Search

ZusammenfassungDie Extrakorporale Photopherese ist ein etabliertes Therapieverfahren für Patienten mit T-Zell vermittelten Erkrankungen. Dabei besteht das Verfahren aus der Gewinnung autologer mononukleärer Zellen, deren Behandlung mit 8-Methoxypsoralen und UVA-Licht und die Retransfusion der behandelten Zellen.Die Wirkmechanismen der Photopherese sind zwar noch nicht vollständig geklärt, ein zentraler Mechanismus stellt jedoch die Apoptose mononukleärer Zellen dar. Das Ziel der Studie war eine Optimierung der Photopherese im Hinblick auf die Behandlung der Zellen mit 8-Methoxypsoralen/UVA und der daraus induzierten verstärkten Apoptose der Lymphozyten. Dabei sind einige Faktoren bekannt, welche die Effektivität der 8-Methoxypsoralen/UVA-Behandlung der Zellen beeinflussen können, wie z.B. der Hämatokrit oder die UVA-Dosis. Unser Fokus lag auf der Verfügbarkeit der photoaktiven Substanz und dem Einfluss der Zellsuspensionsmatrix auf die Apoptose der Lymphozyten.Die Verfügbarkeit von 8-Methoxypsoralen für die Aufnahme in die Zellen reduzierte sich durch Absorption an Kunststoffe der Bestrahlungssysteme sowie durch Bindung an Proteine bei der Verwendung von autologem Plasma bei der Suspendierung der Zellen. Eine Steigerung der Zugabe von 8-Methoxypsoralen auf 340 ng/mL anstelle von 200 ng/mL führte zu einem Anstieg der T-Zell Apoptose, die sich unter Verwendung von physiologischer Kochsalzlösung als Zellsuspensionsmatrix weiter erhöhte. Eine Anpassung des Verfahrens mit NaCl anstelle von Plasma und die Verwendung einer höheren 8-Methoxypsoralen Konzentration führte zu einer gesteigerten Apoptoseinduktion der T-Zellen. Inwiefern sich eine Erhöhung der Apoptose auf die klinische Wirksamkeit auswirkt, bedarf hingegen noch weiterer klinischen Untersuchungen.

Supplemetary Tables 1-3

Purpose:Non–small cell lung cancer (NSCLC) is a fatal disease with poor prognosis. A membrane-bound form of Hsp70 (mHsp70) which is selectively expressed on high-risk tumors serves as a target for mHsp70-targeting natural killer (NK) cells. Patients with advanced mHsp70-positive NSCLC may therefore benefit from a therapeutic intervention involving mHsp70-targeting NK cells. The randomized phase II clinical trial (EudraCT2008-002130-30) explores tolerability and efficacy of ex vivo–activated NK cells in patients with NSCLC after radiochemotherapy (RCT).Patients and Methods:Patients with unresectable, mHsp70-positive NSCLC (stage IIIa/b) received 4 cycles of autologous NK cells activated ex vivo with TKD/IL2 [interventional arm (INT)] after RCT (60–70 Gy, platinum-based chemotherapy) or RCT alone [control arm (CTRL)]. The primary objective was progression-free survival (PFS), and secondary objectives were the assessment of quality of life (QoL, QLQ-LC13), toxicity, and immunobiological responses.Results:The NK-cell therapy after RCT was well tolerated, and no differences in QoL parameters between the two study arms were detected. Estimated 1-year probabilities for PFS were 67% [95% confidence interval (CI), 19%–90%] for the INT arm and 33% (95% CI, 5%–68%) for the CTRL arm (P = 0.36, 1-sided log-rank test). Clinical responses in the INT group were associated with an increase in the prevalence of activated NK cells in their peripheral blood.Conclusions:Ex vivo TKD/IL2-activated, autologous NK cells are well tolerated and deliver positive clinical responses in patients with advanced NSCLC after RCT.

Background: Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy for various diseases. Autologous leukocytes are collected, photoactivated with a photosensitizer (8-methoxypsoralen, 8-MOP) and UVA light, and subsequently reinfused back to the patient. Leukapheresis and UVA irradiation systems can be integrated into one device (inline) or handled by two separate devices (offline). ECP works via intercalation of 8-MOP into DNA helices and UVA-based interactions to inhibit DNA replication. 8-MOP is known to adhere to plastic materials, which might reduce its availability for intercalation. In the present study we examined the bioavailability of 8-MOP when different plastic materials and solvents are used as matrices. Methods: Varying amounts of shredded ethylene vinyl acetate (EVA) and polyvinylchloride (PVC) from the MacoGenic irradiation bag (EVA1), UVA PIT irradiation bag (EVA2), UVA PIT recirculation bag (PVC A) and UVA PIT tubing (PVC B) by MacoPharma and PIT Medical Systems, respectively, were incubated with 200 ng mL−1 8-MOP dissolved in diisopropyl ether (DIPE) plus toluene 90/10 vol%, deionized water or plasma. After 2 h 8-MOP concentrations were determined by GC-MS. Results: After incubation, 8-MOP concentrations varied depending on the amount and type of plastic (PVC > EVA) and solvent (water > plasma > DIPE/toluene). Absorption to 200 mg EVA1 or EVA2 resulted in 8-MOP concentrations of 57% or 32% in water, 91% or 80% in plasma, and 93% or 92% in DIPE/toluene, while 200 mg PVC A and PVC B yielded recovery rates of 26% and 10% in water, 76% and 75% in plasma, and 55% and 30% in DIPE/toluene, respectively. Remaining 8-MOP differed significantly between container materials (EVA vs. PVC; p < 0.022) but not manufacturers (MacoPharma vs. PIT Medical Systems). Conclusion: Absorption loss of 8-MOP depends on the type of plastic and solvent and is more pronounced with water than with plasma. As the DNA binding effect of 8-MOP is dose-dependent, ECP starting doses should be adjusted to ensure that a sufficient concentration of free bioavailable 8-MOP is present during UV irradiation.

