Abstracts

2003 Transfusion Medicine and Hemotherapy  
Recombinant soluble HLA molecules may be useful tools for the detection of HLA-specific alloantibodies in the sera of patients awaiting transplantation or peptide-specific T-cell recognition. The production of heterotrimeric HLA in procaryotic expression systems consists of several steps: expression of the heavy chain, expression of β2-microglobulin (β2M), isolation of the recombinant proteins, synthesis of specific peptide-ligands and a final refolding step to assemble the trimeric construct.
more » ... n order to facilitate these expression and isolation procedures a β2M-HLA-A*0201 fusion protein was constructed and expressed in Escherichia coli. Therefore, the extracellular domains (α1, α2 and α3 domains) of HLA-A*0201, were cloned and physically connected to the C-terminus of β2M by a glycine-rich linker. For the detection of the recombinant protein by western blot and direct ELISA a V5 tag was connected to the C-terminus, followed by a histidine-rich sequence for isolation purposes. The proteins were expressed in the inclusion body compartment of E. coli. After isolation by immobilized metal affinity chromatography via the 6xHis-tag, they were finally refolded. An immunological in vitro refold assay involving the monoclonal antibody W6/32 was used to gauge refolding yielding a similar refolding behavior compared to the heterotrimeric protein. Thus, this straight forward strategy has the potential to simplify considerably large scale production of soluble HLA. The chimeric proteins can be used for the induction of epitope-specific CTL responses, as well as for the detection of HLA-specific alloantibodies in the sera of patients. B 1.05 Purpose: Finding HLA-specific antibodies is of uttermost importance in transplantation of solid organs, transplantation of bone marrow, diagnostics of immunthrombocytopenia, and transfusions of platelets. Methods: We analyzed 1081 sent in samples from Nov. 2001 to Nov. 2002, which were examined for HLA-specific antibodies because of a variety of diagnostic questions. The routine strategies of our laboratory include testing for HLA-Antibodies in patients on the kidney transplantation lists, routinely checks of patients who have reacted on transfusion of blood products, patients with suspected HIT and other causes of thrombocytopenia techniques used at our laboratory for screening and differentiation are commercially available LCT-kits (Biotest lymphoscreen ABC 60 and DR 30*2), ELISA-kits (GTI Quikscreen, GTI B screen, Quik ID) and the MAIPA-technique (platelets from HLA-typed donors of our blood donation service). Results: 173 sample proofed positive in screening of which 82 were positive in LCT and ELISA, 48 negative in LCT and positive in ELISA, 31 positive in LCT but negative in ELISA and in 12 samples LCT and ELISA were negative, but non-HLA-antibodies were found. The non HLA-antibodies were specific for platelet antigens and were found by ELISA (GTI PAK LE and PAK plus) and MAIPA-technique. The case of 48 year old patient who developed vascular rejection of his second transplanted kidney as an example of the relevance of non-complement activating antibodies, which are negative in LCT-technique but positive in ELISA-technique. Immunadsorption by therapeutic apheresis resulted in improvement of kidney functions. In this case HLA-specific antibodies were only detectable in the eluate of therapeutic apheresis. The antibodies apparently were bound to the endothelium and therefore not detectable in the patients serum. These an- The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel blank allele HLA-B*35SRE which was detected in a cadaveric organ donor. The serological HLA class I type, determined by a lymphocytotoxicity test was A11,24; B38; Bw4; Cw-; whereas the molecular genetic type as identified by PCR-SSP, was A*11,*24, B*35,*38; Bw4, Bw6; Cw*12, thus indicating the presence of a non-expressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C→G) was observed, which at the amino acid level results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Since this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. As translation into a polypeptide would result in a complete though non-functional B35 molecule, peptides derived from it might be presented via the indirect allorecognition pathway, and thus impair the outcome of hematopoietic stem cell transplantation. B 1.03 HLA polymorphism is a major barrier for hematopoietic stem cell and solid organ transplantation. To estimate the allogeneic potential between HLA mismatched stem cell donor/recipient pairs, we recently proposed a matching score (dissimilarity index), that is based on structural data of HLA class I molecules, and on functional similarity of amino acids. This first approach revealed new features about presumptive subtype allogenicities within the HLA-A*23 and A*24 groups. We now have developed an internet-based software tool ('HistoCheck') that is capable to assess the allogenicity (matching score) between any pair of HLA class I and also class II alleles. Newly described HLA sequences will be regularly integrated into the database according to the updates of the nomenclature for factors of the HLA system. The software is intended to be a first step for estimating the allogenicity of HLA mismatches in peculiar clinical settings, as