Juana Merino

Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)‐derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function.

Treatment of reperfused ischemia with adipose-derived stem cells in a preclinical Swine model of myocardial infarction.

The aim of the study was to determine the long-term effect of transplantation of adipose-derived stromal cells (ADSCs) in a preclinical model of ischemia/reperfusion (I/R). I/R was induced in 28 Goettingen minipigs by 120 min of coronary artery occlusion followed by reperfusion. Nine days later, surviving animals were allocated to receive transendocardial injection of a mean of 213.6 ± 41.78 million green fluorescent protein (GFP)-expressing ADSCs (n = 7) or culture medium as control (n = 9). Heart function, cell engraftment, and histological analysis were performed 3 months after transplantation. Transplantation of ADSCs induced a statistically significant long-lasting (3 months) improvement in cardiac function and geometry in comparison with control animals. Functional improvement was associated with an increase in angiogenesis and vasculogenesis and a positive effect on heart remodeling with a decrease in fibrosis and cardiac hypertrophy in animals treated with ADSCs. Despite the lack of cell engraftment after 3 months, ADSC transplantation induced changes in the ratio between MMP/TIMP. Our results indicate that transplantation of ADSCs, despite the lack of long-term significant cell engraftment, increases vessel density and prevents adverse remodeling in a clinically relevant model of myocardial infarction, strongly suggesting a paracrine-mediated effect. ADSCs thus constitute an attractive candidate for the treatment of myocardial infarction.

Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction.

Although transplantation of adipose-derived stem cells (ADSC) in chronic myocardial infarction (MI) models is associated with functional improvement, its therapeutic value is limited due to poor long-term cell engraftment and survival. Thus, the objective of this study was to examine whether transplantation of collagen patches seeded with ADSC could enhance cell engraftment and improve cardiac function in models of chronic MI. With that purpose, chronically infarcted Sprague-Dawley rats (n = 58) were divided into four groups and transplanted with media, collagen scaffold (CS), rat ADSC, or CS seeded with rat ADSC (CS-rADSC). Cell engraftment, histological changes, and cardiac function were assessed 4 months after transplantation. In addition, Göttingen minipigs (n = 18) were subjected to MI and then transplanted 2 months later with CS or CS seeded with autologous minipig ADSC (CS-pADSC). Functional and histological assessments were performed 3 months post-transplantation. Transplantation of CS-rADSC was associated with increased cell engraftment, significant improvement in cardiac function, myocardial remodeling, and revascularization. Moreover, transplantation of CS-pADSC in the pre-clinical swine model improved cardiac function and was associated with decreased fibrosis and increased vasculogenesis. In summary, transplantation of CS-ADSC resulted in enhanced cell engraftment and was associated with a significant improvement in cardiac function and myocardial remodeling.

Trial of complete weaning from immunosuppression for liver transplant recipients: Factors predictive of tolerance

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty‐four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4+, CD8+, and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range = 8.5‐22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P = 0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P = 0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n = 7 for an interval > 10 years and an SI < 20), 100% reached tolerance; in the second group (n = 10 for an interval > 10 years and an SI > 20 or an interval < 10 years and an SI < 20), 60% reached tolerance; and in the third group (n = 7 for an interval < 10 years and an SI > 20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables—the time from transplantation and the SI—may help to identify these patients. Liver Transpl 19:937–944, 2013.

Anti-Inflammatory Cytokines Induce Lipopolysaccharide Tolerance in Human Monocytes Without Modifying Toll-Like Receptor 4 Membrane Expression

Toll‐like receptor 4 (TLR4) participates in innate immunity by detecting lipopolysaccharides (LPS) of Gram‐negative bacterial cell walls. TLR4 macrophage expression in mice is modulated by LPS. This fact constitutes, at least partially, the molecular basis for LPS tolerance. Very recently, the effect of interferon‐γ (IFN‐γ), a pro‐inflammatory cytokine, has been described on TLR4 membrane expression of human monocytes. IFN‐γ up‐regulates TLR4 expression and antagonizes the LPS‐induced TLR4 down‐regulation. These data prompted us to study the expression of membrane TLR4 in human mono‐ cytes in which LPS tolerance was induced by LPS and by anti‐inflammatory cytokines [interleukin‐10 (IL‐10) and transforming growth factor β1 (TGFβ1)]. Data concerning this latter model, and more specifically, the effect of anti‐inflammatory cytokines over TLR4 expression, are not available at present. We show here that membrane TLR4 expression in human monocytes falls after LPS exposure. The effect was prolonged for 12 h, but then expression returned to normal levels. The incubation of human monocytes with IL‐10, TGFβ1 or a mixture of both induces no alterations in membrane TLR4 expression. However, these cytokines are able to substitute the tolerizing LPS exposure in order to induce LPS tolerance. Our data help to achieve a better understanding of the way cytokines control the cellular expression of TLR.