PLOS ONE 17(1), e0262110 (2022). doi:10.1371/journal.pone.0262110, Published by PLOS, San Francisco, California, US

R script to analyze blood donors blood counts.

R script to analyze blood donors blood counts.

Objectives This validation study investigated a flow cytometric apoptosis assay according to good manufacturing practice (GMP). Background Extracorporeal photopheresis (ECP) is a treatment for various immunological diseases and cutaneous T‐cell lymphomas. It is based on the induction of apoptosis by 8‐methoxypsoralene and ultraviolet A light. The quantification of apoptosis is therefore essential for ECP improvements. However, despite numerous publications on apoptosis, validated technical details are lacking. Methods and materials Mononuclear cells were collected by apheresis and treated by ECP or camptothecin. Samples taken before and after ECP were cultured for 24, 48 and 72 h and analysed for apoptosis and viability of T cells and monocytes by flow cytometry with Annexin V and 7‐AAD staining. Accuracy of the assay, intra‐ and inter‐assay precision and the pre‐analytical and analytical stability of the analytes were the investigated parameters. Results Our data indicate that the median intra‐ and inter‐assay precision coefficient of variation for T cells was 3.86% and 4.80%, respectively. Pre‐analytical stability of T cells and monocytes was ensured during short‐term storage for up to 2 h on ice. After staining, analytical stability was limited to 30 min, likely because of ongoing apoptosis and loss of monocytes due to plastic adhesion. Conclusion The results of this validation study show that the assay is GMP‐compliant and that its reliability, accuracy and precision are acceptable. While pre‐analytical stability of the cells was compatible with on‐site procedures, our analytical stability data indicate that this assay is not suited for batch mode analysis of ECP products.

Background Plateletpheresis using a leukocyte reduction system (LRS) traps donor WBCs in the LRS chamber, which may lead to lymphopenia, especially in frequent plateletpheresis donors. It seems plausible that this might cause adverse effects. However, current knowledge about potential confounders and donor health impacts is incomplete. Donors and methods Recent platelet donors and donations collected at University Hospital Regensburg from 2016 to 2019 using the Terumo BCT Trima Accel LRS system were retrospectively analyzed and compared with historical platelet donors and donations collected mainly with Fresenius Kabi Amicus non-LRS system from 2010 to 2013. Additionally, recent donors were prospectively surveyed using a health-related topics questionnaire. Results Analysis of 819 recent donors with 11,254 blood counts and 1464 questionnaires and 1011 historical donors with 12,848 blood counts revealed that increased annual platelet donation frequencies were associated with decreased lymphocyte counts in both groups. Median lymphocyte counts in recent donors with no versus ≥24 previous annual donations declined from 2.0 to 1.2 × 103/μL (p

Background Orthotopic liver transplantation (OLT) is the treatment option for early-stage hepatocellular carcinoma (HCC). OLT is often associated with high blood loss, requiring blood transfusion. Retransfusion of autologous blood is a key part of blood conservation. There are, however, concerns that the retransfusion of salvaged blood might cause the spread of cancer cells and induce metastasis. Irradiation of salvaged blood before retransfusion eliminates viable cancer cells. Here, we analyzed the incidence of tumor recurrence in patients with HCC undergoing OLT who received irradiated cell-salvaged blood during transplant surgery. Methods We retrospectively analyzed patients undergoing OLT for HCC between 2002 and 2018 at our center. We compared the tumour recurrence in patients who received no retransfusion of autologous blood with patients who received autologous blood with or without preceding irradiation of the blood. Results Fifty-one (40 male, 11 female) patients were included in the analysis; 10 patients developed tumor recurrence within a time period of 2.45 ± 2.0 years. Statistical analysis revealed that there was no significant difference in tumor recurrence between patients who received autologous blood with or without irradiation. Conclusion Intraoperative transfusion of cell-salvaged blood did not increase tumor recurrence rates. Cell salvage should be used in liver transplantation of HCC patients as part of a blood conservation strategy. The effect of blood irradiation on tumor recurrence could not be definitively evaluated.

Background:Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. Methods:The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered withClinicalTrials.gov,NCT01656135,NCT02252055,NCT02085629,NCT02244801,NCT02371434,NCT02129881, andNCT02091232. Findings:The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2–18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. Interpretation:Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression.

