Ribosomal and Immune Transcripts Associate with Relapse in Acquired ADAMTS13-Deficient Thrombotic Thrombocytopenic Purpura.

Approximately 40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe acquired ADAMTS13 deficiency experience one or more relapses. Risk factors for relapse other than severe ADAMTS13 deficiency and ADAMTS13 autoantibodies are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation. This cross-sectional study asked whether autoantibodies against RNA-binding proteins or peripheral blood gene expression profiles measured during remission are associated with history of prior relapse in acquired ADAMTS13-deficient TTP. Peripheral blood from 38 well-characterized patients with autoimmune ADAMTS13-deficient TTP in remission was examined for autoantibodies and global gene expression. A subset of TTP patients (9 patients, 24%) exhibited a peripheral blood gene signature composed of elevated ribosomal transcripts that associated with prior relapse. A non-overlapping subset of TTP patients (9 patients, 24%) displayed a peripheral blood type I interferon gene signature that associated with autoantibodies to RNA-binding proteins but not with history of relapse. Patients who had relapsed bimodally expressed higher HLA transcript levels independently of ribosomal transcripts. Presence of any one potential risk factor (ribosomal gene signature, elevated HLA-DRB1, elevated HLA-DRB5) associated with relapse (OR = 38.4; p = 0.0002) more closely than any factor alone or all factors together. Levels of immune transcripts typical of natural killer (NK) and T lymphocytes positively correlated with ribosomal gene expression and number of prior episodes but not with time since the most recent episode. Flow cytometry confirmed elevated expression of cell surface markers encoded by these transcripts on T and/or NK cell subsets of patients who had relapsed. These data associate elevated ribosomal and immune transcripts with relapse history in acquired, ADAMTS13-deficient TTP.

Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity

Certain HLA-B antigens have been associated with lack of progression to AIDS. HLA-B alleles can be divided into two mutually exclusive groups based on the expression of the molecular epitopes HLA-Bw4 and HLA-Bw6. Notably, in addition to its role in presenting viral peptides for immune recognition, the HLA-Bw4, but not HLA-Bw6, motif functions as a ligand for a natural killer cell inhibitory receptor (KIR). Here, we show that profound suppression of HIV-1 viremia is significantly associated with homozygosity for HLA-B alleles that share the HLA-Bw4 epitope. Furthermore, homozygosity for HLA-Bw4 alleles was also significantly associated with the ability to remain AIDS free and to maintain a normal CD4 T cell count in a second cohort of HIV-1-infected individuals with well defined dates of seroconversion. This association was independent of the presence of a mutation in CC chemokine receptor 5 (CCR5) associated with resistance to HIV-1 infection, and it was independent of the presence of HLA alleles that could potentially confound the results. We conclude that homozygosity for HLA-Bw4-bearing B alleles is associated with a significant advantage and that the HLA-Bw4 motif is important in AIDS pathogenesis.

Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus

We inoculated BALB/c mice deficient in STAT6 (STAT6−/−) and their wild-type (wt) littermates (STAT6+/+) with the natural mouse pathogen, ectromelia virus (EV). STAT6−/− mice exhibited increased resistance to generalized infection with EV when compared with STAT6+/+ mice. In the spleens and lymph nodes of STAT6−/− mice, T helper 1 (Th1) cytokines were induced at earlier time points and at higher levels postinfection when compared with those in STAT6+/+ mice. Elevated levels of NO were evident in plasma and splenocyte cultures of EV-infected STAT6−/− mice in comparison with STAT6+/+ mice. The induction of high levels of Th1 cytokines in the mutant mice correlated with a strong natural killer cell response. We demonstrate in genetically susceptible BALB/c mice that the STAT6 locus is critical for progression of EV infection. Furthermore, in the absence of this transcription factor, the immune system defaults toward a protective Th1-like response, conferring pronounced resistance to EV infection and disease progression.

Eradication of human hepatic and pulmonary melanoma metastases in SCID mice by antibody-interleukin 2 fusion proteins.

