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Purpose of review Despite decades of suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and fuel viral rebound if therapy is interrupted. The persistence of viral reservoirs in infected individuals is the main obstacle to achieving HIV-1 eradication or a long-term remission. Accurate assessment of the viral reservoir size is necessary for monitoring the effectiveness of the curative interventions. Here, we review the recent progress in the development of assays to measure HIV-1 persistence, highlighting their key advantages and limitations. Recent findings To estimate the viral reservoir size, a number of assays have been developed that assess different aspects of HIV-1 persistence in ART-treated individuals. These include viral outgrowth assays to measure proviral replication competence, sequencing-based assays to measure genetic intactness of HIV-1 proviruses, and diverse techniques that measure the ability of proviruses to produce viral RNA and/or proteins (transcription and translation competence), with or without ex vivo stimulation. Recent years have seen the development of next-generation reservoir assays that, in addition to measuring viral persistence markers, assess the proviral integration sites and characterize the HIV-1 reservoir cells on the single-cell level. Summary Although no assay yet can measure the HIV-1 reservoir with 100% accuracy, recent technical advances allow reliable estimation of its size and composition.

The World Health Organization (WHO) considers cancer as the deadliest disease, due to the increase in death in the 21 century. In the search for new therapeutic molecules, the scientific researches consider the proviral integration moloney (Pim) kinases as promising therapeutic targets for the treatment of hematological cancers. A series of thirty-four 3,5-disubstituted indole derivatives as potent Pim1 kinase inhibitors were studied using 3D-QSAR (CoMFA and CoMSIA) and molecular docking. CoMFA analysis showed Q2 value of 0.56, R2 value of 0.86 and rtest2 value of 0.78, while CoMSIA analysis showed a Q2 value of 0.73, R2 value of 0.93 and rtest2 value of 0.80. The models were generated using 28 compounds as training set and 6 compounds as test set. Furthermore, the contour maps acquired from the CoMSIA and CoMFA models were used to rationalize the principal structural requirement responsible for the activity. As results, four new compounds were designed in silico. Furthermore, the newly designed compound X1 and the most active compound 28 were subjected to molecular docking study in order to validate their stability. Molecule docking shows that compound X1 has greater stability than compound 28. The newly compounds were evaluated for in silico toxicity properties and verified the five Lipinski rules for wet-lab applicability.

Pim-1 is one of the isoforms of pim proteins comprising pim-1, pim-2 and pim-3. It was basically recognized as proviral integration moloney murine leukemia virus which is associated with T-cell lymphomogenesis. Pim-1 is known to play a crucial role in cell cycle progression and acts as downstream target for the JAK/STAT signaling pathway. Recently it has emerged as a hopeful therapeutic target for cancer treatment as deregulation or over expression of pim causes hematologic cancers. In present article molecular docking based three dimensional quantitative structure and activity relationship and molecular dynamics simulation studies have been carried out on indole derivatives reported as pim-1 inhibitors. Initially docking was carried out to obtain the receptor specific orientation of the molecules and later to understand the structural requirements of pim-1 inhibitors robust 3 D QSAR models were built using CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods. The reliability of the models was established from conventional (r

The proviral integration site of the Moloney leukemia virus (PIM) family includes three homologous members. PIM-1 kinase is an important target in effective therapeutic interventions of lymphomas, prostate cancer and leukemia. In the current work, we performed free energy calculations to calculate the binding affinities of several inhibitors targeting this protein. The alchemical method with integration and perturbation-based estimators and the end-point methods were compared. The computational results indicated that the alchemical method can accurately predict the binding affinities, while the end-point methods give relatively unreliable predictions. Decomposing the free energy difference into enthalpic and entropic components with MBAR reweighting enabled us to investigate the detailed thermodynamic parameters with which the entropy-enthalpy compensation in this protein-ligand binding case is identified. We then studied the conformational ensemble, and the important protein-ligand interactions were identified. The current work sheds light on the understanding of the PIM-1-kinase-inhibitor interactions at the atomic level and will be useful in the further development of potential drugs.