Purpose: Non–small cell lung cancer (NSCLC) is a fatal disease with poor prognosis. A membrane-bound form of Hsp70 (mHsp70) which is selectively expressed on high-risk tumors serves as a target for mHsp70-targeting natural killer (NK) cells. Patients with advanced mHsp70-positive NSCLC may therefore benefit from a therapeutic intervention involving mHsp70-targeting NK cells. The randomized phase II clinical trial (EudraCT2008-002130-30) explores tolerability and efficacy of ex vivo–activated NK cells in patients with NSCLC after radiochemotherapy (RCT). Patients and Methods: Patients with unresectable, mHsp70-positive NSCLC (stage IIIa/b) received 4 cycles of autologous NK cells activated ex vivo with TKD/IL2 [interventional arm (INT)] after RCT (60–70 Gy, platinum-based chemotherapy) or RCT alone [control arm (CTRL)]. The primary objective was progression-free survival (PFS), and secondary objectives were the assessment of quality of life (QoL, QLQ-LC13), toxicity, and immunobiological responses. Results: The NK-cell therapy after RCT was well tolerated, and no differences in QoL parameters between the two study arms were detected. Estimated 1-year probabilities for PFS were 67% [95% confidence interval (CI), 19%–90%] for the INT arm and 33% (95% CI, 5%–68%) for the CTRL arm (P = 0.36, 1-sided log-rank test). Clinical responses in the INT group were associated with an increase in the prevalence of activated NK cells in their peripheral blood. Conclusions: Ex vivo TKD/IL2-activated, autologous NK cells are well tolerated and deliver positive clinical responses in patients with advanced NSCLC after RCT.

BACKGROUND Extracorporeal photopheresis (ECP) is an immunosuppressive treatment that involves leukocyte apheresis, psoralen and UV light treatment, and subsequent reinfusion. Patients treated with ECP are usually immunosuppressed. Bacterial contamination therefore poses a much unwanted risk, but incidence data are lacking. PATIENTS AND METHODS We screened all 1922 consecutive ECP procedures scheduled within a roughly 3‐year period for eligibility. Those with missing data on ECP method (inline or offline) or type of venous access (peripheral or central) were excluded. ECPs with complete aerobic and anaerobic microbial testing of baseline patient blood samples (n = 1637) and of ECP cell concentrates (n = 1814) were included in the analysis. RESULTS A test for microbial contamination was positive for 1.82% of the cell concentrates, with central venous access was the most significant risk factor for the contamination (odds ratio = 19). Patient blood samples were positive in 3.85% of cases, but no patients became septic. Staphylococcus spp. were most abundant, and products with bacterial contamination did not cause side effects after reinfusion. There were no significant differences in contamination rates between inline and offline ECP. CONCLUSION These findings stress the importance of sterile procedures and the benefits of using peripheral over central venous access for reducing the risk of bacterial contamination in ECP.

Background To overcome the compromised wound healing in radiation induced chronic wounds platelet-rich plasma (PRP), as therapeutic agent, is current subject of studies. PRP is associated with pro-angiogenic effects. Nevertheless, effects of platelet-rich plasma in cutaneous wound healing processes are poorly understood so far. Methods In this study, the migration of endothelial cells, fibroblasts and keratinocytes in conjunction with platelet-rich plasma treatment is investigated in the context of radiation effects. Additionally, cell proliferation and viability after external radiation was analyzed regarding treatment by platelet-rich plasma. Results All cell cultures showed a trend towards decreasing proliferation and viability after irradiation irrespective of PRP. Upon PRP treatment, irradiated fibroblasts as well as endothelial cells showed an enhanced proliferation whereas proliferation and viability of keratinocytes was reduced after PRP treatment. Scratch assays support the positive effect of PRP on fibroblast and endothelial cell migration, whereas a negative effect on keratinocytes was observed after PRP treatment. Conclusions The present study documents both deleterious effects of external radiation as well as the protective effect of PRP. In summary, increased viability, proliferation and migration are indeed a consequence of the pro-proliferative effect exerted by PRP. Therefore, treatment with PRP products might be useful in the management of chronic radiogenic wounds.

BACKGROUND Granulocyte apheresis requires a sedimentation agent. Usually, hydroxyethyl starch (HES) is administered to donors for this purpose and, as granulocyte concentrate (GC) ingredient, also to patients. Authorities recently recommended suspending market authorizations for starch-containing products due to side effects. Therefore, we tested the efficacy of modified fluid gelatin (MFG, Gelafundin 4%) versus hetastarch (Hespan) for GC apheresis. STUDY DESIGN AND METHODS This retrospective matched-pair analysis compared MFG- and hetastarch-derived GCs. Each group consisted of 15 unrelated male donors mobilized with dexamethasone and granulocyte–colony-stimulating factor for apheresis on 1 or 2 days with the COBE Spectra's PMN program. RESULTS In each group, 24 GCs were collected from 15 male donors and analyzed. None of the HES-derived products, but two of the MFG-derived products (8.3%), had aggregates and could not be used. The HES-derived products had significantly higher neutrophil counts on the first day (7.7 × 1010/unit vs. 4.0 × 1010/unit; p = 0.00005) as well as second day of apheresis (4.0 × 1010/unit vs. 1.1 × 1010/unit; p = 0.0002). Median white blood cell collection efficacies were lower with MFG than with HES on Day 1 (24% vs. 43%) and Day 2 (15% vs. 37%). Twenty-one percent of the MFG-derived products had less than 1 × 1010 granulocytes. CONCLUSION These results indicate that granulocyte apheresis is feasible with MFG as well as with hetastarch and that the latter is superior for GC production, if used in the same dosage. In addition, aggregates in GC from the COBE Spectra were observed in the MFG group but not in the hetastarch group.

