Autologous Blood Doping Induced Changes in Red Blood Cell Rheologic Parameters, RBC Age Distribution, and Performance.

Autologous blood doping (ABD) refers to the transfusion of one's own blood after it has been stored. Although its application is prohibited in sports, it is assumed that ABD is applied by a variety of athletes because of its benefits on exercise performance and the fact that it is not detectable so far. Therefore, this study aims at identifying changes in hematological and hemorheological parameters during the whole course of ABD procedure and to relate those changes to exercise performance. Eight healthy men conducted a 31-week ABD protocol including two blood donations and the transfusion of their own stored RBC volume corresponding to 7.7% of total blood volume. Longitudinal blood and rheological parameter measurements and analyses of RBC membrane proteins and electrolyte levels were performed. Thereby, responses of RBC sub-populations-young to old RBC-were detected. Finally, exercise tests were carried out before and after transfusion. Results indicate a higher percentage of young RBC, altered RBC deformability and electrolyte concentration due to ABD. In contrast, RBC membrane proteins remained unaffected. Running economy improved after blood transfusion. Thus, close analysis of RBC variables related to ABD detection seems feasible but should be verified in further more-detailed studies.

Transregional autologous serum eye drop provision by a large German Red Cross Blood Donation Service.

BACKGROUND: The Blood Donation Service West serves North Rhine-Westphalia (NRW), Rhineland-Palatinate (RP), and Saarland, an area of 56,500 km2. In addition to routine red blood cell concentrates, plasma, and platelets, special products are provided. Since 2014, this has included autologous serum eye drops (ASED) for topical use in patients suffering from different illnesses accompanied by dry eye disease. METHODS: A volume of 250-525 mL of patient blood was collected into an anticoagulant-free blood bag. Laboratory testing included Hepatitis B/C-, HIV 1/2-, and Lues-serology. Coagulation and centrifugation were followed by leukoreduction. Single-use vials were obtained by filling mini-bag systems using a sterile tube welder. Storage at ≤-20 °C enabled a shelf-life of up to 6 months and 30 days at 4 °C after thawing for shipment. RESULTS: Contracts were closed with 15 ophthalmology clinics and medical practices in NRW and RP to supply patients with ASED. The patient pool increased from 19 in 2014 to 46 in 2020, with an average age of 43-55 years. Overall, blood collections almost tripled from 31 to 100 per year, increasing the stock of deliverable single-use vials from 3328 to 13,358. Delivery in a liquid state allowed engagement of 44 pharmacies located in the patient neighborhoods for continuous supply. CONCLUSION: Manufacturing in a closed bag system allowed integration into blood bank operations. However, cost-coverage by health insurance remained a case-by-case decision. Allogeneic application as 'just-another-blood-product' could be an aspiration. Yet, conclusive data from large clinical trials are needed for licensed provision in Germany.

Natural Killer Cells for Therapy of Leukemia.

Clinical application of natural killer (NK) cells against leukemia is an area of intense investigation. In human leukocyte antigen-mismatched allogeneic hematopoietic stem cell transplantations (HSCT), alloreactive NK cells exert powerful anti-leukemic activity in preventing relapse in the absence of graft-versus-host disease, particularly in acute myeloid leukemia patients. Adoptive transfer of donor NK cells post-HSCT or in non-transplant scenarios may be superior to the currently widely used unmanipulated donor lymphocyte infusion. This concept could be further improved through transfusion of activated NK cells. Significant progress has been made in good manufacturing practice (GMP)-compliant large-scale production of stimulated effectors. However, inherent limitations remain. These include differing yields and compositions of the end-product due to donor variability and inefficient means for cryopreservation. Moreover, the impact of the various novel activation strategies on NK cell biology and in vivo behavior are barely understood. In contrast, reproduction of the third-party NK-92 drug from a cryostored GMP-compliant master cell bank is straightforward and efficient. Safety for the application of this highly cytotoxic cell line was demonstrated in first clinical trials. This novel 'off-the-shelf' product could become a treatment option for a broad patient population. For specific tumor targeting chimeric-antigen-receptor-engineered NK-92 cells have been designed.

Selection and expansion of natural killer cells for NK cell-based immunotherapy.

Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine.

NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy.

Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.

