Phylogenetic Analysis of Thecosomata Blainville, 1824 (Holoplanktonic Opisthobranchia) Using Morphological and Molecular Data

Thecosomata is a marine zooplankton group, which played an important role in the carbonate cycle in oceans due to their shell composition. So far, there is important discrepancy between the previous morphological-based taxonomies, and subsequently the evolutionary history of Thecosomata. In this study, the remarkable planktonic sampling of TARA Oceans expedition associated with a set of various other missions allowed us to assess the phylogenetic relationships of Thecosomata using morphological and molecular data (28 S and COI genes). The two gene trees showed incongruities (e.g. Hyalocylis, Cavolinia), and high congruence between morphological and 28S trees (e.g. monophyly of Euthecosomata). The monophyly of straight shell species led us to reviving the Orthoconcha, and the split of Limacinidae led us to the revival of Embolus inflata replacing Limacina inflata. The results also jeopardized the Euthecosomata families that are based on plesiomorphic character state as in the case for Creseidae which was not a monophyletic group. Divergence times were also estimated, and suggested that the evolutionary history of Thecosomata was characterized by four major diversifying events. By bringing the knowledge of palaeontology, we propose a new evolutionary scenario for which macro-evolution implying morphological innovations were rhythmed by climatic changes and associated species turn-over that spread from the Eocene to Miocene, and were shaped principally by predation and shell buoyancy.     

Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.462). IFNAR2c mutants truncated at residues 417 (R2. 417) and 346 (R2.346) or IFNAR2c mutant lacking tyrosine residues in its cytoplasmic domain (R2.Y-F) render the receptor inactive. A similar pattern was observed for IFN-inducible STAT activation, STAT complex formation, and STAT-DNA binding. Consistent with these data, IFN-inducible gene expression was ablated in U5A, R2.Y-F, R2.417, and R2.346 cell lines. The implications are that tyrosine phosphorylation and the 462-417 region of IFNAR2c are independently obligatory for receptor activation. In addition, the distal 53 amino acids of the intracellular domain of IFNAR2c are not required for IFN-receptor mediated STAT activation, ISFG3 or SIF complex formation, induction of gene expression, and inhibition of thymidine incorporation. These data demonstrate for the first time that both tyrosine phosphorylation and a specific domain of IFNAR2c are required in human cells for IFN-dependent coupling of JAK activation to STAT phosphorylation, gene induction, and antiproliferative effects. In addition, human and murine cells appear to require different regions of the cytoplasmic domain of IFNAR2c for regulation of IFN responses.     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

Targeting molecular interactions essential for Plasmodium sexual reproduction

Malaria remains one of the most devastating infectious diseases, killing up to a million people every year. Whereas much progress has been made in understanding the life cycle of the parasite in the human host and in the mosquito vector, significant gaps of knowledge remain. Fertilization of malaria parasites, a process that takes place in the lumen of the mosquito midgut, is poorly understood and the molecular interactions (receptor–ligand) required for Plasmodium fertilization remain elusive. By use of a phage display library, we identified FG1 (Female Gamete peptide 1), a peptide that binds specifically to the surface of female Plasmodium berghei gametes. Importantly, FG1 but not a scrambled version of the peptide, strongly reduces P. berghei oocyst formation by interfering with fertilization. In addition, FG1 also inhibits P. falciparum oocyst formation suggesting that the peptide binds to a molecule on the surface of the female gamete whose structure is conserved. Identification of the molecular interactions disrupted by the FG1 peptide may lead to the development of novel malaria transmission‐blocking strategies.     

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.     

“In vitro” Antifungal activity of protease inhibitors

In the last five years, as HAART has become standard therapy in HIV seropositive or AIDS patients, changes have been noted in the numbers and types of opportunistic fungal infections in these cohorts of patients. Particularly, oropharyngeal candidiasis have become rare in HIV infected patients since the introduction of new anti-HIV drugs of the protease inhibitors type. At the Immunology Institute of the Universidad Central de Venezuela the most frequent protease inhibitors (PIs) used for the treatment of these patients have been: Nelfinavir (Viracept^TM, Roche),Indinavir (Crixivan^® Merck),Ritonavir (Norvir^®, Abbott),Saquinavir (Fortovase^®, Roche).Recently, we observed that recurrent candidiasis was less frequent and no Candidacould be isolated in our patients. A direct relation to the PIs was suspected. In order to assess the in vitro antifungal activity of the afore mentioned protease inhibitors on Candida sp., we used both the well diffusion test and the NCCLS broth microdilution test to assay 100 Candida sp. isolates from HIV seropositive or AIDS patients with syntomatic oropharyngeal Candida infection. In general, the data obtained with the well diffusion test were in agreement with those obtained by the broth microdilution test. All 100 isolates were susceptible to Saquinavir and 32 were susceptible to Indinavir using the NCCLS microdilution test,while 97 were susceptible to Saquinavir and 52 to Indinavir by the well diffusion test. From 17 C. albicans resistant to fluconazole, all were susceptible to Saquinavir by the NCCLS micro method and 16 by the well diffusion test. Our results showed anticandidal activity in vitro of PIs, mainly Saquinavir.This revised version was published online in October 2005 with corrections to the Cover Date.     