BACKGROUND Extracorporeal photopheresis (ECP) is a therapeutic technique that combines leukapheresis and ultraviolet (UV)A irradiation of the leukapheresate after 8-methoxypsoralen treatment with subsequent retransfusion. It can be achieved with a single device (online) or by combining an apheresis machine with a separate UVA light source (offline). The comparability of both established methods is unknown. STUDY DESIGN AND METHODS In a prospective setting, four ECP systems were evaluated: one with integrated UVA irradiation for online ECP (Therakos) and three with external UVA irradiation for offline ECP (Amicus, Optia, and Cobe Spectra). Apheresis variables and cell counts were determined by methods including flow cytometry. RESULTS The duration of apheresis ranged from 120 minutes (Amicus, Optia) to 275 minutes (Therakos). Mononuclear cell (MNC) counts in the treatment bags were comparable between offline ECP methods (median, 57 × 108 – 66 × 108) and lower for online ECP (14 × 108). CD16+ monocytes were abundant in online ECP (82%) but rarer in offline ECP (median, 14% – 19%). Hematocrit ranged from 0.1% (Therakos) to 8% (Amicus). There were no side effects in any patients. DISCUSSION All offline ECP systems studied yielded comparable cellular compositions and highly enriched populations of MNCs. In contrast, white blood cells from online ECP displayed enrichment of nonclassical monocytes. The relevance of these findings is unknown as there is no established biomarker to predict the therapeutic efficacy of these procedures.

Membrane-bound heat shock protein 70 (mHsp70) is indicative for high-risk tumors with negative prognosis but also serves as a target for NK cells that are stimulated with Hsp70 peptide TKD and low dose IL-2 (TKD/IL-2). Herein, the efficacy of ex vivo TKD/IL-2 activated, autologous NK cells was tested in a randomized, investigator initiated phase II clinical trial in patients with mHsp70 positive advanced stage NSCLC after radiochemotherapy (RCT, 60-70 Gy, platinum-based chemotherapy). The interventional (INT) group received 4 cycles TKD/IL-2 activated, autologous NK cells every 2-6 weeks subsequent to standard RCT and the control (CTRL) group received best supportive care. The primary objective of the study was to examine whether the adjuvant treatment of NSCLC patients with TKD/IL-2 activated NK cells is feasible and effective with respect to progression-free survival (PFS). Secondary objectives were the assessment of treatment and biological responses, toxicity, quality-of-life (QoL, QLQ-LC13). Eight patients were randomized into the INT and eight into the CTRL arm. None of the patients died between randomization and final tumor assessment 18 months after randomization. In the INT group one patient had complete response (CR), one patient partial response (PR), two patients stable disease (SD) and one patient progressive disease (PD) at the last documented visit, whereas in the CTRL group only 2 patients showed clinical responses (PR, SD) and five patients had PD. The clinical response of patients in the INT group appeared to be mediated by activated NK cells whereas in the CTRL group by CD8+ T cells. The NK cell therapy after RCT was well tolerated, no differences in QoL were observed between both study groups. Citation Format: Gabriele Multhoff, Stephanie E. Combs, Sophie Seier, Stefan Stangl, Wolfgang Sievert, Maxim Shevtsov, Christiane Blankenstein, Martin Hildebrandt, Konrad Kokowski, Matthias Hautmann, Hubert Hautmann, Claus Roedel, Rainer Fietkau, Rudolf M. Huber, Bernhard Haller, Christina Ertl, Michal Devecka, Robert Offner, Norbert Ahrens. Targeted Natural Killer (NK) cell based adoptive immunotherapy for the treatment of patients with non-small cell lung cancer (NSCLC) after radiochemotherapy - results of a randomized phase II clinical trial (NSCLC-TKD/IL-2) (Eudra-CT Number 2008-002130-30) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-076.

BACKGROUND Patients with neutropenia or granulocyte dysfunction may require granulocyte transfusions for adequate immune restoration. High-molecular-weight hydroxyethyl starch (HES) is the most commonly used sedimentation agent to enhance granulocyte collection efficiency. However, authorities recently restricted the use of HES due to its unfavorable risk-benefit profile. As modified fluid gelatin (MFG) is already used as an alternative sedimentation agent, we tested the hypothesis that MFG is not inferior to HES in terms of the functionality and viability of granulocytes. STUDY DESIGN AND METHODS Granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15%, or 30% MFG (Gelafundin) or HES (Hespan), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ROS) production, neutrophil extracellular trap formation (NETosis), antigen expression, and viability were subsequently investigated in vitro. RESULTS Relative to the controls, all three concentrations of HES compared to only 15% and 30% MFG lowered migration distances, and the 15% and 30% concentrations of both sedimentation agents reduced track straightness. HES resulted in lower CD11b expression and higher CD62L expression compared to MFG and the controls, whereas the differences for CD66b did not reach statistical significance. No significant differences in the timing of ROS production or NETosis, or in neutrophil viability or respiratory burst were observed. CONCLUSION These results indicate that MFG is not inferior to HES in terms of granulocyte function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with HES.

