Acute hantavirus infection presenting as haemolytic-uraemic syndrome (HUS): the importance of early clinical diagnosis.

The European prototype of hantavirus, Puumala virus (PUUV), isolated from a common wild rodent, the bank vole (Myodes glareolus), causes nephropathia epidemica (NE). NE can perfectly mimic haemolytic-uraemic syndrome (HUS), progressing from an aspecific flu-like syndrome to acute kidney injury with thrombocytopaenia, and presenting with some signs of haemolytic anaemia and/or coagulopathy. Moreover, both NE and HUS can occur in local outbreaks. We report an isolated case of NE, initially referred for plasmapheresis for suspected HUS, although signs of overt haemolysis were lacking. Early suspicion of hantavirus infection, later confirmed by serology and reverse transcription polymerase chain reaction (RT-PCR), prevented subsequent excessive treatment modalities.

Characterization of Interleukin-15-Transpresenting Dendritic Cells for Clinical Use.

Personalized dendritic cell- (DC-) based vaccination has proven to be safe and effective as second-line therapy against various cancer types. In terms of overall survival, there is still room for improvement of DC-based therapies, including the development of more immunostimulatory DC vaccines. In this context, we redesigned our currently clinically used DC vaccine generation protocol to enable transpresentation of interleukin- (IL-) 15 to IL-15Rßγ-expressing cells aiming at boosting the antitumor immune response. In this study, we demonstrate that upon electroporation with both IL-15 and IL-15Rα-encoding messenger RNA, mature DC become highly positive for surface IL-15, without influencing the expression of prototypic mature DC markers and with preservation of their cytokine-producing capacity and their migratory profile. Functionally, we show that IL-15-transpresenting DC are equal if not better inducers of T-cell proliferation and are superior in tumor antigen-specific T-cell activation compared with DC without IL-15 conditioning. In view of the clinical use of DC vaccines, we evidence with a time- and cost-effective manner that clinical grade DC can be safely engineered to transpresent IL-15, hereby gaining the ability to transfer the immune-stimulating IL-15 signal towards antitumor immune effector cells.

Medical costs of treatment and survival of patients with acute myeloid leukemia in Belgium.

The advent of new cell-based immunotherapies for leukemia offers treatment possibilities for certain leukemia subgroups. The wider acceptability of these new technologies in clinical practice will depend on its impact on survival and costs. Due to the small patient groups who have received it, these aspects have remained understudied. This non-randomized single-center study evaluated medical costs and survival for acute myeloid leukemia between 2005 and 2010 in 50 patients: patients treated with induction and consolidation chemotherapy (ICT) alone; patients treated with ICT plus allogeneic hematopoietic stem cell transplantation (HCT), which is the current preferred post-remission therapy in patients with intermediate- and poor-risk AML with few co-morbidities, and patients treated with ICT plus immunotherapy using autologous dendritic cells (DC) engineered to express the Wilms' tumor protein (WT1). Total costs including post- consolidation costs on medical care at the hematology ward and outpatient clinic, pharmaceutical prescriptions, intensive care ward, laboratory tests and medical imaging were analyzed. Survival was markedly better in HCT and DC. HCT and DC were more costly than ICT. The median total costs for HCT and DC were similar. These results need to be confirmed to enable more thorough cost-effectiveness analyses, based on observations from multicenter, randomized clinical trials and preferably using quality-adjusted life-years as an outcome measure.

Dendritic cells in multiple sclerosis: key players in the immunopathogenesis, key players for new cellular immunotherapies?

Many studies have demonstrated the role of the adaptive immune system in the pathogenesis of multiple sclerosis (MS). Recent data suggest that dendritic cells (DCs), which are innate immune cells, also contribute to the pathogenesis of MS. In patients with MS, DCs are abundantly present in brain lesions, and display an altered phenotype and/or function as compared with this in healthy controls. DCs are thus in the position to pathologically influence the effector function of (auto-reactive) T and B cells. Interestingly, current first-line immunomodulating therapies for MS have been shown to restore DC phenotype and function, albeit in a non-specific manner. To date, clinical trials using agents specifically targeting DC function are ongoing. Moreover, several studies worldwide are currently investigating possible strategies to develop tolerogenic DCs. This review focuses on the phenotypic and functional alterations of conventional DCs and plasmacytoid DCs in patients with MS. Furthermore, we discuss how existing immunomodulating therapies for MS patients affect DC function and address future perspectives in the development of immunotherapies specifically targeting DCs.

Leukemia-associated antigens and their relevance to the immunotherapy of acute myeloid leukemia.