Combination antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) replication and improves immune function. However, due to the persistence of long-lived HIV reservoirs, therapy interruption almost inevitably leads to a fast viral rebound. A small percentage of individuals who are able to control HIV replication for extended periods after therapy interruption are of particular interest because they may represent a model of long-term HIV remission without ART. These individuals are characterized by a limited viral reservoir and low reservoir measures can predict post-treatment HIV remission. However, most individuals with a low reservoir still experience fast viral rebound. In this Perspective, we discuss the possible reasons behind this and propose to develop an integral profile, composed of viral and host biomarkers, that could allow the accurate prediction of post-treatment HIV remission. We also propose to incorporate information on the chromatin context of the proviral integration sites into the characterization of the HIV reservoir, as this likely influences the reactivation capacity of latent proviruses and, together with the actual number of intact proviruses, contributes to the replication competence of the reservoir.

The combined application of linear amplification-mediated PCR (LAM-PCR) protocols with next-generation sequencing (NGS) has had a large impact on our understanding of retroviral pathogenesis. Previously, considerable effort has been expended to optimize NGS methods to explore the genome-wide distribution of proviral integration sites and the clonal architecture of clinically important retroviruses like human T-cell leukemia virus type-1 (HTLV-1). Once sequencing data are generated, the application of rigorous bioinformatics analysis is central to the biological interpretation of the data. To better exploit the potential information available through these methods, we developed an optimized bioinformatics pipeline to analyze NGS clonality datasets. We found that short-read aligners, specifically designed to manage NGS datasets, provide increased speed, significantly reducing processing time and decreasing the computational burden. This is achieved while also accounting for sequencing base quality. We demonstrate the utility of an additional trimming step in the workflow, which adjusts for the number of reads supporting each insertion site. In addition, we developed a recall procedure to reduce bias associated with proviral integration within low complexity regions of the genome, providing a more accurate estimation of clone abundance. Finally, we recommend the application of a “clean-and-recover” step to clonality datasets generated from large cohorts and longitudinal studies. In summary, we report an optimized bioinformatics workflow for NGS clonality analysis and describe a new set of steps to guide the computational process. We demonstrate that the application of this protocol to the analysis of HTLV-1 and bovine leukemia virus (BLV) clonality datasets improves the quality of data processing and provides a more accurate definition of the clonal landscape in infected individuals. The optimized workflow and analysis recommendations can be implemented in the majority of bioinformatics pipelines developed to analyze LAM-PCR-based NGS clonality datasets., SCOPUS: ar.j, info:eu-repo/semantics/published

Little is known about the emergence and persistence of HIV-infected T cell clones in perinatally-infected children. We analyzed peripheral blood mononuclear cells for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8-17.4 months of age and with viremia suppressed for 6-9 years. We obtained 8,662 HIV-1 integration sites from pre-ART and 1,861 sites on ART. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6-9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMC infected ex-vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

In the development of kinase inhibitors, one of the major concerns is selectivity. An effective strategy to achieve high selectivity is to utilize structural differences among kinases to inform inhibitor design. Here, we set out to improve the PIM (proviral integration site for Moloney murine leukemia virus) kinase-inhibitory selectivity of our previously reported 7-azaindole derivative 2, which has promising ADMET properties, by targeting a unique bulge in the ATP-binding pocket. 6-Substituted 7-azaindoles, especially the 6-chlorinated derivatives, proved to be potent and selective PIM kinase inhibitors and appear to be promising lead compounds for future drug discovery.