Recent introduction of all-oral direct-acting antiviral (DAA) treatment has revolutionized care of patients with chronic hepatitis C virus (HCV) infection. Regrettably, the high cost of DAA treatment is burdensome for healthcare systems and may be prohibitive for some patients who would otherwise benefit. Understanding how patient-related factors influence individual responses to DAA treatment may lead to more efficient prescribing. In this observational study, patients with chronic HCV infection were comprehensively monitored by flow cytometry to identify pretreatment immunological variables that predicted HCV RNA negativity within 4 weeks of commencing DAA treatment. Twenty-three patients [genotype 1a (n = 10), 1b (n = 9), and 3 (n = 4)] were treated with daclatasvir plus sofosbuvir (SOF) (n = 15), ledipasvir plus SOF (n = 4), or ritonavir-boosted paritaprevir, ombitasvir, and dasabuvir (n = 4). DAA treatment most prominently altered the distribution of CD8+ memory T cell subsets. Knowing only pretreatment frequencies of CD3+ and naive CD8+ T cells allowed correct classification of 83% of patients as “fast” (HCV RNA-negative by 4 weeks) or “slow” responders. In a prospective cohort, these parameters correctly classified 90% of patients. Slow responders exhibited higher frequencies of CD3+ T cells, CD8+ TEM cells, and CD5high CD27− CD57+ CD8+ chronically activated T cells, which is attributed to bystander hyperactivation of virus-non-specific CD8+ T cells. Taken together, non-specific, systemic CD8+ T cell activation predicted a longer time to viral clearance. This discovery allows pretreatment identification of individuals who may not require a full 12-week course of DAA therapy; in turn, this could lead to individualized prescribing and more efficient resource allocation.

BACKGROUND: Platelet-rich plasma (PRP) products are currently suggested in the treatment of chronic wounds due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of PRP on irradiated cells of the cutaneous wound healing process are still poorly understood. MATERIAL AND METHODS: Human dermal microvascular endothelial cells (HDMEC) and human adipose derived stem cells (hASC) were cultured and irradiated with doses of 2 to 12 Gy. PRP was activated, characterized and added to the incubation media in different concentrations after external radiation. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (bFGF) and soluble intercellular adhesion molecule-1 (sICAM-1) in the supernatants of HDMEC and hASC co-cultures were determined by enzyme-linked immunosorbent assay (ELISA). Non-irradiated hASC and HDMEC served as controls. RESULTS: The employed PRP preparations were characterized and contained platelet derived growth factor (PDGF-AB), vascular endothelial growth factor (VEGF), bFGF and high levels of sICAM-1. Addition of PRP to irradiated cultures of HDMEC and hASC prevented profound radiation-induced decline in cell numbers. 10% PRP restored cell numbers to levels of untreated, non-irradiated cultures. Basic FGF expression was decreased significantly in hASC monocultures treated with 10% PRP without external radiation and after irradiation with 6 and 12 Gy. These inhibitory effects of PRP were also observed in HDMEC. In contrast, co-cultures of HDMEC-ASC showed a dose-dependent increase in bFGF expression when treated with 5 or 10% PRP. Doses of 6 and 12 Gy increased IL-6 expression in cultures stimulated with 5% PRP. CONCLUSIONS: Use of PRP in co-cultures of hASC and HDMEC restores proliferative defects caused by external radiation probably by induction of bFGF. Under irradiated conditions, PRP might induce pro-inflammatory stimuli which could be beneficial in treatment of chronic wounds where healing processes are defective. Combined use of hASC and PRP products might be helpful in the treatment of radiogenic wounds.

International audience; Background. The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs.Methods. The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry.Results. DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE 2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14 + monocytes and was further enhanced by IFN-γ treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs. Conclusion. DHRS9 is a specific and stable marker of human Mregs.

Administering immunoregulatory cells as medicinal agents is a revolutionary approach to the treatment of immunologically mediated diseases. Isolating, propagating, and modifying cells before applying them to patients allows complementation of specific cellular functions, which opens astonishing new possibilities for gain-of-function antigen-specific treatments in autoimmunity, chronic inflammatory disorders, and transplantation. This critical review presents a systematic assessment of the potential clinical risks posed by cell-based immunotherapy, focusing on treatment of renal transplant recipients with regulatory macrophages as a concrete example.

Introduction Allogeneic haematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for sickle cell disease (SCD) offered to patients with a fully matched sibling (MSD) or matched unrelated donor (MUD). With a MSD/MUD donor availability of below 20% a haploidentical HSCT from a family donor either with post-transplant cyclophosphamide (post-cy) or with a TCRαß/CD19 depleted graft (T-haplo) is an increasingly successful alternative for the majority of patients. In contrast to post-cy where mostly bone marrow is used, T-haploHSCT requires the generation of a G-CSF stimulated peripheral stem cell grafts. Almost uniformly haploidentical relatives of SCD patients are heterozygous carriers of SC trait. For these donors with a genetically heterozygous sickle trait, G-SCF represents a major safety concern with regard to triggering a sickle cell crisis. Therefore, the use of sickle trait donors is prohibited or not recommended in several countries, and guidelines are currently not available due to lack of evidence. Methods Haploidentical related donors with heterozygousSCD (group S, n=13) as well as healthy donors (group O, n=9) were stimulated with G-CSF. In all donors a similar mobilizations schedule using G-CSF was applied. All donors were applied 10µg/kg body weight (Neupogen®or Granocyte®) sub cutaneous daily with first apheresis on day 5 and day 6 when necessary. The amount of mobilized CD34+ cells were assessed after mobilization. In addition, the harvested stem cell preparations were analysed for CD34 content after apheresis and patients monitored for potential serious adverse effects (AE) during and after the apheresis. Results Mobilization was tolerated well with only mild and typical AE related to apheresis procedure such as citrate reactions, joint pain, ostealgia and headache, observed equally in both groups. In most of the donors (n=13/13 and 8/9), we were able to collect a sufficient amount of CD34+peripheral stem cells (mean of 16.3 and 8.2 x 10^6/kg in group S and O, respectively). We observed slight increases in LDH and reticulocyte counts in few donors also without significant differences between both groups. In particular, there were no severe neurologic side effects and no problems like sickle cell crisis despite mean pre-apheresis hemoglobin concentrations of 14.1 (range 11.9-17.5) g/dL in group S. Conclusions Collectively, these data indicate that in the absence of a MSD or MUD donor, haploidentical family donors with heterozygoussickle cell trait provide a safe and effective alternative for collection of peripheral CD34+stem cells for a T-haplo HSCT. Disclosures No relevant conflicts of interest to declare.