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Linfomas com expressão na cavidade oral

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Impacto de la haploinsuficiencia de CD3 en la expresión y función del complejo TCR/CD3 en humanos y ratones

TCR/CD3 (αβTCRand γδTCR) complexes are members of a family of modular biosensors that are responsible for driving T-cell development, activation, and effector functions. They inform essential checkpoint decisions by relaying key information from their ligand-binding modules (TCR chains) to their signaling modules (CD3γε, CD3δε and CD3ζζ) and onto the intracellular signaling apparatus. Their actions shape the T-cell repertoire, as well as T-cell-mediated immunity; yet, the mechanisms that underlie their activity are still to be precisely determined. As with any molecular machine, understanding how they function depends critically on dissecting how their parts fit and work together. T-cell receptor immunodeficiencies (TCRID) are low-prevalence autosomal recessive diseases characterized by impaired surface TCR expression, frequently associated with peripheral blood T lymphocytopenia, severe combined ID (SCID) and (or) autoimmune symptoms, but not associated with B or natural killer (NK) lymphocytopenia. Several CD3, CD247, and TCR deficiencies have been described which can be classified as complete or partial according to the absence or presence of residual levels of the affected protein. Although rare and sometimes based on single cases, TCRID offer rich information about the underpinnings of human TCR structure and function, which in turn impact our understanding of T-cell development and function... NOTA 520 8 Las inmunodeficiencias humanas del receptor de antígeno de los linfocitos T (TCR) son enfermedades autosómicas recesivas con baja prevalencia, caracterizadas por un defecto de expresión del TCR asociado a una linfopenia T selectiva. La ausencia congénita de componentes del TCR tiene un impacto diferencial en el desarrollo y función de los linfocitos T, que depende de la cadena del TCR afectada y de la especie, siendo en algunos casos diferente en los pacientes humanos en comparación con los modelos en ratones. Las inmunodeficiencias del TCR han sido ampliamente estudiadas, en particular las de las sub-unidades CD3 del complejo receptor. En contraste, hasta ahora es muy poca la atención que se le ha prestado a los potenciales efectos de la haploinsuficiencia de componentes del TCR en el desarrollo, fenotipo y función de los linfocitos T. Esto es debido en gran parte al hecho de que los individuos haploinsuficientes (portadores de mutaciones nulas en heterocigosis) no presentan alteraciones obvias de la inmunidad mediada por células T o signos de enfermedad. En este trabajo se analiza por primera vez, en humanos y modelos de ratones, el impacto de la haploinsuficiencia de CD3 y CD3 en la expresión y funcionalidad del TCR, y su potencial relevancia en el desarrollo y función de los linfocitos T. Los resultados indican que la haploinsuficiencia de CD3 y, en menor medida, la de CD3causa una disminución en la expresión del TCR en la superficie de las células T y T, y provoca alteraciones claras en el desarrollo y función de los linfocitos T en ambas especies...

Estudo funcional e fenotípico das células NK em esclerose sistémica

Introdução: A esclerose sistémica (ES) é uma doença reumática rara, onde se verifica uma reacção auto-imune, afectando o tecido conjuntivo. De etiologia desconhecida, esta patologia, dividide-se em dois subtipos: limitada e difusa, tendo como processos característicos e inter-relacionados: a lesão vascular, a reacção autoimune e a fibrose. As células NK modulam as doenças auto-imunes através das suas capacidades citotóxicas e de produção de citocinas. Deste modo procedeu-se ao estudo imunofenotípico e funcional de duas sub-populações de células Natural Killer (NK) (NK CD56dim e NK CD56bright). Métodos: Recorrendo à citometria de fluxo, caracterizamos fenotipicamente as células NK, com base na expressão de CD49e, CD29 e LAIR-1; funcionalmente, com base nas citocinas TNF-α e IFN-γ e citotoxicamente, com base na granzima B e na perforina. Resultados: Os nossos resultados sugerem uma diminuição da percentagem e valor absoluto de células NK totais e suas subpopulações, NK CD56dim e NK CD56bright, em doentes com ES, sobretudo na presença de úlceras digitais (UD) e fibrose pulmonar (FP). Observamos ainda uma diminuição da expressão de CD49e e um aumento da expressão de LAIR-1, nas células NK, em doentes com ES, principalmente nos que não apresentavam UD e FP. Num modo geral observou-se um aumento da frequência de células NK a expressar granzima B e perforina e um aumento da expressão destas duas proteínas. Conclusão: Concluímos que as células NK poderão ter um papel importante na fisiopatologia da ES, pelas alterações fenotípicas e funcionais, podendo, futuramente vir a ser utilizados na monitorização da severidade desta doença.

The deterministic Dendritic Cell Algorithm

The Dendritic Cell Algorithm is an immune-inspired algorithm originally based on the function of natural dendritic cells. The original instantiation of the algorithm is a highly stochastic algorithm. While the performance of the algorithm is good when applied to large real-time datasets, it is difficult to analyse due to the number of random-based elements. In this paper a deterministic version of the algorithm is proposed, implemented and tested using a port scan dataset to provide a controllable system. This version consists of a controllable amount of parameters, which are experimented with in this paper. In addition the effects are examined of the use of time windows and variation on the number of cells, both which are shown to influence the algorithm. Finally a novel metric for the assessment of the algorithms output is introduced and proves to be a more sensitive metric than the metric used with the original Dendritic Cell Algorithm.

