CD11c+ dendritic cells mediate antigen-specific suppression in extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) represents one of the most widespread and effective cell therapies for graft-versus-host disease and other T cell-mediated disorders. However, the key factors affecting the therapeutic efficacy of ECP remain unclear. We hypothesized that therapeutic effects are mediated by ECP-treated antigen-presenting dendritic cells (DC). To test this hypothesis, we used the experimental model of contact hypersensitivity (CHS). The ECP's therapeutic activity improved when the total cell dose of the ECP-treated cells was increased. We used different haptens during sensitization to demonstrate that the anti-inflammatory activity of ECP is antigen-specific. This confirmed the hypothesis that professional antigen-presenting cells are involved in the mode of action. Also, the ECP's therapeutic activity was abrogated by the depletion of CD11c+ DC, which represents fewer than 1% of all the ECP-exposed cells. Finally, we confirm the critical importance of CD11c+ DC for ECP activity by showing that only a few purified CD11c+ DC are sufficient to mediate its therapeutic effect. The finding that ECP-treated, physiological antigen-presenting DC alone mediate antigen-specific modulation of a pathological immune response may result in better-targeted interventions when treating patients.

Prospective quality control study of a novel gravity-driven whole blood separation system suitable for humanitarian crises.

Centrifugation-based whole blood (WB) separation represents the worldwide standard but it depends on electricity and infrastructure. We have prospectively evaluated a novel hollow-fibre WB separation system that does not require manual priming or blood flow regulation (n = 29). RBC units contained sufficient Hb (50·4 g ± 4·3), low leucocytes (90 000 ± 0·008), exhibited low haemolysis (0·57% ± 0·49) and robust ATP content (51·47% ± 8·2) after 43 days storage. Plasma units contained low leucocytes and mean coagulation factor activities for FV, FVIII and FXI were 47%, 90% and 68%, respectively. RBC met quality specifications but plasma units exhibited reduced FV and FXI activity.

Report on the 15th International Society of Blood Transfusion platelet immunology workshop.

BACKGROUND AND OBJECTIVES: The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet-reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti-HPA-1a and serologically difficult-to-assess antibodies against HPA-3, to identify whether anti-HPA-1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. MATERIALS AND METHODS: Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. RESULTS: Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA-1a, in contrast, gave largely congruent results. Serologically difficult-to-assess antibodies recognizing HPA-3a and HPA-3b were not detected by many laboratories. For genotyping, good agreement was achieved. CONCLUSIONS: Detection of HPA-1a antibodies, titration of anti-HPA-1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA-3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.

Impact of pretransplant intravenous immunoglobulin administration on anti-AB0 antibody levels in AB0-incompatible living donor kidney transplantation.

BACKGROUND: From March 2007 to July 2010, we performed 14 AB0-incompatible (AB0i) living kidney transplantations using donor blood group-specific immunoadsorption (IA), anti-CD20 monoclonal antibody, and intravenous immunoglobulin (IVIG) pretreatment. METHODS: To analyze the effect of a presumed anti-donor blood group-specific antibody transfer by IVIG administration (0.5 g/kg; 5.4 ± 0.9 days pretransplant), we assessed AB0i antibody titers in different IVIG preparations and evaluated their impact on patient AB0i antibody titers. RESULTS: AB0i antibody IgG titers before treatment ranged from 8 to 1024. We performed 6.9 ± 1.1 IA procedures pretransplant to reach AB0i antibody titers ≤4, which enabled successful transplantation in all pretreated patients. Their mean serum creatinine at discharge was 1.5 ± 0.1 mg/dL. IVIG preparations differed profoundly in their AB0i antibody titers: The lowest titers were observed in Sandoglobulin preparations (1-8) compared with Intratect (2-128), Octagam (4-32) and Gamunex (2-512). Usually, administration of the IVIG preparation containing the lowest isoagglutinin titer resulted in low AB0i antibody titer increments in patient sera: Sandoglobulin, 2 titer steps (n = 2), 1 titer step (n = 1), and 0 titer steps (n = 5). In contrast, Octagam showed 0 titer steps (n = 2) and Intratect, 0 titer steps (n = 3). However, after Gamunex administration, the AB0i antibody titer of 8 and the AB0i antibody titer rose 3 titer steps (16 to 128; n = 1), which could not be explained by passive transfer of isoagglutinin alone. CONCLUSION: Our data showed that the choice of IVIG preparation with the lowest AB0i antibody levels is a time- and cost-sparing step in the pretreatment of AB0i living donor kidney recipients. Posttransplant, a high isoagglutinin content within the IVIG preparation has the potential to induce antibody-mediated rejection.