The graft-versus-leukemia effect of allogeneic hematopoietic stem cell transplantation (HSCT) has shown that the immune system is capable of eradicating acute myeloid leukemia (AML). This knowledge, along with the identification of the target antigens against which antileukemia immune responses are directed, has provided a strong impetus for the development of antigen-targeted immunotherapy of AML. The success of any antigen-specific immunotherapeutic strategy depends critically on the choice of target antigen. Ideal molecules for immune targeting in AML are those that are: (1) leukemia-specific; (2) expressed in most leukemic blasts including leukemic stem cells; (3) important for the leukemic phenotype; (4) immunogenic; and (5) clinically effective. In this review, we provide a comprehensive overview on AML-related tumor antigens and assess their applicability for immunotherapy against the five criteria outlined above. In this way, we aim to facilitate the selection of appropriate target antigens, a task that has become increasingly challenging given the large number of antigens identified and the rapid pace at which new targets are being discovered. The information provided in this review is intended to guide the rational design of future antigen-specific immunotherapy trials, which will hopefully lead to new antileukemia therapies with more selectivity and higher efficacy.

A hypogranular variant of acute promyelocytic leukaemia showing a heterogenic immunophenotype with CD34, CD2, HLA-DR positivity: a case report and review of the literature.

We report the case of a 46-year-old man who presented with the hypogranular variant of an acute promyelocytic leukaemia (APL). RT-PCR analysis for detection of the t(15;17) fusion transcript confirmed the cytological findings by demonstrating a bcr-3 type PML/RARalpha rearrangement. According to the WHO-classification, this leukaemia fulfilled the criteria for 'Acute promyelocytic leukaemia with t(15;17)(q22;q12)'(1). Immunophenotyping revealed a leukaemic population with an aberrant expression of markers. Besides the presence of the expected immunophenotypic markers in APL (CD45 with low density, CD13+, CD33+, CD15-, CD117+ and MPO+), the population showed a positivity for CD34, CD2 and HLA-DR for at least a part of the malignant promyelocytes. Since the expression of these three markers, all together, is rather unique, we reviewed the literature to prove the relationship of this specific immunophenotype with morphology, clinical and molecular findings.

Natural killer cell immune escape in acute myeloid leukemia.

As central players of the innate immune system, natural killer (NK) cells can exert direct and indirect anti-tumor effects via their cytotoxic and immune regulatory capacities, pivotal in the induction of an effective adaptive anti-tumor immune response. Hence, NK cells are considered to be important in the immune surveillance of cancer. In acute myeloid leukemia (AML) patients, however, significantly impaired NK cell functions can facilitate escape from immune surveillance and affect patient outcome. Here, we review various NK cell defects and AML evasion mechanisms to escape from NK cell-mediated immune surveillance and we discuss NK cell-related parameters as prediction factors of AML patient outcome. On the basis of these observations, novel immunotherapeutic strategies capitalizing on the potentiation of NK cell functions have emerged in AML immunotherapy, as discussed in this review. Increased knowledge on AML escape routes from NK cell immune surveillance will further aid in the design of novel NK cell-based immunotherapy approaches for the treatment of AML.

Immunotherapy in leukaemia.

Therapeutic cancer vaccination, e.g. by using tumour antigen-presenting dendritic cells (DCs) that 'educate' the immune system to recognise and attack tumour cells, represents a new concept of treatment in oncology. DCbased immunotherapy elicits both innate (NK) and adaptive (T cells) cellular responses correlated with clinical benefit. WT1 mRNA-transfected DCs emerge as a feasible and effective strategy to control residual disease in acute myeloid leukaemia (AML), in particular as a post-remission treatment to prevent full relapse. This innovative approach takes advantage of the intrinsic potential of the immune system to eradicate malignant disease.

Interferon-α in acute myeloid leukemia: an old drug revisited.

Interferon-α (IFN-α), a type I IFN, is a well-known antitumoral agent. The investigation of its clinical properties in acute myeloid leukemia (AML) has been prompted by its pleiotropic antiproliferative and immune effects. So far, integration of IFN-α in the therapeutic arsenal against AML has been modest in view of the divergent results of clinical trials. Recent insights into the key pharmacokinetic determinants of the clinical efficacy of IFN along with advances in its pharmaceutical formulation, have sparked renewed interest in its use. This paper reviews the possible applicability of IFN-α in the treatment of AML and provides a rational basis to re-explore its efficacy in clinical trials.

Antibiotic management of febrile neutropenia: current developments and future directions.

Mortality due to febrile neutropenia has decreased since the concept of empiric therapy became standard care. However, infectious complications remain the most common adverse events of chemotherapy. bacterial epidemiology has changed during the past decades. There is currently an increasing trend in infections due to Gramnegative bacteria which have higher rates of resistance for a variety of reasons.The use of biomarkers for diagnosis remains a domain of further investigation. Since the patient population with febrile neutropenia is very heterogeneous, models of risk assessment have been developed with the most commonly used today being the mASCC score.Oral antibiotic treatment seems to be appropriate in low-risk patients. In moderate or high-risk patients monotherapy is the most common option. However, due to emerging resistance this could change by next year. Some new antibiotics have been developed, but experience in the treatment of neutropenic fever is limited. The use of antibiotics for prophylaxis remains controversial, although recent studies suggest a reduction in death from all causes.

