Prevalence of feline leukemia virus infection in domestic cats in Rio de Janeiro

Peripheral blood smears of 1094 domestic cats were collected and tested by indirect immunofluorescence antibody assay for p27 antigen in cells to study the prevalence and risk factors for feline leukemia virus (FeLV) in the state of Rio de Janeiro. Sex, age, breed, outdoor access, neutering status, type of habitation (household, shelter, veterinary clinics and other places), number of household cats and clinical signs were registered on a form. Among the tested samples, 11.52% were positive. Risk factors for FeLV infection included outdoor access, age range between 1 and 5 years old, and cohabitation with numerous cats.

Exploring the Role of Soluble Factors Associated with Immune Regulatory Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are characterized as multipotent stromal cells with the capacity for both self-renewal and differentiation into mesodermal cell lineages. MSCs also have a fibroblast-like phenotype and can be isolated from several tissues. In recent years, researchers have found that MSCs secrete several soluble factors that exert immunosuppressive effects by modulating both innate (macrophages, dendritic and NK cells) and adaptive (B cells and CD4+ and CD8+ T cells) immune responses. This review summarizes the principal trophic factors that are related to immune regulation and secreted by MSCs under both autoimmune and inflammatory conditions. The understanding of mechanisms that regulate immunity in MSCs field is important for their future use as a novel cellular-based immunotherapy with clinical applications in several diseases.

Monoclonal B-cell lymphocytosis (MBL), CD4(+)/CD8(weak) T-cell large granular lymphocytic leukemia (T-LGL leukemia) and monoclonal gammopathy of unknown significance (MGUS): molecular and flow cytometry characterization of three concomitant hematological disorders

The diagnosis of T-cell large granular lymphocytic leukemia in association with other B-cell disorders is uncommon but not unknown. However, the concomitant presence of three hematological diseases is extraordinarily rare. We report an 88-year-old male patient with three simultaneous clonal disorders, that is, CD4+/CD8(weak) T-cell large granular lymphocytic leukemia, monoclonal gammopathy of unknown significance and monoclonal B-cell lymphocytosis. The patient has only minimal complaints and has no anemia, neutropenia or thrombocytopenia. Lymphadenopathy and hepatosplenomegaly were not present. The three disorders were characterized by flow cytometry analysis, and the clonality of the T-cell large granular lymphocytic leukemia was confirmed by polymerase chain reaction. Interestingly, the patient has different B-cell clones, given that plasma cells of monoclonal gammopathy of unknown significance exhibited a kappa light-chain restriction population and, on the other hand, B-lymphocytes of monoclonal B-cell lymphocytosis exhibited a lambda light-chain restriction population. This finding does not support the antigen-driven hypothesis for the development of multi-compartment diseases, but suggests that T-cell large granular lymphocytic expansion might represent a direct antitumor immunological response to both B-cell and plasma-cell aberrant populations, as part of the immune surveillance against malignant neoplasms.

Relationship between B-Cell-specific moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase III beta (topoIII beta) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIII beta, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA1 (p=0.003), H2AX (p=0.024) and topoIII beta (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 mu mol/mL and < 6.0 mu mol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL. (Blood. 2012;120(1):207-213)

Differential MiRNA expression in childhood acute lymphoblastic leukemia and association with clinical and biological features

The present study aimed to analyze the expression profile of the microRNAs previously described as associated with childhood ALL, miR-92a, miR-100, miR-125a-5p, miR-128a, miR-181b, miR-196b and let-7e, and their association with biological/prognostic features in 128 consecutive samples of childhood acute lymphoblastic leukemia (ALL) by quantitative real-time PCR. A significant association was observed between higher expression levels of miR-196b and T-ALL, miR-100 and patients with low white blood cell count at diagnosis and t(12;21) positive ALL. These findings suggest a potential activity of these microRNAs in pediatric ALL biology. (C) 2011 Elsevier Ltd. All rights reserved.

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a protooncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.

A new dic(7;12)(p12.21;p12.2) and i(12)(q10) during the lymphoid blast crisis of patient with Ph plus chronic myeloid leukemia

Chronic myelogenous leukemia (CML) is a common myeloproliferative disease that is characterized by the clonal expansion of marrow stem cells, and is associated with the Philadelphia chromosome. As the disease progresses, additional chromosome abnormalities may arise. The prognostic impact of secondary chromosomal abnormalities in CML is complex, heterogeneous, and sometimes related to previous treatment. Here, we describe a CML patient in lymphoid blast crisis associated with a new chromosomal abnormality identified, dic(7;12)(p12.21;p12.2) and i(12)(q10) using classical cytogenetics and spectral karyotype analysis. To the best of our knowledge, this is the first report of t(7;12)(p11.1;q11.1) and i(12)(q10) in a CML patient with lymphoid evolution.