PCA 4 DCA: the application of principal component analysis to the Dendritic Cell Algorithm

As one of the newest members in the field of articial immune systems (AIS), the Dendritic Cell Algorithm (DCA) is based on behavioural models of natural dendritic cells (DCs). Unlike other AIS, the DCA does not rely on training data, instead domain or expert knowledge is required to predetermine the mapping between input signals from a particular instance to the three categories used by the DCA. This data preprocessing phase has received the criticism of having manually over-fitted the data to the algorithm, which is undesirable. Therefore, in this paper we have attempted to ascertain if it is possible to use principal component analysis (PCA) techniques to automatically categorise input data while still generating useful and accurate classication results. The integrated system is tested with a biometrics dataset for the stress recognition of automobile drivers. The experimental results have shown the application of PCA to the DCA for the purpose of automated data preprocessing is successful.

Role of chemerin and its receptors in mouse models of tumorigenesis

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

The ascidian natural product eusynstyelamide B is a novel topoisomerase II poison that induces DNA damage and growth arrest in prostate and breast cancer cells

As part of an anti-cancer natural product drug discovery program, we recently identified eusynstyelamide B (EB), which displayed cytotoxicity against MDA-MB-231 breast cancer cells (IC50 = 5 μM) and induced apoptosis. Here, we investigated the mechanism of action of EB in cancer cell lines of the prostate (LNCaP) and breast (MDA-MB-231). EB inhibited cell growth (IC50 = 5 μM) and induced a G2 cell cycle arrest, as shown by a significant increase in the G2/M cell population in the absence of elevated levels of the mitotic marker phospho-histone H3. In contrast to MDA-MB-231 cells, EB did not induce cell death in LNCaP cells when treated for up to 10 days. Transcript profiling and Ingenuity Pathway Analysis suggested that EB activated DNA damage pathways in LNCaP cells. Consistent with this, CHK2 phosphorylation was increased, p21CIP1/WAF1 was up-regulated and CDC2 expression strongly reduced by EB. Importantly, EB caused DNA double-strand breaks, yet did not directly interact with DNA. Analysis of topoisomerase II-mediated decatenation discovered that EB is a novel topoisomerase II poison.

A novel structural derivative of natural alkaloid ellipticine, MDPSQ, induces necrosis in leukemic cells

DNA intercalating molecules are promising chemotherapeutic agents. In the present study, a novel DNA intercalating compound of pyrimido4',5':4,5]selenolo(2,3-b)quinoline series having 8-methyl-4-(3 diethylaminopropylamino) side chain is studied for its chemotherapeutic properties. Our results showed that 8-methyl-4-(3 diethylaminopropylamino) pyrimido 4',5':4,5] selenolo(2,3-b)quinoline (MDPSQ) induces cytotoxicity in a time- and concentration-dependent manner on leukemic cell lines. Both cell cycle analysis and tritiated thymidine assays revealed that MDPSQ affects DNA replication. Treatment with MDPSQ resulted in both elevated levels of DNA strand breaks and repair proteins, further indicating its cytotoxic effects. Besides, Annexin V/PI staining revealed that MDPSQ induces cell death by triggering necrosis rather than apoptosis.

A natural compound, methyl angolensate, induces mitochondrial pathway of apoptosis in Daudi cells

Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt's lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells.

Natural convection in metal foams with open cells

This paper presents a combined experimental and numerical study on natural convection in open-celled metal foams. The effective thermal conductivities of steel alloy (FeCrAlY) samples with different relative densities and cell sizes are measured with the guarded-hot-plate method. To examine the natural convection effect, the measurements are conducted under both vacuum and ambient conditions for a range of temperatures. The experimental results show that natural convection is very significant, accounting for up to 50% of the effective foam conductivity obtained at ambient pressure. This has been attributed to the high porosity (ε > 0.9) and inter-connected open cells of the metal foams studied. Morphological parameters characterizing open-celled FeCrAlY foams are subsequently identified and their cross-relationships are built. The non-equilibrium two-equation energy transfer model is employed, and selected calculations show that the non-equilibrium effect between the solid foam skeleton and air is significant. The study indicates that the combined parameter, i.e., the porous medium Rayleigh number, is no longer appropriate to correlate natural convection by itself when the Darcy number is sufficiently large as in the case of natural convection in open-celled metal foams. Good agreement between model predictions and experimental measurements is obtained. © 2005 Elsevier Ltd. All rights reserved.