Efficient mRNA electroporation of peripheral blood mononuclear cells to detect memory T cell responses for immunomonitoring purposes.

To date, the number of immunotherapy vaccines in clinical trials increases exponentially. To evaluate the efficacy of these clinical vaccination trials, we fervently need cellular immunomonitoring tools. In this study, we present a newly developed short-time assay which allows direct ex vivo analysis of multi-epitope antigen-specific T cell immune responses. This novel method is based on mRNA electroporation of isolated peripheral blood mononuclear cells (PBMC). Fresh and cryopreserved PBMC of both healthy volunteers as well as of allogeneic stem cell transplanted patients enrolled in a cytomegalovirus (CMV) dendritic cell vaccination trial were electroporated with CMV pp65-encoding mRNA. Using a direct IFN-gamma EliSPOT and intracellular cytokine flow cytometry we detected significantly higher numbers of CMV pp65-specific IFN-gamma-secreting T cells as compared to the assay with non-treated PBMC and as compared to PBMC electroporated with mRNA coding for an irrelevant protein. Compared to conventional methods to evaluate T cell-mediated immune responses, this method is time-saving and less labor-intensive because it obviates the need for in vitro cultured antigen-presenting cells and because an overnight incubation is sufficient for activation of T cells. Moreover, the use of CMV pp65-encoding mRNA will broaden the immune response because it covers every epitope with potential relevance. This is a major advantage compared to the recognition of a single epitope covered by a HLA-restricted peptides. In summary, we developed a highly efficient mRNA electroporation protocol for fresh and cryopreserved PBMC. This novel method is a rapid and elegant tool and will be convenient for monitoring the cellular immune status of patients in a clinical vaccination setting.

Quantification of IFN-gamma produced by human purified NK cells following tumor cell stimulation: comparison of three IFN-gamma assays.

Interferon (IFN)-gamma released by natural killer (NK) cells has become a subject of major interest, given its importance in bridging the innate and adaptive immune system. Interestingly, reports concerning tumor cell stimulation of NK cells show divergent data on which stimuli induce IFN-gamma production. Here, the question remains whether tumor cell recognition is sufficient to trigger IFN-gamma or whether a second signal is required such as type I IFN. While IFN-gamma detection methods are abundantly used with peripheral blood mononuclear cells or purified T cell fractions as responder populations, only limited data is available about comparison of these assays with purified NK cells. In this study, we assessed the relationship between stimulation of human purified resting peripheral blood NK cells with one (tumor cell or IFN-alpha) and two (tumor cell+IFN-alpha) signals by measuring IFN-gamma using three different assays. We performed the enzyme-linked immunosorbent assay (ELISA), the enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining (ICS) assay in parallel per donor and determined whether there was a correlation between these assays. Our results show that two-signal stimulation of human resting NK cells induces significantly more IFN-gamma as compared to one-signal stimulation, readily picked up by all assays. Moreover, statistical analysis points towards a positive correlation between these assays for IFN-gamma produced following two-signal stimulation. Importantly, we show that tumor cell stimulation alone is enough to trigger secretion of IFN-gamma, but this finding was only evidenced by ELISPOT. These results reveal that the choice of IFN-gamma detection method can markedly influence the outcome regarding induction of NK cell IFN-gamma by tumor cells.

Cefepime and amikacin as empirical therapy in patients with febrile neutropaenia: a single-centre phase II prospective survey.

The aim of the survey was to prospectively evaluate the effectiveness of the combination therapy cefepime and amikacin in the initial treatment of haematology patients with febrile neutropaenia. Two hundred twenty (220) episodes of febrile neutropaenia were analysed in 54 males and 82 females (median age 58 years), most patients had a severe neutropaenia with in 72% of all periods a neutrophil count of less than 100. Microbiological infection was confirmed in 72 cases (32.8%). Sixty-one (61) bacteria were isolated from blood cultures of which 22 were identified as Gram-negative bacteria and 38 as Gram-positive bacteria. Sixty-three (63) episodes (28.6%) were clinically documented, 85 episodes (38.6%) were fever of unknown origin. Clinical cure was achieved in 123 febrile episodes (56%) after initiation of the current antibiotic protocol; another 22 patients (10%) became afebrile after modifying the initial antibiotic regimen 48 hours or longer after treatment initiation. In 61 cases (27.7%) there was persistent fever or re-occurrence of fever, these cases were considered as treatment failure. Eight patients (3.6%) died during the study. This survey has demonstrated that the combination therapy with cefepime and amikacin can be considered as an effective treatment for febrile neutropaenia in high-risk haematological patients in our centre with a high incidence of resistance to Gram-negative bacteria.