Parvovirus B19 acute infection and a reactivation of cytomegalovirus and herpesvirus 6 in a chronic myeloid leukemia patient during treatment with dasatinib (BMS-354825).

T lymphocyte activation is controlled by the T cell antigen receptor (TCR) in combination with additional signals triggered by accessory molecules present on the surface of the antigen-presenting cells (APC). Several studies have reported that treatment with imatinib can induce inhibition of the T-cell mediated immune response [1,2]. Imatinib inhibits T cell receptor-mediated T cell proliferation and activation in a dose-dependent manner and reduces tyrosine phosphorylation of ZAP-70 and LAT in response to activation through TCR [1]. Studies conducted in a mouse model indicate that imatinib treatment also leads to a selective inhibition of memory CTLs without affecting primary T or B cells responses [2]. Recent reports describing the development of severe viral infections such as varicella-zoster [3] and fatal hepatitis B virus reactivation in patients with CML treated with imatinib along with in vitro data suggest that the potential for immune suppression and viral reactivation in patients treated with abl and src kinase inhibitors should be considered [4]. We describe the development of a cytomegalovirus and herpes 6 virus associated with treatment with dasatinib (BMS354825) in a patient with diagnosis of CML. A 53-year-old woman was diagnosed with CML in November 2003. Treatment with imatinib was initiated at doses of 400 mg/day and further increased to 800 mg/day due to the persistence of BCR-ABL chromosome after 1 year. Treatment with imatinib was discontinued 2 years after diagnosis (September 2005) due to the development of grade III/IV cutaneous and gastrointestinal complications. In March 2006 treatment with dasatinib (BMS354825) was started at doses of 140 mg/day. One month after initiation of treatment with dasatinib, the patient was admitted to the hospital with atypical pneumonia (bilateral infiltrates in chest radiography, fever, dry cough, dyspnea and pleuritic pain). Despite extensive work-up, only three positive citomegalovirus cells were identified by the rapid viral culture (shell vial method) from peripheral blood. The patient was treated with antiviral (Ganciclovir) and antibiotic therapy (TeicoplaninMeropenem) with good response and was discharged after 15 days. In May 2006, the patient presented with a generalized cutaneous rash with positive serological test for parvovirus B19 (IgG titer 512 and IgM titer 40) and herpesvirus 6 (IgG titer 80). This pattern is compatible with an acute infection from parvovirus B19 because detectable low levels of B19 specific IgM can be found within 7 – 10 days of the exposure. We excluded the possibility to herpesvirus 6 infection because the patient had low levels of specific herpesvirus 6 IgG and a percentage of healthy adults are IgM positive at any time, making

Splenic marginal zone lymphoma with Evans’ syndrome, autoimmunity, and peripheral gamma/delta T cells