Background: Extracorporeal photopheresis (ECP) is an efficient method to treat various autoimmune diseases, cutaneous T-cell lymphoma, and graft-versus-host disease. It is based on the ex vivo inactivation of lymphocytes by 8-methoxypsoralen (8-MOP)/UV light treatment. Despite the adhesive, lipophilic nature of 8-MOP, no quality control is established for the ECP procedure. Methods: We developed a sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to monitor residual 8-MOP concentration after UVA irradiation in the whole blood supernatant after acetonitrile precipitation. Results: The preanalytical stability of 8-MOP exceeded 7 days, allowing batch mode analysis. Linearity was determined with R2 above 0.99. The 8-MOP concentrations decreased exponentially after UV exposure, with decay constants of 0.0259 in plasma and 0.0528 in saline. The recovery of 8-MOP in photopheresates was about 68%, indicating binding to DNA as well as to plastic structures. UVA induced no 8-MOP fragmentation, but caused self-adducts under extreme conditions (10-fold UV dosage). Conclusions: Detection of 8-MOP proved to be feasible and demonstrated that the doses were in the pharmaceutically active range.

ABO blood group antigen incompatibility (ABO mismatch) is not an obstacle to allogeneic stem cell transplantation (allo-SCT). However, the impact on clinical outcome after allo-SCT remains controversial. We analyzed 512 patients after allogeneic peripheral blood SCT (allo-PBSCT) for an association of ABO mismatch with transfusion requirements, myeloid and platelet engraftment, the incidence of GvHD, relapse, transplant-related mortality (TRM), and overall survival (OS). A total of 260 patients underwent ABO-mismatched transplantation and the control group consisted of 252 patients with ABO-matched allo-PBSCT. We found a significant association between major-0 ABO mismatch (group 0 recipient/group A, B, or AB donor) and increased red blood cell (RBC) and platelet transfusion requirements (both P

Extracorporeal photopheresis (ECP) is a therapeutic technique that combines leukapheresis and ultraviolet (UV)A irradiation of the leukapheresate after 8-methoxypsoralen treatment with subsequent retransfusion. It can be achieved with a single device (online) or by combining an apheresis machine with a separate UVA light source (offline). The comparability of both established methods is unknown.In a prospective setting, four ECP systems were evaluated: one with integrated UVA irradiation for online ECP (Therakos) and three with external UVA irradiation for offline ECP (Amicus, Optia, and Cobe Spectra). Apheresis variables and cell counts were determined by methods including flow cytometry.The duration of apheresis ranged from 120 minutes (Amicus, Optia) to 275 minutes (Therakos). Mononuclear cell (MNC) counts in the treatment bags were comparable between offline ECP methods (median, 57 × 10All offline ECP systems studied yielded comparable cellular compositions and highly enriched populations of MNCs. In contrast, white blood cells from online ECP displayed enrichment of nonclassical monocytes. The relevance of these findings is unknown as there is no established biomarker to predict the therapeutic efficacy of these procedures.

BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC-EVs), and plasma. STUDY DESIGN AND METHODS: Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC-EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC-EV protein expression. RESULTS: During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC-EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine-1-phosphate (−53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC-EVs were enriched in lysophosphatidic acid (60-fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A-I, E, and J/clusterin. CONCLUSION: This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC-EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease.

The phenotypic diversity of familial hypercholesterolemia (FH) and lipoprotein(a) hyperlipidemia (Lp(a)-HLP), as defined risks for coronary artery disease with genetic background, and their frequent co-incidence with additional cardiovascular risk factors require a critical revisiting of the current diagnostic and screening criteria as well as therapeutic recommendations established for FH or isolated Lp(a)-HLP, since there is no clear guidance for patient stratification and disease management for combined cases. Further evaluation of the recent biomarkers and establishment of novel biomarkers are necessary for extended risk assessment of cardiovascular disease in FH or Lp(a)-HLP and to better understand the pathophysiology of these syndrome complexes. Lipoprotein apheresis is used as long-term treatment to reduce circulating lipoproteins in patients with severe FH and/or Lp(a)-HLP, particularly with multiple cardiovascular risks who are intolerant or insufficiently responsive to lipid-lowering drugs. Recent sophisticated analyses of molecular lipid species (lipidome) extended with transcriptomic and/or proteomic approaches may provide further lipid biomarkers for disease management of FH and/or Lp(a)-HLP, and relevant data for optimization of apheresis treatment. This review summarizes current studies investigating FH and Lp(a)-HLP as independent and combined cardiovascular risk factors, and some promising biomarker candidates for these entities.