HLA-B*9553: a novel HLA allele shown in three generations of a family.

Human leukocyte antigen (HLA)-B*9553, a novel HLA-B allele, was identified in a volunteer hematopoietic stem cell donor. HLA-B*9553 differs from the closely related allele HLA-B*1518 in one single nucleotide substitution resulting in an amino acid substitution.

DQB1*0323 is the result of a gene conversion event between a DQB1*06 and a DQB1*030303 allele.

A novel human leukocyte antigen-DQB1 allele, DQB1*0323, was identified in a volunteer hematopoietic stem cell donor. DQB1*0323 differs from the closely related allele DQB1*030303 in five nucleotide positions.

Prospective evaluation of PF4/heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies that recognize platelet factor 4-heparin (PF4/hep) complexes leading to platelet activation. Several methods are available for the identification of HIT antibodies. OBJECTIVES: To evaluate the clinical usefulness of different antigen-binding assays for detection of antibodies against PF4/hep complexes in a prospective study. PATIENTS/METHODS: A prospective cohort of 500 surgical and medical patients suspected of having HIT was evaluated. The laboratory assessment included particle gel immunoassay (PaGIA), polyspecific ELISA recognizing IgG, IgM and IgA antibodies (Poly-ELISA), IgG-specific ELISA (IgG-ELISA) and the HIPA test. The pretest probability of HIT was determined using the 4T's model. Positive and negative predictive values (PPV, NPV) of each immunoassay were determined depending upon the heparin-induced platelet activation (HIPA) results and the clinical scoring. The operating characteristics of each immunoassay were determined using the receiver-operation characteristic (ROC) curve. RESULTS: Platelet-activating antibodies were identified in 35/500 patients, all of whom had intermediate to high clinical probability. PF4/hep antibodies were detected in 124, 86 and 90 sera using Poly-ELISA (PPV = 28), IgG-ELISA (PPV = 40.6) and PaGIA (PPV = 36.6). NPV was 100% for Poly- and IgG-ELISA, but only 99.5% for PaGIA. ROC analysis revealed that PaGIA is less informative than ELISA. The IgG-ELISA provides better diagnostic information than the other assays. In addition, there is a clear correlation between optical density (OD) value and the probability of having HIT. CONCLUSIONS: Our observation indicates that an IgG-ELISA provides the best diagnostic information of all antigen-binding assays.

Screening of multiparous women to avoid transfusion-related acute lung injury: a single centre experience.

The aim of this study was to investigate which approach for serological testing of multiparous donors might be feasible and effective to reduce the risk of transfusion-related acute lung injury (TRALI). TRALI is a serious adverse event of blood transfusion. Antibodies to granulocytes and human leucocyte antigens (HLAs) are frequently detected in sera of implicated donors. These donors are often multiparous women. A general deferral of female plasma or screening strategies for leucocyte antibodies has been proposed to increase blood safety. A prospective study was initiated in 2003. Until 2006, serum samples from all female donors reporting three or more pregnancies (n = 229) were screened for the presence of antibodies against granulocytes and HLAs by immunofluorescence and agglutination tests as well as by a commercial HLA enzyme immunoassay. In total, 40% of all multiparous women were reactive in one of the assays. Twenty-nine percent of the reactive sera contained antibodies to granulocytes but not to HLAs. During the observation period, three TRALI reactions occurred in our hospital, two of which would have been prevented if the screening program had been extended to all previously pregnant donors. We conclude from these data that, not unexpectedly, the number of previous pregnancies is not a reliable indicator for the likelihood of inducing TRALI. More importantly, screening strategies for antibodies that might induce TRALI should probably not be reduced to HLA antibody screening. This finding awaits further research.

Topical use of platelet-rich plasma to influence bone volume in maxillary augmentation: a prospective randomized trial.