Immunosuppression induced by immature dendritic cells is mediated by TGF-beta/IL-10 double-positive CD4+ regulatory T cells.

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

Can cell therapy heal a spinal cord injury?

STUDY DESIGN: Literature survey. OBJECTIVES: To summarize and discuss current possibilities and success rates for the treatment of spinal cord injury in animal models. SETTINGS: University of Antwerp, Belgium. METHODS: We searched Pubmed for publications from 1997 onwards. Seven older papers were used for completion of data. RESULTS: Despite major progress in pharmacological and surgical approaches, a spinal cord injury still remains a very complex medical and psychological challenge, both for the patients and their relatives, as well as for the involved physicians, with currently no existing curative therapy. For a future efficient treatment, one has to consider and combine four main approaches: (1) tissue or cell transplantation, (2) providing growth-stimulating factors (neurotrophic factors), (3) blocking factors which inhibit neural regeneration and (4) modulation of inflammatory response following spinal cord injury. CONCLUSIONS: Although different treatment options have proven to be successful in animal models, they also provide a realistic view on a complex therapeutical approach, which needs to be further investigated in many carefully designed animal studies before human applications can be considered.

Proinflammatory response of human leukemic cells to dsRNA transfection linked to activation of dendritic cells.

Leukemic cells exert immunosuppressive effects that interfere with dendritic cell (DC) function and hamper effective antileukemic immune responses. Here, we sought to enhance the immunogenicity of leukemic cells by loading them with the double-stranded (ds) RNA Toll-like receptor 3 (TLR3) ligand polyriboinosinic polyribocytidylic acid (poly(I:C)), mimicking viral infection of the tumor cells. Given the responsiveness of DC to TLR ligands, we hypothesized that the uptake of poly(I:C)-loaded leukemic cells by immature DC (iDC) would lead to DC activation. Primary acute myeloid leukemia (AML) cells and AML cell lines markedly responded to poly(I:C) electroporation by apoptosis, upregulation of TLR3 expression, enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules and by production of type I interferons (IFN). Upon phagocytosis of poly(I:C)-electroporated AML cells, DC maturation and activation were induced as judged by an increased expression of MHC and costimulatory molecules, production of proinflammatory cytokines and an increase of T helper 1 (T(H)1)-polarizing capacity. These immune effects were suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of poly(I:C) transfection over pulsing. Our results demonstrate that poly(I:C) electroporation is a promising strategy to increase the immunogenicity of AML cells and to convert iDC into activated mature DC following the phagocytosis of AML cells.

Acute myeloid leukaemia of mixed megakaryocytic and erythroid origin: a case report and review of the literature.

We report the case of a 78-year-old man who presented with acute myeloid leukaemia showing subpopulations of cells expressing platelet-associated markers and the presence of a pan-myeloid component, besides glycophorin A-positive cells. Most of the immature cells had a proerythroblast-like morphology and we classified this case as an FAB-M6 variant, as suggested by Bain (1). According to the WHO classification, this leukaemia fulfilled the criteria of'AML with multilineage dysplasia' (2). Immunophenotyping characteristics showed two distinct aberrant subpopulations, a young pan-myeloid (CD45+ with low density, CD34+, CD117+, CD13+, CD33+, partial cytoplasmic myeloperoxidase (MPO)+) population with platelet-associated markers (CD41+, CD42+, CD61+) and a CD45+, CD117+, CD34- population with partial CD235a positivity indicative for erythroid maturation. This case belongs to the group of 'early' erythroblastic leukaemias where a subset of progenitor cells present with erythroid-megakaryocyte bipotentiality or are blocked at an early BFU-E (burst-forming unit erythrocyte)-like stage of erythroid differentiation (11, 12, 13).

Messenger RNA electroporation: an efficient tool in immunotherapy and stem cell research.

Over the last decades medicine has developed tremendously, but still many diseases are incurable. The last years, cellular (gene) therapy has become a hot topic in biomedical research for the potential treatment of cancer, AIDS and diseases involving cell loss or degeneration. Here, we will focus on two major areas within cellular therapy, cellular immunotherapy and stem cell therapy, that could benefit from the introduction of neo-expressed genes through mRNA electroporation for basic research as well as for clinical applications. For cellular immunotherapy, we will provide a state-of-the-art on loading antigen-presenting cells with antigens in the mRNA format for manipulation of T cell immunity. In the area of stem cell research, we will highlight current gene transfer methods into adult and embryonic stem cells and discuss the use of mRNA electroporation for controlling guided differentiation of stem cells into specialized cell lineages.