Feasibility of peripheral blood stem cell collection from sickle cell trait donors with an intensified G-CSF regimen.

OBJECTIVES: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCD and bone marrow from an HLA-matched sibling is currently the standard of care. Haploidentical HSCT from a family donor with a TCR αß/CD19 depleted graft (T-haplo) is an increasingly successful alternative, which requires the generation of G-CSF stimulated peripheral stem cell (PBSC) from haploidentical relatives. These sickle cell trait (SCT) donors reported to develop SCD-related complications in conditions of severe stress. METHODS: In this retrospective analysis, we compared the safety and efficacy of PBSC mobilization with a G-CSF intensified mobilization regimen in SCT donors with a conventional G-CSF mobilization regimen in healthy donors. RESULTS: The reported adverse events were similar during intensified G-CSF mobilization, apheresis, and shortly after stem cell apheresis in SCT and control donors. In SCT and control donors, we were able to mobilize high yields of CD34+ stem cells and the harvested CD34+ cell count was comparable with control donors. CONCLUSIONS: Peripheral stem cell mobilization using an intensified G-CSF regimen is safe, and well tolerated among SCT donors. SCT donors are a valid alternative for collection of peripheral CD34+ stem cells for T-cell-depleted haploidentical stem cell transplantation.

Human Infections with Borna Disease Virus 1 (BoDV-1) Primarily Lead to Severe Encephalitis: Further Evidence from the Seroepidemiological BoSOT Study in an Endemic Region in Southern Germany.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

IFN-γ-Based ELISpot as a New Tool to Detect Human Infections with Borna Disease Virus 1 (BoDV-1): A Pilot Study.

More than 40 human infections with the zoonotic Borna disease virus 1 (BoDV-1) have been reported to German health authorities from endemic regions in southern and eastern Germany. Diagnosis of a confirmed case is based on the detection of BoDV-1 RNA or BoDV-1 antigen. In parallel, serological assays such as ELISA, immunoblots, and indirect immunofluorescence are in use to detect the seroconversion of Borna virus-reactive IgG in serum or cerebrospinal fluid (CSF). As immunopathogenesis in BoDV-1 encephalitis appears to be driven by T cells, we addressed the question of whether an IFN-γ-based ELISpot may further corroborate the diagnosis. For three of seven BoDV-1-infected patients, peripheral blood mononuclear cells (PBMC) with sufficient quantity and viability were retrieved. For all three patients, counts in the range from 12 to 20 spot forming units (SFU) per 250,000 cells were detected upon the stimulation of PBMC with a peptide pool covering the nucleocapsid protein of BoDV-1. Additionally, individual patients had elevated SFU upon stimulation with a peptide pool covering X or phosphoprotein. Healthy blood donors (n = 30) and transplant recipients (n = 27) were used as a control and validation cohort, respectively. In this pilot study, the BoDV-1 ELISpot detected cellular immune responses in human patients with BoDV-1 infection. Its role as a helpful diagnostic tool needs further investigation in patients with BoDV-1 encephalitis.

Granulocyte concentrate splitting does not affect phenotype and function.

BACKGROUND: More granulocyte concentrates (GCs) could be produced for more patients from the same donor if apheresis bags were split and stored for longer periods of time. Hence, we tested the hypothesis that splitting and extension of storage of GCs do not impair granulocyte function or viability. STUDY DESIGN AND METHODS: Granulocyte apheresis concentrates were produced using modified fluid gelatin as a separation enhancer, split into two portions, and stored for 24 and 48 h. Granulocyte function, represented by cell migration, reactive oxygen species (ROS) production, and neutrophil extracellular trap formation (NETosis), was measured by live-cell imaging. ROS production, adhesive surface protein expression, and viability were measured by flow cytometry. RESULTS: Splitting had no effect on any of the tested parameters. After 24 h of storage, live-cell imaging showed no significant difference in migration, time to maximum ROS production, time to half-maximum NETosis, viability, or CD11b expression, but ROS production induced by phorbol 12-myristate 13-acetate (PMA) decreased from an initial median fluorescence intensity of 1775-590 artificial units. After 48 h, PMA-induced ROS production, viability, and migration declined, as reflected by decreases in median total distance (119 vs. 63.5 µm) and median Euclidean distance (30.75 vs. 14.3 µm). CONCLUSION: Splitting GC products has no effect on granulocyte viability or function, but extended storage >24 h does compromise granulocyte function. The findings confirm that GCs should be transfused within 24 h of collection. Longer storage cannot be recommended.