BACKGROUND AND OBJECTIVES: The atrophic posterior maxilla often requires restoration of the alveolar ridge due to a lack of bone quantity and quality before dental implant placement. The aim of this study was to verify the hypothesis that platelet-rich plasma (PRP) has an influence on bone density in the maxilla after sinus floor elevation in combination with autologous cancellous bone from the iliac crest. Therefore, a randomized, prospective, controlled trial was set up in two centres. STUDY DESIGN AND METHODS: Fifty-three patients who underwent osteoplastic bone grafting for sinus floor elevation were included. The intervention group was treated with defined concentrations of PRP in addition to transplanted bone. Bone biopsy was performed 4 months after augmentation. Bone volume was then measured using the histomorphometric parameter bone volume. RESULTS: The histomorphometric evaluation of the biopsies did not indicate superiority of any of the treatments in terms of bone volume. CONCLUSION: Topical use of PRP did not improve maxillary bone volume either clinically relevant or statistically significant compared to that in conventionally treated patients. The use of PRP to support bone regeneration cannot be recommended as a standard method for maxillary augmentation.

Association of polymorphisms in the mannose-binding lectin gene and pulmonary morbidity in preterm infants.

Deficiency in the collectin mannose-binding lectin (MBL) increases the risk for pulmonary and systemic infections and its complications in children and adults. The aim of this prospective cohort study was to determine the genetic association of sequence variations within the MBL gene with systemic infections and pulmonary short- and long-term complications in preterm infants below 32 weeks gestational age (GA). Three single-nucleotide polymorphisms (SNPs) in the coding region and one SNP in the promotor region of MBL2 were genotyped by direct sequencing and with sequence-specific probes in 284 newborn infants <32 weeks GA. Clinical variables were comprehensively monitored. An association was found between two SNPs and the development of bronchopulmonary dysplasia (BPD), defined as persistent oxygen requirement at 36 weeks postmenstrual age, adjusting for covariates GA, grade of respiratory distress syndrome and days on mechanical ventilation (rs1800450 (exon 1 at codon 54, B variant): odds ratio dominant model (OR)=3.59, 95% confidence interval (CI)=1.62-7.98; rs7096206 (-221, X variant): OR=2.40, 95% CI=1.16-4.96). Haplotype analyses confirmed the association to BPD, and a single haplotype (frequency 56%) including all SNPs in their wild-type form showed a negative association with the development of BPD. We detected no association between the MBL gene variations and the development of early-onset infections or further pulmonary complications. Frequent variants of the MBL gene, leading to low MBL concentrations, are associated with the diagnosis of BPD in preterm infants. This provides a basis for potential therapeutic options and further genetic and proteomic analysis of the function of MBL in the resistance against pulmonary long-term complications in preterm infants.

Sanglifehrin a blocks key dendritic cell functions in vivo and promotes long-term allograft survival together with low-dose CsA.

Sanglifehrin A (SFA) is a novel compound with unsurpassed cyclophilin-binding affinity, but relatively low direct antilymphocytic activity. Here, we report the capacity of SFA to selectively inhibit key dendritic cell (DC) functions in vivo and to suppress acute and chronic heart allograft rejection. We show that in vivo, SFA profoundly decreases DC receptor-mediated endocytosis and macropinocytosis and DC-T-cell allostimulatory activity. Furthermore, SFA abrogates >90% of IL-12p70 production in vivo while having no significant effect on IL-10 and TNF-alpha production. In a rat vascularized heart transplant model, SFA alone did not prevent graft rejection and rejection occurred within 23 days after low-dose CsA, whereas addition of SFA to low-dose CsA promoted long-term graft survival (median survival time >100 days; p = 0.0007). With respect to chronic rejection, SFA + CsA almost completely prevented graft arteriosclerosis compared to animals treated with CsA alone and controls. We propose that SFA represents a novel class of immunophilin-binding immunosuppressants with high activity against DCs and the development of graft vasculopathy in CsA-treated recipients.

Dendritic cell deficiency in the blood of kidney transplant patients on long-term immunosuppression: results of a prospective matched-cohort study.

