmed. Our results demonstrate the importance of testing large sample sizes and of performing confirmation studies to validate genetic associations.

Sequential analysis by immunoprecipitation-MALDI-TOF: a novel method for detection and identification of alloantibody specificities.

Alloantibodies are known to influence transplant outcomes. Apart from human leukocyte antigens (HLA), non-HLA targets have been suggested to play a significant role, but little is known about their nature. Here, we present a novel method for identification and characterization of cell surface antigens bound by alloreactive antibodies. Our method consists of 2 consecutive steps: first, immunoprecipitation of cell surface proteins is carried out with serum and, second, matrix-assisted laser desorption/ionization-time-of-flight is used to fingerprint the precipitated cell-surface proteins. As an example, we performed immunoprecipitation with peripheral blood lymphocytes, which had been incubated with an alloreactive serum; immune complexes were coupled to protein-G beads and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; differential protein fractions were then analyzed by matrix-assisted laser desorption/ionization-time-of-flight. The method was validated with serum as well as with plasmapheresis material, which contained antibodies of known HLA specificities, demonstrating its applicability for clinical use.