A phase I/II clinical trial of autologous cytokine-induced killer cells as adjuvant immunotherapy for acute and chronic myeloid leukemia in clinical remission.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells have shown remarkable cytotoxicity against various tumors in vitro and in animal studies. We report on the clinical outcome of autologous CIK cells for patients with acute (AML) and chronic (CML) myeloid leukemia in remission. METHODS: Eleven of the 13 recruited AML patients undergoing autologous peripheral blood stem cell transplant (autoPBSCT) were given autologous CIK cell infusion upon engraftment post-transplant and followed-up for disease relapse. Eleven CML patients on Imatinib with residual disease detectable by polymerase chain reaction (PCR) were given infusion and monitored by quantitation of the bcr-abl transcript. RESULTS: Despite the presence of interferon (IFN)-γ-secreting T cells against various AML- and CML-associated peptides at sporadic time-points and demonstration of in vitro cytotoxicity of CIK cells against autologous and allogeneic AML targets, there was no survival benefit in AML patients post-autoPBSCT given CIK cells compared with historical controls. For CML patients, all continued to have a detectable bcr-abl transcript fluctuating within a range comparable to their pre-treatment baseline, although two had a transient but non-sustainable disappearance of bcr-abl transcript. There were no adverse reactions except for fever within the first day of infusion. CONCLUSIONS: Our small series, while confirming safety, failed to demonstrate a clinical benefit of autologous CIK cells given in its current form for AML and CML. Further manipulation of CIK cells to improve anti-leukemic potency and specificity, together with the preparation of patients to create a more conducive milieu for in vivo expansion and persistence of infused CIK cells, should be explored.

Cell therapy: promise fulfilled?

Cellular immunotherapy has been widely accepted as a new powerful modality of cancer treatment. The last 2 decades have seen impressive results in its application against haemato-oncologic malignancies, melanomas and prostate carcinoma. Cellular immunotherapy has since found applicability beyond cancer into autoimmunity and continues to expand in its clinical applicability. The discovery that stem cells have the ability to differentiate into more mature cell types, like neurones and myocardium, has focused research on using exogenous cells to repair damaged tissues. This led to numerous clinical trials using stem cells in myocardial infarction, cardiomyopathy and spinal cord damage. Results have ranged from modest to significant clinical outcomes with continuing debate on the exact process of regeneration achieved. The intertwining between cell therapy and transfusion medicine now includes research on progenitor cells for the production of mature red cells. It is also clear that cell therapy has enabled an improved understanding of the pathogenesis and clinical course of many diseases, while perhaps its role in regenerative medicine is most enticing. However, the critical role of manufacturing in terms of cost, complexity, reproducibility, and regulatory matters remains a central issue in the consideration of whether cell therapy has met all of its promise.

Interleukin-15 supports generation of highly potent clinical-grade natural killer cells in long-term cultures for targeting hematological malignancies.

OBJECTIVE: Interleukin (IL)-15 is a promising novel cytokine for natural killer (NK) cell activation and survival. We studied the effects of IL-15 compared to IL-2 on NK cells in long-term cultures for clinical translation. MATERIALS AND METHODS: CD56(+)CD3(-) NK cells were expanded with IL-2 or IL-15 for 2 to 4 weeks within lymphokine-activated killer (LAK) cell cultures (LAK-NK) in serum-enriched AIM V or CellGro Stem Cell Growth Medium (SCGM). Cell growth, viability, and NK cell content were monitored and cytotoxicity assessed in a flow cytometric cytotoxicity assay. RESULTS: IL-15 (100-1000 U/mL) could replace IL-2 (1000 U/mL) in AIM V cultures to achieve efficient LAK cell expansion. However, IL-15-stimulated LAK cells exceeded cytotoxicity of IL-2-stimulated LAK cells against K562, notably at later culture points. In the powerful CellGro SCGM, LAK cells expanded over 28 days an average of 905-fold ± 320-fold standard error of the mean (SEM) for IL-2 (500 U/mL) and 484-fold ± 98-fold SEM for IL-15 (500 U/mL), and NK cells within such LAK cultures expanded an average of 2320-fold ± 975-fold SEM for IL-2 and 1084-fold ± 309-fold SEM for IL-15. Importantly, such IL-15-activated LAK-NK cells retained enhanced cytotoxicity at later culture points against K562 as well. IL-15-stimulated effectors were also highly cytotoxic against hematological targets MOLT-4 and KU812 and nontoxic against autologous nonmalignant cells. Interestingly, IL-15-LAK-NK cells showed overall significant upregulation of the main activating and inhibitory NK cell receptors after long-term cytokine stimulation. CONCLUSIONS: Our results demonstrate the potential for IL-15 to support large-scale expansion of clinical-grade LAK-NK effectors, which could retain enhanced longer-term potency and preserve activation receptors in therapy of hematological malignancies. Protocols are readily clinically translatable.