Evidence from in vitro studies suggests that immunosuppressive drugs interfere with key functions of dendritic cells (DCs), but the in vivo relevance of these findings is elusive. We prospectively analyzed the major DC precursor subsets in the blood of kidney transplant recipients on long-term immunosuppression (> or =1 year). A total of 87 patients were compared to 87 age- and sex-matched controls. Total DC numbers and the precursor subsets, myeloid type 1 DCs, myeloid type 2 DCs (mDC1, mDC2) and plasmacytoid DCs (pDCs) were identified by four color flow cytometry. Long-term immunosuppression was associated with significant reduction of all major DC subsets in comparison to healthy controls (mDC1 p < 0.001; mDC2 p < 0.0001; two-tailed Mann-Whitney U-test) with the strongest negative impact on pDCs (p < 0.00001). In contrast, total leukocyte numbers were not significantly affected. Analysis of the relative impact of different agents revealed a significant impact of prednisolone on pDCs (p = 0.009) and mDCs2 (p = 0.006). The functional relevance of pDC deficiency was confirmed independently by Interferon-alpha analysis after Toll-like receptor 7 (p < or = 0.001) and 9 (p < 0.05) stimulation. These results indicate for the first time a profound negative impact of long-term immunosuppression on major DC subsets in kidney transplant recipients. DC deficiency may have important implications with respect to viral infections and tumor development.

Common human Toll-like receptor 9 polymorphisms and haplotypes: association with atopy and functional relevance.

BACKGROUND: Toll-like receptor 9 (TLR9) is a pattern-recognition receptor that detects unmethylated CpG motifs prevalent in bacterial and viral DNA. TLR9 stimulation is a key event after bacterial infection, triggering innate immunity and T-helper type 1 skewed adaptive immunity. Synthetic CpG-oligodeoxynucleotides (CpG-ODNs) represent a promising and novel class of immune adjuvants for allergy treatment, vaccination, and cancer therapy. However, common functional TLR9 gene variants could interfere with the clinical utilization of CpG-ODN in immunotherapy. Recently, a possible association of TLR9 polymorphism C-1237T with asthma has been reported. OBJECTIVE: The aim of the present study was to investigate whether TLR9 polymorphisms or haplotypes have functional relevance and are associated with atopy. METHODS: We genotyped five common TLR9 single-nucleotide polymorphisms (SNPs) in promoter, exon, and intron regions of the gene in 527 healthy blood donors, and estimated four common haplotypes. The total IgE and specific IgE levels against the most common aeroallergens were measured (n=303). IFN-alpha production by plasmacytoid dendritic cells (pDCs) was analysed after stimulation with TLR9 ligand CpG-ODN (n=220). RESULTS: No significant influence of common TLR9 polymorphisms and haplotypes on the total and specific IgE levels was found. Functional analysis of CpG-ODN-induced IFN-alpha did not indicate a significant role for common TLR9 gene polymorphisms in TLR9 function. CONCLUSION: We conclude that common genetic differences in the TLR9 gene exert no major influence on allergy susceptibility, and are unlikely to have on impact on clinical application of CpG-ODNs.

Genotyping of human neutrophil antigen-5a (Ond).

Antibodies to human neutrophil alloantigens (HNA) can cause immune-mediated neutropenias and transfusion complications. The diagnosis of antibodies to the HNA-5a (Ond) isoform of the alphaLbeta2 integrin is hampered by the lack of reliable methods for HNA-5a antigen typing. We have devised a polymerase chain reaction sequence-specific primer method (PCR-SSP) and used it to determine the HNA-5a gene frequencies in 320 individuals from different ethnicities. 15.3% were found to be HNA-5a negative, with no significant deviation between the populations. Results of HNA-5a genotyping were in accordance with phenotyping. Availability of HNA-5a PCR-SSP will facilitate the diagnosis of Ond antibody-mediated clinical conditions.

A promoter polymorphism in the CD14 gene is associated with elevated levels of soluble CD14 but not with IgE or atopic diseases.

BACKGROUND: A polymorphism in the promoter region of the CD14 gene, C-159T, has been shown to be associated with increased levels of soluble CD14 (sCD14) and decreased serum immunoglobulin E (IgE) and the expression of a more severe atopic phenotype in previous studies. METHODS: To test if these associations are consistently found in different populations and different age groups, we genotyped 2048 children of different age groups as well as 888 adults from different regions of Germany for the CD14 C-159T polymorphism. RESULTS: While an association between this promoter polymorphism and levels of sCD14 could be confirmed in our study population (CC: 1017 ng/ml vs TT: 1370 ng/ml, P = 0.03), no association between CD14 C-159T genotypes and IgE levels or the prevalence of atopic diseases was seen. CONCLUSIONS: The lack of association between CD14 genotypes and IgE as well as atopic outcomes in this large German study population seems to indicate that CD14 genotypes may not directly be involved in the development of allergies during childhood.