Multistep screening and selection of COVID-19 convalescent plasma donors at the early stage of the SARS-CoV-2 pandemic: A retrospective analysis.

Background and Aims: The COVID-19 pandemic reached Bavaria in February 2020. Almost simultaneously, Chinese physicians published reports on the first successful treatments with plasma from COVID-19 convalescent donors. With these silver linings on the horizon, we decided to establish the manufacturing of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody-containing plasma from COVID-19 convalescent donors at our site. Here we describe our donor selection process, built from the ground up, which enabled us to cope with the immense resonance after our social media call for donors. Methods: As a first step, we created a specific questionnaire for telephone interviews applied by trained students to filter the wave of callers interested in plasma donation. Afterward, the medical staff evaluated the hotline questionnaires and chose eligible donors to be invited for on-site donor evaluation. Data documentation was performed with MS Excel, and statistical analyses were calculated with GraphPad Prism 8. A quantitative in-house ELISA was used to detect anti-SARS-CoV-2 antibodies and determine specific titers. Results: Out of 1465 calls from potential plasma donors, we could register 420 persons with a completed questionnaire. Evaluation of questionnaires identified 222 of 420 persons as eligible for donation, and 55 were directly asked for on-site donor qualification. Subsequently, as anti-SARS-CoV-2 antibody titers ≥1:800 were required, we invited 89 of 222 potential donors for an antibody screening. This procedure resulted in another 28 potential donors for an on-site evaluation. Finally, 12 donors qualified with a titer of 1:400 and 24 with ≥1:800. Conclusion: Identifying suitable COVID-19 convalescent plasma donors was expected to be highly time-consuming. Implementing a screening procedure with our hotline questionnaire helped us streamline the donor selection process and reduce the workload for the staff. We propose combining the described selection process with the later introduced on-site antibody screening as an effective strategy.

Apheresis for chimeric antigen receptor T-cell production in adult lymphoma patients.

BACKGROUND: To date, in-depth analysis of leukapheresis products as starting material for CAR T-cell manufacturing, specifically Tisagenlecleucel production, are scarce. In this study, we report on lymphapheresis data for production of Tisagenlecleucel for elderly and pretreated lymphoma patients. STUDY DESIGN AND METHODS: Spectra Optia from Terumo BCT, Lakewood, CO, was employed for apheresis using the cMNC program. Apheresis success was defined as meeting a target total nucleated cell (TNC) count of ≥2 × 109 , a CD3-positive lymphocyte count of ≥1 × 109 and an overall viability of ≥70% in the lymphapheresis product. RESULTS: Twenty-three patients (age 37-77 years) and 24 apheresis runs were evaluated. The median CD3-positive lymphocyte count in peripheral blood at the beginning of apheresis was 565 cells/µl (range: 70-1345 cells/µl). Circulating lymphoma cells were detected in one patient prior to apheresis. Target criteria were met in 21 of 23 patients. The median TNC count in the apheresate was 11.2 × 109 (range: 2.9 × 109 -47.4 × 109 ). The median CD3-positive lymphocyte count in the apheresate was 2.55 × 109 (range: 0.370 × 109 -6.915 × 109 ), which resulted in a median collection efficiency for CD3-positive lymphocytes of 63.7% (range: 9.56%-93.6%). No adverse events associated with the apheresis process were observed. CONCLUSIONS: Lymphapheresis with the Spectra Optia cMNC program provided a sufficient quantity of CD3-positive lymphocytes for CAR T-cell manufacturing for the majority of patients despite their heavy pretreatment and advanced age. Moreover, we are the first to advocate early pre-emptive lymphocyte collection in DLBCL-NOS patients intended to undergo treatment with Tisagenlecleucel.

Emergency Use of Convalescent Plasma: Perception of the Regulatory Framework from a Clinical Perspective.

The pandemic spread of an infectious disease poses a plethora of challenges to society, clinicians, health care providers and regulating authorities. In order to mount a rapid response and to provide hope in a potentially catastrophic situation as the current COVID-19 pandemic, emergency plans, regulations and funding strategies have to be developed on regional, national and international levels. The speed needed to establish rapid response programs is challenged by the dynamics of the spread of the disease, the concurrent and competing development of different and potentially more effective treatment options, and not the least by regulatory uncertainty. Convalescent plasma, that is plasma collected from patients who have recovered from COVID-19 infections, has emerged as one of the first potential treatment options in the absence of drugs or vaccines with proven efficacy against SARS-CoV-2. The societal aspects of convalescent plasma and the public awareness gave an additional boost to the rapid employment of convalescent plasma donation platforms immediately after the SARS-CoV-2 outbreak. At the same time, uncertainty remains as to the efficacy of convalescent plasma. With evidence mostly limited to empirical reports, convalescent plasma has been used for decades for the prophylaxis and treatment of various infectious diseases. Clinical trials have addressed different infectious agents, stages of disease, target groups of patients and yielded sometimes inconclusive results. The aim of this short review is to delineate the regulatory background for the emergency use of convalescent plasma in the USA, in the European Union and in Germany, and the transition to the setting of clinical trials. In addition, we describe observations made in the process of collecting COVID-19 convalescent plasma (herein referred to as CCP), and formulate proposals to further improve the framework for rapid responses in future emergency situations.