A New Algorithm to Diagnose Atrial Ectopic Origin from Multi Lead ECG Systems - Insights from 3D Virtual Human Atria and Torso

Rapid atrial arrhythmias such as atrial fibrillation (AF) predispose to ventricular arrhythmias, sudden cardiac death and stroke. Identifying the origin of atrial ectopic activity from the electrocardiogram (ECG) can help to diagnose the early onset of AF in a cost-effective manner. The complex and rapid atrial electrical activity during AF makes it difficult to obtain detailed information on atrial activation using the standard 12-lead ECG alone. Compared to conventional 12-lead ECG, more detailed ECG lead configurations may provide further information about spatio-temporal dynamics of the body surface potential (BSP) during atrial excitation. We apply a recently developed 3D human atrial model to simulate electrical activity during normal sinus rhythm and ectopic pacing. The atrial model is placed into a newly developed torso model which considers the presence of the lungs, liver and spinal cord. A boundary element method is used to compute the BSP resulting from atrial excitation. Elements of the torso mesh corresponding to the locations of the placement of the electrodes in the standard 12-lead and a more detailed 64-lead ECG configuration were selected. The ectopic focal activity was simulated at various origins across all the different regions of the atria. Simulated BSP maps during normal atrial excitation (i.e. sinoatrial node excitation) were compared to those observed experimentally (obtained from the 64-lead ECG system), showing a strong agreement between the evolution in time of the simulated and experimental data in the P-wave morphology of the ECG and dipole evolution. An algorithm to obtain the location of the stimulus from a 64-lead ECG system was developed. The algorithm presented had a success rate of 93%, meaning that it correctly identified the origin of atrial focus in 75/80 simulations, and involved a general approach relevant to any multi-lead ECG system. This represents a significant improvement over previously developed algorithms.     

Combinatorial treatments including vaccines,chemotherapy and monoclonal antibodies for cancer therapy

Accumulating evidence suggests that despite the potency of cytotoxic anticancer agents, and the great specificity that can be achieved with immunotherapy, neither of these two types of treatment by itself has been sufficient to eradicate the disease. Still, the combination of these two different modalities holds enormous potential for eliciting therapeutic results. Indeed, certain chemotherapeutic agents have shown immunomodulatory activities, and several combined approaches have already been attempted. For instance, chemotherapy has been proven to enhance the efficacy of tumor cell vaccines, and to favor the activity of adoptively transferred tumor-specific T cells. A number of mechanisms have been proposed for the chemotherapy-triggered enhancement of immunotherapy response. Thus, chemotherapy may favor tumor cell death, and by that enhance tumor-antigen cross-presentation in vivo. Drug-induced myelosuppression may induce the production of cytokines favoring homeostatic proliferation, and/or ablate immunosuppression mechanisms. Furthermore, the recently reported synergy between monoclonal antibodies and chemotherapy or peptide vaccination is based upon the induction of endogenous humoral and cellular immune responses. This would suggest that monoclonal antibodies may not only provide passive immunotherapy but can also promote tumor-specific active immunity. This article will review several strategies in which immunotherapy can be exploited in preclinical and clinical studies in combination with other agents and therapeutic modalities that are quite unique when compared with “conventional” combination therapies (ie, treatments with chemotherapeutic drugs or chemotherapy and radiotherapy based protocols). The results from these studies may have significant implications for the development of new protocols based on combinatorial treatments including vaccines, chemotherapy and monoclonal antibodies, suggesting an exciting potential for therapeutic synergy with general applicability to various cancer types. Given the complicity of immune-based therapies and cancer pharmacology, it will be necessary to bring together cancer immunologists and clinicians, so as to provide a robust stimulus for realizing the successful management of cancer in the near future.     

Pyridones as glucokinase activators: Identification of a unique metabolic liability of the 4-sulfonyl-2-pyridone heterocycle

A promising area of novel anti-diabetic therapy involves identification of small molecule activators of the glucokinase enzyme to reduce blood glucose and normalize glucose stimulated insulin secretion. Herein, we report the identification and optimization of a series of 4-sulfonyl-2-pyridone activators. The activators were evaluated for in vitro biochemical activation and pharmacokinetic properties. As part of these efforts, a unique metabolic liability of the 4-sulfonyl-2-pyridone ring system was identified wherein this heterocycle readily undergoes conjugation with glutathione under non-enzymatic conditions.