Clinical scale expansion of cytokine-induced killer cells is feasible from healthy donors and patients with acute and chronic myeloid leukemia at various stages of therapy.

OBJECTIVE: In our clinical studies involving cytokine-induced killer (CIK) cells for patients with hematological malignancies, starting cells came from a heterogeneous group of patients and donors. Here we study the feasibility of expansion and analyzed the characteristics of the end product from starting cells derived from different sources and at different disease states. MATERIALS AND METHODS: Seventy-five clinical scale cultures were grown from 28 patients and 20 donors in Good Manufacturing Practices facilities under CIK condition. RESULTS: CIK cells could be successfully expanded from healthy donors, patients with acute myeloid leukemia recovering from chemotherapy, untreated patients with acute myeloid leukemia or myelodysplastic syndrome with circulating leukemic blasts, and patients with chronic myeloid leukemia on imatinib. Furthermore, CIK cells of donor origin could be expanded from leukapheresis product collected from patients who relapsed post-allogeneic transplantation, thereby offering a useful method of obtaining activated donor cells in patients for whom further donor cells were unavailable. Interestingly, CIK cells cultured from patients with untreated acute myeloid leukemia and myelodysplastic syndrome had a significantly higher proportion of CD3(+)CD56(+) subset and higher fold expansion of CD3(+) cells as compared to other groups of patients or healthy donors. Multivariate analysis showed that fresh starting cells expanded better than frozen-thawed cells, while prior exposure to granulocyte colony-stimulating factor or imatinib before harvesting did not adversely affect CIK cell expansion. CONCLUSIONS: Clinical scale expansion of CIK cells is feasible from both healthy donors and leukemia patients at various stages of treatment. This robust system allows clinical translation using CIK cells as immunotherapy in various clinical settings.

KHYG-1 and NK-92 represent different subtypes of LFA-1-mediated NK cell adhesiveness.

Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.

Emerging natural killer cell immunotherapies: large-scale ex vivo production of highly potent anticancer effectors.

Natural killer (NK) cell therapies are emerging worldwide as promising anticancer treatments, exploiting the fast cytolytic action of NK effectors and their potentially broad applicability against a wide range of malignancies. Until recently, clinical protocols have mainly involved freshly isolated NK cells or short- term activated NK cells or lymphokine-activated killer (LAK) cells. However, overall effector numbers and their anticancer potencies remained restricted, which poses a limiting factor to clinical efficacy. Recent developments in the field aim to improve clinical trial designs by increasing effector to target cell ratios in vivo and by application of superior cytotoxic NK effectors. Large-scale production of clinical grade NK cells through long-term activation in ex vivo cultures are another novel means in achieving these goals. However, such procedures require compliance with the strict Good Manufacturing Practice (GMP) regulations to ensure quality and safety of the NK cell product. Although the overall number of new protocols still remains comparably low, some of the protocols are already translated into clinical use. Also striking is the diversity of the different protocols proposed. We highlight in this review the most recent developments in the NK cell field with a focus on long-term NK cell expansion. Critical issues relating to this novel and promising type of therapy are highlighted and discussed.

Novel approaches using natural killer cells in cancer therapy.

Strategies are emerging to apply natural killer (NK) cells as therapeutic agents against a broad range of malignancies. Novel clinical approaches aim to overcome limitations of original therapies, which have utilized lymphokine activated killer cells or systemic cytokine treatments. Remarkable results, including survival improvements and amelioration of graft versus host disease, were obtained with alloreactive NK cells in some cases. Other approaches in clinical evaluation include targeting heat-shock protein (Hsp) 70 expressing tumors with pre-stimulated autologous NK cells or the application of an NK cell line, NK-92, with enhanced cytolytic activity. Further mechanistic insights into NK cell cytotoxicity are a prelude to improved clinical cancer therapies.

Constitutively polarized granules prime KHYG-1 NK cells.

The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.

B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase.

Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.

KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.