Objective: Proviral integration site for moloney murine leukemia virus (PIM) kinases are among the contemporary targets for cancer chemotherapy as they play a pivotal role in inhibition of apoptosis and oncogenic signaling. Owing to their structural and functional dissimilarity to other kinases, they can be specifically targeted. The present study is an attempt to establish three dimensional quantitative structure activity relationship (3D QSAR) between some 5-(1 H -Indol-5-yl)-1,3,4-thiadiazol-2-amines for their inhibitory activity against PIM by partial least squares regression (PLSR) analysis. Method: PLSR coupled with different variable selection methods such as stepwise (forward-backward) (SW), genetic algorithm (GA) and simulated annealing (SA) was performed to derive QSAR models and these models were validated for statistical significance internally and externally and robustness. Results: Two most significant models were generated through GA with optimum values of validation parameters. Among these model 1 exhibited q 2 and pred_r 2 values of 0.7523 and 0.8714 and model 2 evinced 0.6577 and 0.7675 respectively. The steric field point in model 1 suggests the need of more bulky group at this position and the positive coefficients of electrostatic field points at positions E_999, E_1162 respectively indicate the need of electronegative groups for favorable biological activity. Model 2 suggests the requirement of a positive hydrophobic group at H_564 and less electronegative and more electronegative group at E_157 and E_630 respectively for more potent biological activity. Conclusion: More bulky and/or hydrophobic R groups apart from the electrostatic contribution may be favorable for better PIM-1 inhibition of 5-(1 H -indol-5-yl)-1,3,4-thiadiazol-2 amines. Key words: 1,3,4-Thiadiazole-2-amine, PIM, QSAR, Anticancer, PLSR.

Based on the up-regulation of the proviral integration site of the Moloney murine leukemia virus (Pim) kinase family (Pim1, 2, and 3) observed in several types of leukemias and lymphomas, the development of pan-Pim inhibitors is an attractive therapeutic strategy. While only PIM447 and AZD1208 have entered the clinical stages. To elucidate the interaction mechanisms of three Pim kinases with PIM447 and AZD1208, six Pim/ligand systems were studied by homology modeling, molecular docking, molecular dynamics (MD) simulation and molecular mechanics/generalized Born surface area (MM/GBSA) binding free energy calculation. The residues of the top group (Leu44, Val52, Ala65, Lys67, and Leu120 in Pim1) dominated the pan-Pim inhibitors binding to Pim kinases. The residues of the bottom group (Gln127, Asp128, and Leu174 in Pim1) were crucial for Pims/PIM447 systems, while the contributions of these residues were decreased sharply for Pims/AZD1208 systems. It is likely that the more potent pan-Pim inhibitors should be bound strongly to the top and bottom groups. The residues of the left, right and loop groups were located in the loop regions of the binding pocket, however, the flexibility of these regions triggered the protein interacting with diverse pan-Pim inhibitors efficiently. We hope this work can provide valuable information for the design of novel pan-Pim inhibitors in the future.

Communicated by Ramaswamy H. Sarma.

Background: Currently, cancer continues being a dramatically increasing and serious threat to public health. Although many anti-tumor agents have been developed in recent years, the survival rate of patients is not satisfactory. The poor prognosis of cancer patients is closely related to the occurrence of drug resistance. Therefore, it is urgent to develop new anti-tumor agents to make up for the deficiency. Indazoles is an important class of heterocyclic compounds possessing a variety of biological activities, such as anti-tumor, anti-bacterial, antiinflammatory, anti-depressant and anti-hypertensive. This review focuses on the recent research advances of indazole derivatives in the aspect of anti-tumor. Methods: We have searched the recent literatures about indazole derivatives from the online resources and databases, such as pubmed, scifinder and google scholar. Results: In the recent years, many efforts have been taken to develop indazole derivatives as fibroblast growth factor receptor (FGFR) inhibitors, indoleamine-2,3-dioxygenase1 (IDO1) inhibitors, proviral integration site MuLV (Pim) kinase inhibitors, aurora kinases inhibitors, Bcr-Abl inhibitors, hypoxia inducible factor-1 (HIF-1) inhibitors and carbonic anhydrase (CA) inhibitors. Most compounds display good anti-tumor activities. Conclusion: Developing new anti-cancer agents with new scaffolds and high efficiency is a big challenge for researchers. Indazole derivatives are a class of important bioactive compounds. Making structural modifications on active indazole derivatives according to the corresponding structure-activity relationships is of benefit to obtain more potent anti-cancer leads or clinical drugs. This review will be useful for further development of new indazole-based derivatives as anti-cancer agents.

Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.

The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's genome. This integrated proviral DNA can remain replication silent, but a small part of it is fully competent to restart viral replication when treatment is interrupted. Hence, this replication-competent provirus is the cause of viral rebound and is called the viral reservoir. The exact site of proviral integration within the host's cellular chromosome may affect the transcriptional activity of HIV. Thanks to recent technological advances, HIV-1 integration site analysis has been used to assess HIV-1 reservoirs in HIV-infected individuals. Analysis of HIV-1 integration sites in infected individuals undergoing suppressive ART led to identification of expanded clonal cell populations, indicating that clonal proliferation of the proviral reservoir may contribute to the long-term persistence of viral reservoirs. Here we describe the findings of several clinical studies, where a comprehensive HIV-1 integration site analysis was performed.