Dear Editor, Several theories have tried to explain the occurrence of autoantibodies in lymphoid malignancies. Autoimmune phenomena like Evans’ syndrome, lupus anticoagulants, autoimmune thrombocytopenia, and autoimmune hemolytic anemias are frequently associated with lymphomas [4, 5]. Recently, systemic lupus erythematosus (SLE) was associated with an increased risk of diffuse large B cell and marginal zone lymphomas [3]. Splenic marginal zone lymphoma (SMZL) is an indolent B cell lymphoma, which generally presents splenomegaly and involvement of both bone marrow and peripheral blood. In this report, we describe a patient with autoimmunity and bone marrow infiltration that probably broke the mechanisms of central tolerance in bone marrow producing the emergency of new auto-reactive clones and autoantibodies. A 60-year-old man had a 12-month history of B symptoms. Physical examination was unremarkable, except for the presence of massive splenomegaly. A computerized tomograhpy (CT) scan of the abdomen confirmed splenomegaly and visualized enlarged lymph nodes in retroperitoneum (Fig. 1). Blood smear and peripheral blood immunophenotype suggest SMZL with villous lymphocytes. The patient underwent laparotomy for splenectomy, lymph node, and bone marrow biopsies. Histology corresponds to SMZL. Follow-up studies detected the persistence of lymphoid neoplastic cells (1.4%) and a γδ T cell population (TCR γδ CD2+, CD3+, CD5 low+, CD7+, CD8 low+/−, CD16 −/+ CD56+, CD57 +/−) that represented 12% of the cells in peripheral blood. Five months after diagnosis, the patient presented with abdominal discomfort. On a CT scan, abdominal adenopatic progression was observed and the two populations (3.4% of SMZL and 4.9% of γδ T cells) persisted in peripheral blood. The patient received eight cycles of chemotherapy with cyclophosphamide–doxorubicin–vincristin–prednisone regime, resulting in radiological remission. At this moment, flow cytometry of peripheral blood revealed 0.0026% of tumor cells and 13.75% of γδ T cells. Four months after chemotherapy, the patient was admitted to the hospital with a recurrence of SZML. He showed, besides, respiratory insufficiency and Evans’ syndrome, associated with high titers of antiphospholipid antibodies, lupus anticoagulant and prolonged partial tromboplastin time, high levels of immune complexes, and low levels of both immunoglobulins and C3 and C4 fractions of complement. Antinuclear, antineutrophil cytoplasmic, anticardiolipin, antiplatelets, anti-HLA I, and antiEPCR antibodies were also detected. CT scan ascertained the presence of pulmonary emphysema and hepatomegaly, without associated enlarged lymph nodes. Peripheral blood flow-cytometry indicated an increase in the neoplastic B cell population (50%) and a decrease of γδ T cells (5%). The bone marrow biopsy confirmed lymphoid infiltration (SMZL 35% and γδ T cells 4%) by immunophenotype. Ann Hematol (2009) 88:177–178 DOI 10.1007/s00277-008-0555-z

Enhanced sensitivity of flow cytometry for routine assessment of minimal residual disease

In a recent paper by Bene and Kaeda,[1][1] technical approaches for minimal residual disease (MRD) assessment are extensively reviewed. PCR-based studies have proved to be 1-log more sensitive than flow cytometry (FC). For this reason, they are increasingly being preferred for MRD analysis,

BY55/CD160 cannot be considered a cytotoxic marker in cytomegalovirus-specific human CD8 + T cells

CD160/BY55 is a glucosyl‐phosphatidylinositol (GPI)‐anchored cell membrane receptor that is expressed primarily in natural killer (NK) cells. Its presence in CD8+ T lymphocytes is considered to be a marker of cytotoxic activity, although there are few data in this regard. In the present work, we analysed the expression of CD160 in subpopulations of cytomegalovirus (CMV)‐specific CD8+ T cells. Subpopulations were defined by CD28 and CD57 expression and exhibited varying degrees of differentiation and cytotoxic potential, as evaluated by the expression of perforin, interferon (IFN)‐γ and interleukin (IL)‐7Rα/CD127. We included subjects with different intensities of anti‐viral immune response. Results showed that the terminally differentiated CD28– CD57+ subset displaying the highest level of perforin expressed CD160 at a level similar to that of memory CD28+ CD57–perforin– cells. A comparison of the expression of perforin in CD160+ cells versus CD160– cells showed that expression was significantly higher in the absence of CD160. Interestingly, the CMV‐specific CD8+ T cell subset from a patient with ongoing CMV reactivation did not begin to express CD160 until day +92 of the follow‐up period. Taken together, our data show that CD160 cannot be considered a cytotoxic marker in CMV‐specific CD8+ T cells.

Utility of flow cytometry studies in the management of patients with multiple myeloma.

Purpose of review Although the input of multiparameter flow cytometry (MFC) into the clinical management of multiple myeloma patients has faced some reluctance, continuously growing evidence supports the utility of MFC in this disease. Recent findings MFC immunophenotyping of bone marrow and peripheral blood plasma cells affords cost-effective assessment of clonality, and provides prognostic information on the risk of progression in smoldering multiple myeloma, and the identification of active multiple myeloma patients with dismal outcome (e.g., high numbers of circulating tumor cells) or long-term survival despite suboptimal responses through the characterization of monoclonal gammopathy of undetermined significance-like phenotypes. Extensive data indicate that minimal residual disease (MRD) monitoring can be used as biomarker to evaluate treatment efficacy and act as surrogate for survival. The time has come to address within clinical trials the exact role of baseline risk factors and MRD monitoring for tailored therapy in multiple myeloma, which implies systematic usage of highly sensitive cost-effective, readily available, and standardized MRD techniques such as MFC. Summary Next-generation MFC should be considered mandatory in the routine evaluation of multiple myeloma patients both at diagnosis and after therapy, and represents an attractive technique to integrate with high-throughput DNA and RNA-seq methods to help in understanding the mechanisms behind dissemination and chemoresistance of multiple myeloma.