OBJECTIVE: To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy. MATERIALS AND METHODS: NK/NK T-cell lines KHYG-1, NK-92, YT, and SNT-8 were compared with a novel flow cytometric cytotoxicity assay under different culture conditions. Transcription, expression, and phosphorylation studies were performed using polymerase chain reaction sequence-specific primers, reverse transcription polymerase chain reaction, immunoblotting, and flow cytometry. RESULTS: KHYG-1 is a highly cytotoxic cell line, exceeding the cytolytic capacity of the other cell lines against K562. KHYG-1 is also highly cytotoxic against the leukemia cell lines EM2, EM3, and HL60. The novel activation receptor NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1. This cell line most likely mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin, which is constitutively fully cleaved to the 60-kD form, in contrast to the other cell lines. CONCLUSION: KHYG-1 is a valuable model for the study of enhanced cytotoxicity by NK cells. In addition to the activation of NKp44, KHYG-1 may induce apoptosis of tumor cells by the newly described granzyme M/perforin pathway. Targeted modifications of effector molecules demonstrated in this model could generate NK cells with even greater killing ability that may be particularly attractive for clinical application. Moreover, our demonstration of greater cytotoxicity of KHYG-1 versus NK-92 cells, already in clinical trials, suggests a direct therapeutic role for KHYG-1.

Chemical compounds that target thiol-disulfide groups on mononuclear phagocytes inhibit immune mediated phagocytosis of red blood cells.

BACKGROUND: Patients having immune cytopenias produce antibodies that target hematopoietic cells resulting in their phagocytosis and intracellular destruction. Early reports suggested that phagocytosis could be inhibited by interfering with membrane thiol (SH) groups on phagocytes. Thus, whether chemical compounds that interact with SH or disulfide (SS) groups on mononuclear phagocytes can inhibit phagocytosis of antibody-coated cells was examined. STUDY DESIGN AND METHODS: A monocyte monolayer assay (MMA), which examines the in vitro monocyte-macrophage (Mphi) interaction with anti-Rh(D)-coated red cells (RBCs), was used to study the ability of different SH and SS chemicals to inhibit the Fc receptor-mediated phagocytosis of sensitized RBCs. The compounds examined included thimerosal, dithiothreitol (DTT), pentane-1-thiol, and two recently described SH and two SS chemicals that have been synthesized. RESULTS: All compounds were found to be able to inhibit phagocytosis to varying degrees correlating to the structure of the molecule. In general, those compounds that interact with free SH groups to inhibit phagocytosis were found better than SH-containing compounds that interact with SSs. Thimerosal and p-nitrophenyl methyl disulfide were the most effective compounds inhibiting phagocytosis. Both chemicals showed greater than 50 percent inhibition at concentrations as low as 10(-9) mol per L. DTT was the least effective compound tested. Only thimerosal showed significant toxicity, as determined by decreased cell viability and increased apoptosis, but only at concentrations of 10(-8) mol per L. The effect of chemical treatment was on attachment rather than on phagocytosis itself. Fcgamma receptor-independent endocytosis was not affected by the chemical treatment. CONCLUSION: These studies indicate that pharmacologic strategies that target SH groups on mononuclear phagocytes may have future efficacy for the treatment of immune cytopenias.

Epstein-Barr virus (EBV) SM protein induces and recruits cellular Sp110b to stabilize mRNAs and enhance EBV lytic gene expression.

Promyelocytic leukemia protein (PML) nuclear bodies or nuclear domain 10s (ND10s) are multiprotein nuclear structures implicated in transcriptional and posttranscriptional gene regulation that are disrupted during replication of many DNA viruses. Interferon increases the size and number of PML nuclear bodies and stimulates transcription of several genes encoding PML nuclear body proteins. Moreover, some PML nuclear body proteins colocalize at sites of viral DNA synthesis and transcription. In this study, the relationship between lytic Epstein-Barr virus (EBV) replication and Sp110b, a PML nuclear body protein, was investigated. Sp110b is shown to physically and functionally interact with the EBV protein SM. SM is expressed early in the EBV replicative cycle and posttranscriptionally increases the level of target EBV lytic transcripts. SM bound to Sp110b via two distinct sites in Sp110b in an RNA-independent manner. SM also specifically induced expression of Sp110b during lytic EBV replication and in several cell types. Exogenous expression of Sp110b synergistically enhanced SM-mediated accumulation of intronless and lytic viral transcripts. This synergistic effect was shown to be promoter independent, posttranscriptional, and the result of increased stabilization of target transcripts. Finally, inhibiting Sp110b expression decreased accumulation of an SM-responsive lytic EBV transcript in EBV-infected cells. These findings imply that SM induces Sp110b expression, binds to Sp110b, and utilizes the recruited Sp110b protein to increase the stability of lytic EBV transcripts, indicating that Sp110b is a component of the cellular machinery that EBV utilizes to enhance lytic EBV replication.