Continuous cytomegalovirus seroconversion in a large group of healthy blood donors.

BACKGROUND AND OBJECTIVES: Transmission of cytomegalovirus (CMV) to seronegative, immunocompromised recipients can cause serious and fatal complications. Although the seroprevalence of CMV is high, the risk of primary CMV infection among healthy blood donors has not yet been analysed in a large population. MATERIALS AND METHODS: We developed an algorithm to determine the rate of CMV seroconversion in an overall cohort of 24,260 subjects who donated 176,474 blood units during an 11-year observation period. RESULTS: We detected CMV seroconversion in all relevant age groups (18-60 years) with an overall seroconversion rate of 0.55% per year. Both CMV seroconversion and seroprevalence occurred more frequently in female donors (P = 0.02 and P < 0.001, respectively). We identified 30-35-year-old blood donors as the group with the highest rate of CMV seroconversion per year (1.33% vs. 0.46%; P < 0.0001). CONCLUSIONS: We conclude that the risk of primary CMV infection is a continuous lifelong event and correlates with age and female gender.

Th2-mediated atopic disease protection in Th1-mediated rheumatoid arthritis.

OBJECTIVE: The balance between CD4+ T-helper (h) cell subsets (Th1 and Th2) plays an important role in the pathogenesis of rheumatoid arthritis (RA) and atopy. While RA is believed to be a Th1 mediated disease, Th2 cells predominate in atopic disorders. The purpose of this study was to investigate differences in the occurrence of allergy, hay fever, house dust mite sensitivity and asthma, as well as total serum IgE levels in RA patients and controls. METHODS: The case history of atopic disorders was assessed in 134 RA patients and compared to those found in 305 healthy blood donors. RA patients also answered clinical questions concerning disease activity and severity. Total serum IgE levels were measured in both groups, taking into consideration disease modifying therapy. RESULTS: A significantly lower occurrence of medical history of hay fever (2.3%) and house dust mite sensitivity (3.1%) was found among RA patients compared to controls (24.2% and 12.2%, respectively; p < 0.0001 and p < 0.003 respectively). Moreover, RA patients had significantly lower total serum IgE levels than control subjects (p < 0.0001). RA was less severe in patients with atopy compared to non-atopic RA patients. CONCLUSION: These results support the concept that RA and atopy antagonize each other and that a change in the cytokine patterns of Th1 and Th2 cells could provide an indication for curative effects on RA.

Novel genetic variation of human interleukin-21 receptor is associated with elevated IgE levels in females.

The interleukin-21 receptor (IL21R) was recently discovered as a novel member of the class-I-cytokine-receptor family and is selectively expressed in lymphoid tissues. IL21R shows strong sequence homologies to the interleukin-4 receptor alpha chain gene (IL4RA). In addition, both genes are adjacent and share structural similarity. We analyzed all the exons of the human IL21R gene and its 5' flanking region for sequence variation. We identified four novel single nucleotide polymorphisms (SNPs) and genotyped 300 healthy blood donors. Total serum IgE levels were measured in all subjects and associated with IL21R SNPs. Results revealed a significant association of one IL21R polymorphism (T-83C) with elevated IgE levels (>100 kU/I) in females (OR=3.000, CI=[1.163;8.385], P=0.015, n=138). This was confirmed in a second prospectively collected group of female blood donors (OR=2.535, CI=[0.927;6.733], P=0.046, n=123). In contrast, no effects were observed in male subjects in either population. These findings identify IL21R as a possible novel target locus influencing IgE synthesis in female individuals.

No linkage of the interleukin-4 receptor locus on chromosome 16p11.2-12.1 with sarcoidosis in German multiplex families.

We typed 241 members of 62 sarcoidosis families with 136 affected siblings for three single nucleotide polymorphisms of the interleukin-4 receptor alpha-chain gene (IL4R). Allele frequencies in patients were compared to those of healthy unrelated control individuals. The segregation of the three-point IL4R haplotypes completed by two flanking highly polymorphic microsatellite polymorphisms revealed no evidence for linkage of the IL4R gene locus with sarcoidosis.