The colony of wild mice, named Jyg, has been shown to express an exogenous mouse mammary tumor virus (Jyg-MMTV). This virus induces mammary tumors in its natural host at a high incidence (≈ 80%) resulting from insertion mutations in Notch-4 (43%), Wnt-1 (26%), and Fgf-3 (13%). Since the activation of Notch-4 is not common in mammary tumors of standard laboratory strains of mice infected with various MMTV strains, we examined the consequences of Jyg-MMTV infection in BALB/c and C57BL/6 mice. The results show that Jyg-MMTV induces mammary tumors in both mouse strains, but the incidence of mammary tumors in BALB/c mice is greater than in C57BL/6 mice. Surprisingly, however, none of the 75 mammary tumors, analyzed both by Southern and Northern hybridizations, showed insertion mutations in or expression of Notch-4 . In contrast, both Wnt-1 and Fgf-3 were found to be involved in these tumors. Our findings may suggest, among other possibilities, the existence of a structural difference(s) between laboratory and wild mice at the Notch-4 locus that regulates the integration of Jyg-MMTV proviral DNA.

We report here the topics discussed during the round table of the 2nd European Conference & Practical Course: Towards Clinical Gene Therapy: Preclinical Gene Transfer Assessment, held in Bellaterra (Spain), 1-14 February, 2004. First, how to predict the risk of pathologies generated by changes of the gene expression after proviral genome integration. In the light of the scientific information that emerged after the SAEs occurred in three X-SCID patients treated in France, (a) it is necessary to take into the account the dose of vector used in transduction protocols, in order to minimize the risk to target potentially pathogenic loci. Namely, low vector doses are recommended to minimize the number of vector genomes inserted per cell. (b) The potency of vector elements (ie promoter and transgene), in terms of activation of undesired cell function(s), should be elucidated to devise safe transduction protocols. (c) Target cells should be better characterized before and after transduction to avoid reinfusion into patients' cells, with proviral integration that may be pathogenic. (d) The possibility of replacing onco-retroviruses with other vector systems should be envisaged, for example, nonintegrative gene correction strategies. Second, adequate animal models are required in preclinical experimentation before going to clinics. Although animal models are not yet predictive for risk assessment of proviral insertion, they allow validation of the proof of principle of gene therapy strategies and pharmacological characterization of gene transfer products. Third, a dialogue between researchers and members of regulatory agencies is necessary to implement the regulatory frame where gene therapy products are to be used as new bio-pharmaceuticals. This will implement the whole gene therapy process development at both preclinic (research, development and clinical designs) and postclinic (follow-up of patients) stages. Hence, a European cooperation between professionals (researchers, physicians, industries, patients' associations, investors, etc) will allow implementation of gene therapy regulation in Eastern European countries.

In this issue of Blood , Lu et al shed light on the role of proviral integration sites of Moloney 2 (Pim2) kinase in multiple myeloma and delineate the mechanisms by which it mediates clonal plasma cells proliferation. The authors identify the tumor suppressor tuberous sclerosis 2TSC2 as the direct

We recently developed a high-throughput sequencing method for analysis and quantification of HTLV-1 integration sites in the host genome (Gillet et al, 2011, Blood). Using this method we investigated the effect of the genomic environment on integration targeting, clonal expansion and spontaneous HTLV-1 proviral expression (Gillet et al, 2011, Blood, Melamed et al, 2013, PLoS Pathogens). HTLV-2 preferentially infects CD8+ T cells, with a minority of the proviral load in CD4+ T cells. Here we describe the use of our high-throughput technique to investigate the distribution of HTLV-2 proviral integration sites in the host genome, in peripheral blood mononuclear cell (PBMC) DNA of HTLV-2 infected individuals (n=28). We also mapped and quantified proviral integration sites separately in flow-sorted CD4+CD8- and CD4-CD8+ populations. We quantified the clone frequency distribution and clonal survival over time in 10 individuals, using samples from 2 time points separated by a median of 10 years. The results show that the clone frequency distribution of HTLV-2 in PBMCs is distinct from that of HTLV-1 and resembles that of HTLV-1-infected CD8+ T cells. These results suggest that in both HTLV-1 and HTLV-2 infections, there is a greater degree of selective oligoclonal clonal expansion in infected CD8+ T cells than in CD4+ T cells. We are now investigating the selection forces that underlie this dichotomy between T cell lineages.