long as there are no reliable in vitro or clinical studies available. The clinical data of the 13th IHWC are currently under evaluation using the HistoCheck algorithm. The algorithm can later be modified according to functional data, e.g. peptide binding specificities. With the extension of the sequence similarity concept to all relevant HLA class I and II loci, the HistoCheck software may contribute to prevent HLA mismatching being a matter of chance. tibodies were donor-specific against HLA-antigens of the first and the second transplanted kidney. Conclusion: We consider the ELISA-technique an useful and necessary addition but not a replacement of the LCT-technique for the investigation of HLA-specific antibodies. Introduction: As a severe side effect of heparinisation, some patients develop an antibody-mediated heparin-induced thrombocytopenia type II (HIT II). For these patients an alternative anticoagulant is required. Recombinant Hirudin (rHirudin) are increasingly used in the management of HIT II patients. Heparin in combination with sodium citrate is often used for anticoagulation during PA-IA. Experience with rHirudin anticoagulation for PA-IA is very limited. Material and Methods: We performed a total of 23 PA-IA sessions (Immunosorba  , Fresenius Hemo-Care), using rHirudin (Lepirudin) as a secondary anticoagulant for two HIT II patients with renal impairment. Patient A: Vascular rejection after 2nd kidney transplant with serum creatinine level of 11.2 mg/dl and remaining diuresis of 180 ml/24h, 90 kg body weight (bw). Patient B: SLE, Lupus nephritis, serum creatinine level of 6.57 mg/dl, normal diuresis, 50 kg bw. Patients received a bolus of 1.875 mg Lepirudin; ECT was measured 2 hours later. Plasmapheresis: Cobe Spectra cell separator (blood flow 30-45 ml/min, ACD-A: WB 1:20-1:25). Plasma flow rate for PA-IA: 20-25 ml/min. Results: Patient A: Bolus of 1.875 mg Lepirudin was given at the beginning of the PA-IA, when Lepirudin pre PA-IA was already <100 ng/ml (Lepirudin was used for hemodialysis too; a level <100 ng/ml was reached at <5.44 mg/dl serum creatinine). After 5 PA-IA and 5 dialysis treatments, the level of serum creatinine was decreased from 11.2.mg/dl to 5.44 mg/dl. After 8 PA-IA a second bolus of Lepirudin was necessary because the level of Lepirudin was <100 ng/ml after 2 hours of PA-IA with a serum creatinine of >3.25 mg/dl and <3.62 mg/dl. Diuresis improved from an initial level of 180 ml/24h to 3700 ml/24h. Thrombin time was constant at >180 sec throughout the treatment. Patient B: Bolus of 1.875 mg Lepirudin was given at the beginning of the PA-IA. Lepirudin level measured after 2 hours varied in a range of 409 ng/ml to 135 ng/ml and correlated positively with the level of serum creatinine of 6.57 mg/dl to 1.94 mg/dl. Thrombin time was constantly >180 sec and aPTT ranged from 50.2 sec to 71.4 sec. Conclusion: rHirudin seems to be an effective anticoagulant for PA-IA. Clotting problems or other side effects did not occur. The Lepirudin dose depends on kidney function and half-life. The clinical results of the PA-IA with Immunosorba were very satisfactory. Purpose: To assess the ability of circulating myeloid blasts/monocytes to differentiate into antigen presenting cells (APC) i.e. dendritic cells (DC), their expression of leukemia-associated antigens (LAA) and the effect of autologous DC-treatment in AML patients (pts.). Methods: Autologous DC were generated from circulating blasts in GM-CSF, IL-4 and TNF-α containing medium. The expression of surface antigens essential for APC / T-cell interaction was assessed by flow cytometry. Leukemic blasts and the generated DC were analyzed for WT-1, PRAME and RHAMM. After successful generation of DC, pts. with refractory AML, in stable condition and adequate increment to transfused blood products were included in an ongoing monocenter trial on an out-patient basis. MNC were collected by a single leukapheresis with subsequent GMP-conform generation of APC/DC in the clean room. After cryopreservation and stringent quality controls, 5 × 10E6 cells were thawed and injected s.c. in the vicinity of the inguinal lymph nodes every other week, for 4 times. Results: DC from 19/25 pts. with refractory AML could be generated, expressing HLA-ABC, -DR, CD40, CD80, CD83 and CD86. Preservation of at least one LAA, e.g. WT-1, PRAME or RHAMM, was detectable by RT-PCR in all DC. Till now 3 pts. in relapse of disease underwent leukapheresis for DC generation. 5 × 10E6 cells were injected s.c. according to the study protocol. No serious adverse events were observed after DC vaccination. However, 2 pts. succumbed to an intracranial hemorrhage due to AML-related thrombocytopenia prior to the second DC vaccination. An allosensitization against platelets or HLA-antigens could not be detected. The third patient required repeated blood transfusions and remained in stable condition for several months, but died from pneumonia 13 months after DC vaccination. After completion of DC vaccination ELISPOT analysis for IFN-γ detected a twofold increase of T-cells recognizing primary autologous leukemic blasts. Conclusions: DC can be generated from AML blasts in 75% of AML pts. and preserve LAA profile. In vivo DC vaccination results in a demonstrable immunological anti-leukemic response in vitro.
doi:10.1159/000073466 fatcat:bk7t26z64ze4xmk27mu6aa2pum