Triple apheresis platelet concentrate quality after pneumatic tube system, conveyor box, and courier transport: An observational study.

Background and Aims: Platelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function. Methods: Fourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high-frequency acceleration data logger. Shock index scores were calculated as shock intensity (g-force) times frequency. Results: The shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor-activating peptide-6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes. Conclusion: Though the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.

Granulocyte transfusions made with modified fluid gelatin in pediatric and adolescent patients with prolonged neutropenia.

BACKGROUND: Granulocyte transfusions (GT) are used to treat progressive systemic or local infections in prolonged neutropenic patients with antibiotic or antifungal resistance. Granulocytes are most commonly collected from whole blood by apheresis using hydroxyethyl starch (HES) as the red blood cell (RBC) sedimentation agent. This is the first study on the safety and efficacy of transfusing granulocytes collected with modified fluid gelatin (MFG) instead of HES to pediatric patients. METHODS: Clinical data from 46 pediatric and adolescent patients receiving at least one MFG-based granulocyte transfusion and in total 295 granulocyte concentrates from July 2013 to August 2019 at our local university medical center were evaluated retrospectively. RESULTS: Forty-one patients (89%) survived at least 21 days after their last granulocyte transfusion. These survivors had lower CRP values and higher leukocyte counts after GT than non-survivors (mean delta of -5.34 mg/dl vs. -11.99 mg/dl and + 0.62 × 103 /µl vs. +0.18 × 103 /µl of all GT, respectively). The neutrophil corrected count increment (CCI) was 68.72 mm2 /ml in survivors versus 28.00 mm2 /ml in non-survivors. There were no major or severe adverse events. CONCLUSION: This study suggests that modified fluid gelatin is a safe and effective alternative to hydroxyethyl starch for the collection of granulocytes for transfusion to prolonged neutropenic patients with progressive systemic or local infections refractory to antibiotic or antifungal therapy.

Total platelet donation count and donation frequency are determinants of plateletpheresis-associated lymphopenia.

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 < 2.2 × 10-16 ), and those in historical donors with no versus ≥24 previous annual donations decreased from 2.0 to 1.5 × 103 /µL (p = 6 × 10-4 ), respectively. The questionnaire results showed that donation frequency and lymphopenia were not associated with upper respiratory tract infection (URTI) incidence or duration, but platelet donors who concomitantly donated granulocytes had significantly shorter URTI durations than those who did not (p = .008). CONCLUSION: This study confirmed that plateletpheresis-associated lymphopenia occurs in LRS and to a lesser degree in non-LRS platelet donors, but revealed no evidence of a negative impact on donor health.

Apoptosis induction by extracorporeal photopheresis is enhanced by increasing the 8-methoxypsoralen concentration and by replacing plasma with saline.

BACKGROUND: Extracorporeal photopheresis (ECP), an apheresis-based therapy for various immunological diseases, works mainly by inducing apoptosis in lymphocytes. Several factors influence the efficacy of ECP with the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA). This study aimed to optimize treatment by varying the 8-MOP starting concentration and the cell suspension medium. MATERIALS AND METHODS: All patients (n = 13) included in this study received photopheresis as medically indicated. Cells collected with a Spectra Optia apheresis system were suspended in plasma or physiological saline (NaCl) and incubated with 200 ng/ml versus 340 ng/ml photosensitizer before UVA irradiation (Macogenic G2 or UVA PIT system). Lymphocyte apoptosis and caspase activity were analyzed by flow cytometry and fluorimetry, and residual 8-methoxypsoralen concentrations by liquid chromatography-mass spectrometry. RESULTS: Raising the 8-MOP starting concentration significantly increased lymphocyte apoptosis, with values of 22% versus 35% (plasma) and 28%-46% (NaCl) at 24 h post-ECP and 37% versus 86% (plasma) and 74% versus 97% (NaCl) at 48 h for 200 ng/ml versus 340 ng/ml. Pre-transfusion residual 8-MOP levels were 168 ng/ml (plasma) and 162 ng/ml (NaCl) versus 290 ng/ml (plasma) and 266 ng/ml (NaCl) for the lower versus higher dose, respectively. DISCUSSION: Hence, 8-MOP concentration influences the efficacy of photopheresis as lymphocyte apoptosis rates were significantly higher with the higher starting concentration and with NaCl versus plasma. This indicates that increased 8-MOP starting doses and saline as additional suspension medium could help in improving ECP's efficacy.