The retroviral attachment ( att ) sites at viral DNA ends are cis -acting regions essential for proviral integration. To investigate the sequence features of att important for human immunodeficiency virus type 1 (HIV-1) integration in vivo, we generated a series of 25 att mutants of HIV-1 by mutagenesis of the U3, U5, or both boundaries of att . Our results indicated that the terminal 11 or 12 bp of viral DNA are sufficient for specific recognition by HIV-1 integrase (IN) and suggested that IN might recognize each att site independently in vivo.

Background The HTLV-1 proviral load set point is the strongest correlate of the inflammatory and malignant diseases associated with HTLV-1. This set point appears to be determined by an equilibrium between virus-driven proliferation and CTL-mediated killing of HTLV-1-infected T cells. However, we do not know what determines the number, the abundance or the pathogenic potential of HTLV-1-infected T cell clones. In addition, the contribution of de novo infection to HTLV-1 persistence in the host remains uncertain. We hypothesize that the genomic integration site (IS) of the HTLV-1 provirus determines the pattern and intensity of spontaneous proviral expression; the viral gene products in turn determine the rate of proliferation of the infected cells, and the rate of CTL-mediated killing. We aim to identify the factors that determine the integration site targeting, expression and abundance of the HTLV-1 provirus in natural infection.

International audience; Human Immunodeficiency virus type 1 (HIV-1), as well as many other viruses that depend on nuclear entry for replication, has developed an evolutionary strategy to dock and translocate through the nuclear pore complex (NPC). In particular, the nuclear pore is not a static window but it is a dynamic structure involved in many vital cellular functions, as nuclear import/export, gene regulation, chromatin organization and genome stability. This review aims to shed light on viral mechanisms developed by HIV-1 to usurp cellular machinery to favor viral gene expression and their replication. In particular, it will be reviewed both what is known and what is speculated about the link between HIV translocation through the nuclear pore and the proviral integration in the host chromatin.

Texto completo. Acesso restrito. p. 163–166 Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-07-08T12:21:22Z No. of bitstreams: 1 6666666666666666666.pdf: 739213 bytes, checksum: 6e039d1e6abe7b04a48451c821f6b2d1 (MD5) Approved for entry into archive by Rodrigo Meirelles (rodrigomei@ufba.br) on 2013-11-19T20:55:00Z (GMT) No. of bitstreams: 1 6666666666666666666.pdf: 739213 bytes, checksum: 6e039d1e6abe7b04a48451c821f6b2d1 (MD5) Made available in DSpace on 2013-11-19T20:55:00Z (GMT). No. of bitstreams: 1 6666666666666666666.pdf: 739213 bytes, checksum: 6e039d1e6abe7b04a48451c821f6b2d1 (MD5) Previous issue date: 2013 Few cases of acute adult T-cell leukemia/lymphoma (ATL) have been diagnosed in young patients. This report is the first to describe a young girl with infective dermatitis associated with HTLV-1 that progressed to acute ATL with Southern blot hybridization and gamma-TCR-rearrangement revealing a monoclonal pattern with two copies of the provirus. Salvador

Human T lymphotropic virus type 1 (HTLV-1) appears to persist in the chronic phase of infection by driving oligoclonal proliferation of infected T cells. Our recent high-throughput sequencing study revealed a large number (often > 104) of distinct proviral integration sites of HTLV-1 in each host that is greatly in excess of previous estimates. Here we use the highly sensitive, quantitative high-throughput sequencing protocol to show that circulating HTLV-1+ clones in natural infection each contain a single integrated proviral copy. We conclude that a typical host possesses a large number of distinct HTLV-1–infected T-cell clones.