BACKGROUND: Blood samples were referred over a 10-year period from five patients whose serum samples contained antibodies to unidentified high-incidence antigens. Three patients (A, B, C) were of Moroccan origin and their antibodies and red blood cells (RBCs) were mutually compatible, but incompatible with those of the other two patients (D, E), who were of Pakistani origin. The antibodies and RBCs of D and E were mutually compatible, but incompatible with those of Patients A, B, and C. All the antibodies were detected during pregnancy. STUDY DESIGN AND METHODS: Serologic tests, including the use of enzyme-treated and chemically modified RBCs, suggested a relationship to CD44 (Indian blood group system). The monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay with monoclonal CD44 antibodies, immunoblotting of RBC membranes, and CD44 gene sequencing were carried out. RESULTS: Positive reactions in the MAIEA assay confirmed that the patients' antibodies are directed at CD44. Immunoblotting with two of the antibodies gave positive reactions of identical size to monoclonal anti-CD44 and failed to react with the RBCs of a CD44-deficient patient. One of the antibodies reacted with purified CD44. Sequencing of Exons 1 to 5 of CD44 revealed 255C>G in Exon 3 for A, B, and C encoding H85Q and 488C>A in Exon 5 for D and E encoding T163R. CONCLUSION: Two novel CD44 antigens of high incidence have been identified: IN3 (INFI) and IN4 (INJA) in the IN (Indian) blood group system. Lack of IN3 and IN4 results from homozygosity for mutations encoding H85Q and T163R in CD44, respectively.

BACKGROUND: Besides ABO and RH, the KEL blood group system, including the two antithetical antigens KEL1 and KEL2, is the most important owing to the frequent appearance of anti-KEL alloantibodies and their considerable clinical significance. So far, only limited information was available on KEL variant alleles determining the rare silent KELnull and KELel phenotypes with absent or diminished KEL antigen expression detected only by adsorption-elution techniques, respectively. STUDY DESIGN AND METHODS: For a systematic investigation of the KELnull and KELel phenotypes, 401 KEL:1,-2 samples (representing 2.6% of all Austrian KEL:1,-2 samples) and 811 KEL:1,2 samples were genotyped for the KEL*1/KEL*2-specific single-nucleotide polymorphism. All heterozygous KEL*1/KEL*2 and 4 additional KELnull samples were subjected to detailed immunohematologic examination and allele-specific sequencing. RESULTS: In 14 KEL:1,-2 samples, discrepant KEL*1/KEL*2 heterozygosity was observed, indicating the presence of silent or barely expressed KEL*2 alleles, whereas all KEL:1,2 individuals were homozygous for KEL*2. In the course of further molecular analysis, 8 novel KEL*2null and 2 KEL*2el alleles were discovered, representing 67 and 33 percent of previously known KEL*2null- and KEL*2el-encoding alleles, respectively. In addition, two different known KEL*2null and KEL*2el alleles each were confirmed. The immunohematologic properties of KEL variant red blood cells were defined by extended KEL phenotyping and flow cytometric KEL1, KEL2, KEL4, and KEL7 antigen as well as total Kell protein quantification. CONCLUSION: For the first time, exact KELnull and KELel population frequencies could be established in this population.

Mesenchymal stem cells and multipotent adult progenitor cells (MAPCs) have been proposed as novel therapeutics for solid organ transplant recipients with the aim of reducing exposure to pharmacological immunosuppression and its side effects. In the present study, we describe the clinical course of the first patient of the phase I, dose-escalation safety and feasibility study, MiSOT-I (Mesenchymal Stem Cells in Solid Organ Transplantation Phase I). After receiving a living-related liver graft, the patient was given one intraportal injection and one intravenous infusion of third-party MAPC in a low-dose pharmacological immunosuppressive background. Cell administration was found to be technically feasible; importantly, we found no evidence of acute toxicity associated with MAPC infusions. Significance Liver transplantation is the only definitive treatment for liver failure. However, in order to prevent rejection of the graft, patients must receive lifelong pharmacological immunosuppression, which itself causes clinically significant side effects. This study provides preclinical evidence that mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) can prolong allogeneic solid organ transplant survival in animals by switching the host response toward operational tolerance. To examine the safety and feasibility of MAPC therapy in patients receiving a living-related or dead-before-donation unrelated donor liver graft, the MiSOT-I (Mesenchymal Stem Cells in Solid Organ Transplantation Phase I) trial was designed. The first study patient, a 27-year-old male with liver cirrhosis of unknown etiology, received a living-related adult right liver graft from his brother. MAPC administration in both the operating room (day 0) and intensive care unit (day 2) was feasible, and no evidence was seen of acute complications associated with the cell infusion. The absence of any acute clinical complications of cell infusion is reassuring for the future administration of MAPCs.

The horseradish peroxidase (HRP) catalyzed oxidative coupling of 2-hydrazono-4-thiazolines with α-naphthol using aqueous hdrogen peroxide. These transformations allow an environmentally benign synthesis of p-naphthoquinone-thiazol-2-on-azines under mild conditions.