ECMO in COVID-19-prolonged therapy needed? A retrospective analysis of outcome and prognostic factors.

BACKGROUND: The role of venovenous extracorporeal membrane oxygenation (VV ECMO) in patients with COVID-19-induced acute respiratory distress syndrome (ARDS) still remains unclear. Our aim was to investigate the clinical course and outcome of those patients and to identify factors associated with the need for prolonged ECMO therapy. METHODS: A retrospective single-center study on patients with VV ECMO for COVID-19-associated ARDS was performed. Baseline characteristics, ventilatory and ECMO parameters, and laboratory and virological results were evaluated over time. Six months follow-up was assessed. RESULTS: Eleven of 16 patients (68.8%) survived to 6 months follow-up with four patients requiring short-term (<28 days) and seven requiring prolonged (⩾28 days) ECMO support. Lung compliance before ECMO was higher in the prolonged than in the short-term group (28.1 (28.8-32.1) ml/cmH2O vs 18.7 (17.7-25.0) ml/cmH2O, p = 0.030). Mechanical ventilation before ECMO was longer (19 (16-23) days vs 5 (5-9) days, p = 0.002) and SOFA score was higher (12.0 (10.5-17.0) vs 10.0 (9.0-10.0), p = 0.002) in non-survivors compared to survivors. Low viral load during the first days on ECMO tended to indicate worse outcomes. Seroconversion against SARS-CoV-2 occurred in all patients, but did not affect outcome. CONCLUSIONS: VV ECMO support for COVID-19-induced ARDS is justified if initiated early and at an experienced ECMO center. Prolonged ECMO therapy might be required in those patients. Although no relevant predictive factors for the duration of ECMO support were found, the decision to stop therapy should not be made dependent of the length of ECMO treatment.

Secondary hemophagocytic lymphohistiocytosis and severe liver injury induced by hepatic SARS-CoV-2 infection unmasking Wilson's disease: Balancing immunosuppression.

A 21-year-old woman was hospitalized due to coronavirus disease 2019 (COVID-19)-associated respiratory and hepatic impairment concomitant with severe hemolytic anemia. Upon diagnosis of secondary hemophagocytic lymphohistiocytosis, immunosuppression with anakinra and steroids was started, leading to a hepatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and viremia. Subsequent liver biopsy revealed virus particles in hepatocytes by electron microscopy and SARS-CoV-2 virus could be isolated and cultured. Immunosuppression was stopped and convalescent donor plasma given. In the differential diagnosis, an acute crisis of Wilson's disease was raised by laboratory and genetic testing. This case highlights the complexity of balancing immunosuppression to control hyperinflammation versus systemic SARS-CoV-2 dissemination.

A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization.

OBJECTIVE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. METHODS: In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. RESULTS: The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. CONCLUSIONS: With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.

Manufacturing of convalescent plasma of COVID-19 patients: Aspects of quality.

The ongoing coronavirus disease 2019 (COVID-19) pandemic emerged in December 2019. Convalescent plasma represents a promising COVID-19 treatment. Here, we report on the manufacturing of a plasma-based product containing antibodies specific to SARS-CoV-2 obtained from recently recovered COVID-19 patients. Convalescent plasma donors were screened as follows: 1) previously confirmed SARS-CoV-2 infection (by real-time PCR (RT-PCR)); 2) a subsequent negative PCR test followed by a 2-week waiting period; 3) an additional negative PCR test prior to plasmapheresis; and 4) confirmation of the presence of SARS-CoV-2 specific antibodies. Convalescent plasma was stored fresh (2-6°C) for up to 5 days or frozen (-30°C) for long-term storage. Donor peripheral blood and final plasma product were assayed for binding antibodies targeting the SARS-CoV-2 S-protein receptor-binding domain (RBD) and their titers measured by an enzyme-linked immunosorbent assay (ELISA). We performed 72 plasmaphereses resulting in 248 final products. Convalescent plasma contained an RBD-specific antibody titer (IgG) ranging from 1:100 to 1:3200 (median 1:800). The titer was congruent to the titer of the blood (n = 34) before collection (1:100-1:6400, median 1:800). Levels of IL-8 and LBP of donors were slightly increased. Therapeutic products derived from a human origin must undergo rigorous testing to ensure uniform quality and patient safety. Whilst previous publications recommended RBD-specific binding antibody titers of ≥ 1:320, we selected a minimum titer of 1:800 in order to maximize antibody delivery. Production of highly standardized convalescent plasma was safe, feasible and was readily implemented in the treatment of severely ill COVID-19 patients.