Prion potency in stem cells biology

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

Profiling Three-Dimensional Nuclear Telomeric Architecture of Myelodysplastic Syndromes and Acute Myeloid Leukemia Defines Patient Subgroups

Purpose: Myelodysplastic syndromes (MDS) are a group of disorders characterized by cytopenias, with a propensity for evolution into acute myeloid leukemias (AML). This transformation is driven by genomic instability, but mechanisms remain unknown. Telomere dysfunction might generate genomic instability leading to cytopenias and disease progression. Experimental Design: We undertook a pilot study of 94 patients with MDS (56 patients) and AML (38 patients). The MDS cohort consisted of refractory cytopenia with multilineage dysplasia (32 cases), refractory anemia (12 cases), refractory anemia with excess of blasts (RAEB) 1 (8 cases), RAEB2 (1 case), refractory anemia with ring sideroblasts (2 cases), and MDS with isolated del(5q) (1 case). The AML cohort was composed of AML-M4 (12 cases), AML-M2 (10 cases), AML-M5 (5 cases), AML-M0 (5 cases), AML-M1 (2 cases), AML-M4eo (1 case), and AML with multidysplasia-related changes (1 case). Three-dimensional quantitative FISH of telomeres was carried out on nuclei from bone marrow samples and analyzed using TeloView. Results: We defined three-dimensional nuclear telomeric profiles on the basis of telomere numbers, telomeric aggregates, telomere signal intensities, nuclear volumes, and nuclear telomere distribution. Using these parameters, we blindly subdivided the MDS patients into nine subgroups and the AML patients into six subgroups. Each of the parameters showed significant differences between MDS and AML. Combining all parameters revealed significant differences between all subgroups. Three-dimensional telomeric profiles are linked to the evolution of telomere dysfunction, defining a model of progression from MDS to AML. Conclusions: Our results show distinct three-dimensional telomeric profiles specific to patients with MDS and AML that help subgroup patients based on the severity of telomere dysfunction highlighted in the profiles. Clin Cancer Res; 18(12); 3293-304. (C) 2012 AACR.

Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.

Increased expression of miR-221 is associated with shorter overall survival in T-cell acute lymphoid leukemia

Background: CD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome. Methods: The gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples. Results: miR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56- vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate. Conclusions: miR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

In vitro generation and characterization of acute myeloid leukemia-reactive CD8 + cytotoxic T-lymphocyte clones from healthy donors

Donor-derived CD8+ cytotoxic T lymphocytes (CTLs) eliminating host leukemic cells mediate curative graft-versus-leukemia (GVL) reactions after allogeneic hematopoietic stem cell transplantation (HSCT). The leukemia-reactive CTLs recognize hematopoiesis-restricted or broadly expressed minor histocompatibility and leukemia-associated peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on recipient cells. The development of allogeneic CTL therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of current techniques for generating leukemia-reactive CTLs from unprimed healthy donors in vitro. In this work, a novel allogeneic mini-mixed lymphocyte/leukemia culture (mini-MLLC) approach was established by stimulating CD8+ T cells isolated from peripheral blood of healthy donors at comparably low numbers (i.e. 10e4/well) with HLA class I-matched primary AML blasts in 96-well microtiter plates. Before culture, CD8+ T cells were immunomagnetically separated into CD62L(high)+ and CD62L(low)+/neg subsets enriched for naive/central memory and effector memory cells, respectively. The application of 96-well microtiter plates aimed at creating multiple different responder-stimulator cell compositions in order to provide for the growth of leukemia-reactive CTLs optimized culture conditions by chance. The culture medium was supplemented with interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-2. In eight different related and unrelated donor/AML pairs with complete HLA class I match, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines of donor and patient origin nor primary B cell leukemias expressing the corresponding HLA restriction element. CTLs expressed T cell receptors of single V-beta chain families, indicating their clonality. The vast majority of CTL clones were obtained from mini-MLLCs initiated with CD8+ CD62L(high)+ cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10e8-10e10 cells within six to eight weeks. The capability of mini-MLLC derived AML-reactive CTL clones to inhibit the engraftment of human primary AML blasts was investigated in the immunodeficient nonobese diabetic/severe combined immune deficient IL-2 receptor common γ-chain deficient (NOD/SCID IL2Rγnull) mouse model. The leukemic engraftment in NOD/SCID IL2Rγnull was specifically prevented if inoculated AML blasts had been pre-incubated in vitro with AML-reactive CTLs, but not with anti-melanoma control CTLs. These results demonstrate that myeloid leukemia-specific CTL clones capable of preventing AML engraftment in mice can be rapidly isolated from CD8+ CD62L(high)+ T cells of healthy donors in vitro. The efficient generation and expansion of these CTLs by the newly established mini-MLLC approach opens the door for several potential applications. First, CTLs can be used within T cell-driven antigen identification strategies to extend the panel of molecularly defined AML antigens that are recognizable by T cells of healthy donors. Second, because these CTLs can be isolated from the stem cell donor by mini-MLLC prior to transplantation, they could be infused into AML patients as a part of the stem cell allograft, or early after transplantation when the leukemia burden is low. The capability of these T cells to expand and function in vivo might require the simultaneous administration of AML-reactive CD4+ T cells generated by a similar in vitro strategy or, less complex, the co-transfer of CD8-depleted donor lymphocytes. To prepare clinical testing, the mini-MLLC approach should now be translated into a protocol that is compatible with good manufacturing practice guidelines.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.