Background and objectives. ABO-incompatible red blood cell (RBC) transfusions are a major risk in transfusion medicine. Identification of factors leading to this hazard is important to improve transfusion safety. Material and methods. All consecutive erroneous ABO-incompatible transfusions occurring from January 1997 to December 2004 at the Charite University Hospital in Berlin, Germany were analysed. Results. A total of 343,432 RBC units were transfused, and eight patients erroneously received 13 ABO-incompatible RBC concentrates. The most frequent error was incorrect bedside testing ( n =7). Intensive care treatment was required in two cases, but there were no fatal mistransfusions. Four patients had no or only mild reactions. Conclusion. Mistransfusions are still a considerable risk in transfusion medicine despite quality control systems and electronic data processing. An increase in transfusion safety may require the introduction of further systems, e.g. radio-frequency identification (RFID) tags.

BACKGROUND: Oxaliplatin, a third-generation platinum compound, has been implicated in isolated cases of immune hemolytic anemia and/or immune thrombocytopenia. The first case of severe immune pancytopenia related to oxaliplatin is described. PATIENT AND METHODS: A 79-year-old woman with colorectal cancer was initially treated with 5-fluorouracil and she later received oxaliplatin and leucovorin every 2 to 4 weeks. During the 15th and 17th cycles of chemotherapy she developed thrombocytopenia, hemolysis, and neutropenia. No problems occurred during the 16th cycle without oxaliplatin. Serologic testing including detection of drug-dependent antibodies and autoantibodies was performed with standard techniques. RESULTS: Serologic findings included a positive immunoglobulin G direct antiglobulin test; nonreactive red blood cell (RBC) eluates; platelet (PLT)-bound antibodies to glycophorin (GP) IIb-IIIa, GPIb-IX, and GPIa-IIa; and oxaliplatin-dependent antibodies to PLTs, RBCs, and neutrophils. CONCLUSION: Oxaliplatin may lead to the production of ddabs to RBCs, PLTs, and neutrophils. Thus the risk of immune cytopenias should always be considered in patients treated with oxaliplatin.

Background and Objectives Administration of diclofenac may lead to immune haemolytic anaemia (IHA) owing to the presence of drug-dependent antibodies and/or autoantibodies. A relationship with oral or intramuscular drug administration is unknown. Here, we describe a patient who apparently tolerated oral diclofenac but developed severe IHA following intramuscular injection of the drug. Patients and Methods A 66-year-old-female was admitted to hospital because of jaundice and nausea, which were initially presumed to be manifestations of a postcholecystectomy syndrome. The patient soon developed haemolysis and renal failure. Although the symptoms and signs were suggestive of autoimmune haemolytic anaemia (AIHA), the patient had diclofenac-induced IHA. Results Serological testing, including detection of drug-dependent antibodies, was performed using standard techniques. The patient's serum was found to contain a highly reactive diclofenac-dependent red cell antibody of the immune complex type (titre 256 000). She recovered after 7 weeks of treatment with prednisolone, blood transfusions, haemodialysis and plasma exchange. Conclusions Diclofenac-induced IHA should always be considered when a patient on diclofenac develops haemolysis.

BACKGROUND : Autologous peripheral blood progenitor cells (PBPCs) are usually collected after the administration of conventional-dose chemotherapy (CDCT) and growth factors. However, there are no data available concerning the collection of PBPCs after high-dose chemotherapy (HDCT) and autologous hematopoietic transplantation in a larger series. STUDY DESIGN AND METHODS : Patients (n = 30) underwent leukapheresis for PBPC harvest after CDCT. After HDCT and autografting, the collection of a second PBPC autograft was attempted. RESULTS : Leukapheresis was performed after CDCT in all cases at a median of 118 CD34+ cells per μL (range, 18-589) and resulted in 6.4 × 10 6 CD34+ cells per kg (range, 1.7-29.0). After HDCT and autografting, 24 patients (80%) underwent secondary leukapheresis, although they had a significantly lower median of peripheral blood (PB) CD34+ cells (30/μL; range, 10-171; p < 0.001). In these patients a median of 3.6 × 10 6 CD34+ cells per kg (range, 1.6-10.1) was collected in the post-transplantation course. In the remaining six patients (20%) with PB CD34+ cells < 10 per μL, no PBPC harvesting was performed. These so-called poor mobilizers had received significantly less CD34+ cells for autologous transplantation than patients with successful post-HDCT mobilization (median, 2.5 × 10 6 /kg [range, 1.7-3.0] vs. 6.5 × 10 6 /kg [range, 3.2-19.6]; p < 0.001). CONCLUSION : Collection of PBPCs is possible in most patients during the recovery phase of hematopoiesis after HDCT plus autografting, and the number of circulating PBPCs may be related to the CD34+ cell dose transfused by the preceding autograft.

Rituximab, a human-mouse chimeric monoclonal antibody against the CD20 antigen on B lymphocytes, is increasingly used for treatment of autoimmune diseases. We report on the outcome of rituximab therapy in 1 patient with decompensated idiopathic autoimmune hemolytic anemia (AIHA) and in 2 patients with severe immune thrombocytopenia (ITP) in combination with compensated AIHA (Evans’ syndrome). All 3 patients were refractory to conventional therapies and received rituximab in standard doses. While the drug led to a partial response in the patient with AIHA for roughly 8 months, it was largely ineffective in the 2 patients with Evans’ syndrome. Our results and those of all hitherto reported cases indicate that rituximab therapy is most effective in children with AIHA. It is also effective in 83% of adult patients with cold agglutinin disease (CAD), secondary AIHA and/or ITP, but in only 46% of adults with idiopathic AIHA and/or ITP.