The patterns of mouse mammary tumor virus (MMTV) integration in the DNA of spontaneous-mammary tumors, salivary glands and livers of DD/Tbr mice were examined using MMTV env, int -1c and int-2c probes. The MMTV env probe revealed 1 to 7 new proviral insertions in all mammary tumors. MMTV integration into int-1 was observed in 10 of 18 mammary tumors, whereas that into int-2 was seen in only 2 of 18 tumors. Of the 13 salivary glands examined, only 3 showed new MMTV proviral integrations, but rearrangement in int-1 or int-2 loci by MMTV was not observed. Immuno-collidal gold electron microscopy revealed the presence of MMTV particles both in mammary tumors and in salivary glands, but no tumors were found to be developed in salivary glands. Taken together these results suggest that salivary glands support MMTV replication, but the virions thus produced may not lead to salivary gland tumorigenesis. It is suggested that the salivary gland is the source of horizontally transmitted MMTV in DD/Tbr mice.

Mouse models of human disease are valuable tools for studying pathogenesis and for evaluating novel therapies. T-cell lymphoma is a relatively rare disease in humans, affecting 100-150 persons yearly in Sweden. It exists in both aggressive and more indolent forms. We have established a mouse model for an aggressive T-cell lymphoma, the T-cell lymphoma/leukemia (TLL) mouse. In the present thesis, the TLL mouse model was characterized and used for experimental therapeutic and primary prevention studies. The TLL mouse was established unintentionally in our laboratory during work on VH-gene replacement in a “knock-in” mouse experimental setting. The generated chimeras all developed aggressive T-cell lymphomas affecting the lymphoid organs, lungs, kidneys and liver. The lymphoma phenotype segregated from the targeted locus and we could demonstrate the presence of Moloney murine leukemia virus (MMLV) in the germline of the affected mice. MMLV is a retrovirus known to induce T-cell lymphomas when inoculated in newborn mice. We further characterized two TLL substrains; TLL-2 and TLL-14 carrying the proviral integrations on chromosomes 2 and 14 respectively. Significant differences were found between the substrains regarding lymphoma frequency and immunophenotype, the TLL-14 substrain developing tumors with higher frequency than TLL-2 and with a more mature immunophenotype. A transfer model was developed in which TLL cells could be readily transferred intravenously to syngenic recipients causing aggressive lymphomas. The transfer model was used in a therapeutic study where the selective COX-2 inhibitor celecoxib was evaluated as a single agent and in combination with the established anti-tumor agent cyclophosphamide. The study was based on results from other tumor types that have indicated celecoxib, originally an anti-inflammatory and analgetic drug, to have possible anti-tumor effects. In our TLL model, however, we could not demonstrate any benefit of celecoxib monotherapy or any additive effect to cyclophosphamide. Dietary fatty acids, in particular omega-3 fatty acids, have been a focus of public and scientific interest due to observed effects on the prevention of cardiovascular disease, cancer and inflammatory conditions. In addition, omega-3 fatty acids inhibit T-cell proliferation in vitro. We supplemented the diet of TLL mice with omega-3 and omega-6 fatty acids respectively and could demonstrate a significant delay in lymphoma onset between 5-8 months of age in the group receiving an omega-3 rich diet.

SummaryThis review is comprized of two sections. The first discusses the life-cycle of HIV and potential target sites for anti-HIV drugs. The second reviews anti-HIV agents, which are limited to proteins and peptides in this article. Agents that interfere with events early in the HIV life-cycle are divided into two categories, those that interfere with events prior to viral entry and those that are active after viral entry but prior to proviral integration. These agents can abort virus infection if appropriately used. In contrast, agents interfering with events late in the HIV life-cycle do not abort HIV infection, rather, they suppress events during viral replication such as gene transcription, translation and/or virus assembly and maturation. It is likely that combination therapy with agents inhibiting an event early and another event late in the HIV life-cycle will be most efficacious.Soluble CD4, chimaeric molecules of CD4 and IgG, neutralizing antibodies and N-carbomethoxy-carbonyl-prolyl-phenylalany...

Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14% (8/14) HAM/TSP patients and 27.28% (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.