Targeted Natural Killer Cell-Based Adoptive Immunotherapy for the Treatment of Patients with NSCLC after Radiochemotherapy: A Randomized Phase II Clinical Trial.

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.

Granulocytapheresis with modified fluid gelatin versus high-molecular-weight hydroxyethyl starch: a matched-pair analysis.

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.

First-in-Human Case Study: Multipotent Adult Progenitor Cells for Immunomodulation After Liver Transplantation.

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.

Heat Shock Protein 70 (Hsp70) Peptide Activated Natural Killer (NK) Cells for the Treatment of Patients with Non-Small Cell Lung Cancer (NSCLC) after Radiochemotherapy (RCTx) - From Preclinical Studies to a Clinical Phase II Trial.

Heat shock protein 70 (Hsp70) is frequently overexpressed in tumor cells. An unusual cell surface localization could be demonstrated on a large variety of solid tumors including lung, colorectal, breast, squamous cell carcinomas of the head and neck, prostate and pancreatic carcinomas, glioblastomas, sarcomas and hematological malignancies, but not on corresponding normal tissues. A membrane (m)Hsp70-positive phenotype can be determined either directly on single cell suspensions of tumor biopsies by flow cytometry using cmHsp70.1 monoclonal antibody or indirectly in the serum of patients using a novel lipHsp70 ELISA. A mHsp70-positive tumor phenotype has been associated with highly aggressive tumors, causing invasion and metastases and resistance to cell death. However, natural killer (NK), but not T cells were found to kill mHsp70-positive tumor cells after activation with a naturally occurring Hsp70 peptide (TKD) plus low dose IL-2 (TKD/IL-2). Safety and tolerability of ex vivo TKD/IL-2 stimulated, autologous NK cells has been demonstrated in patients with metastasized colorectal and non-small cell lung cancer (NSCLC) in a phase I clinical trial. Based on promising clinical results of the previous study, a phase II randomized clinical study was initiated in 2014. The primary objective of this multicenter proof-of-concept trial is to examine whether an adjuvant treatment of NSCLC patients after platinum-based radiochemotherapy (RCTx) with TKD/IL-2 activated, autologous NK cells is clinically effective. As a mHsp70-positive tumor phenotype is associated with poor clinical outcome only mHsp70-positive tumor patients will be recruited into the trial. The primary endpoint of this study will be the comparison of the progression-free survival of patients treated with ex vivo activated NK cells compared to patients who were treated with RCTx alone. As secondary endpoints overall survival, toxicity, quality-of-life, and biological responses will be determined in both study groups.

McLeod phenotype without the McLeod syndrome.

BACKGROUND: McLeod neuroacanthocytosis syndrome is a late-onset X-linked multisystem disorder affecting the peripheral and central nervous systems, red blood cells (RBCs), and internal organs. A variety of mutations have been found in the responsible gene (XK) including single nonsense and missense mutations, nucleotide mutations at or near the splice junctions of introns of XK, and different deletion mutations. To date no clear phenotype-genotype correlation is apparent. The clinical details of one case of McLeod phenotype without apparent neuromuscular abnormalities have been reported. Here the clinical details of two additional cases are presented, of which the genetic details have previously been published. STUDY DESIGN AND METHODS: Two asymptomatic or minimally symptomatic cases at ages expected to manifest the McLeod syndrome (MLS) were evaluated. The first case had been authenticated as a genuine McLeod both by serology and by genotyping (R222G missense mutation) and the second case had a mutation in XK (IVS2+5G>A) and by serology exhibited very weak Kx antigen and no detectable Kell antigens, except extremely low k antigen by adsorption-elution technique. The patients were examined for hematologic, neurologic, and other clinical abnormalities. RESULTS: Despite documented McLeod phenotype on RBCs, and identified mutations of XK, neurologic and other clinical findings were minimal at ages expected to manifest MLS. CONCLUSIONS: The different XK mutations may have different effects upon the XK gene product and thus may account for the variable phenotype.