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Bovine leukemia virus (BLV) and human T-lymphotrophic virus type-1 (HTLV-1) are related retroviruses associated with persistent and lifelong infections and a low incidence of lymphomas within their hosts. Both viruses can be spread through contact with bodily fluids containing infected cells, most often from mother to offspring through breast milk. Each of these complex retroviruses contains typical gag, pol, and env genes but also unique, nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the pathogenesis of each virus. Comparisons of BLV and HTLV-1 provide insights into mechanisms of spread and tumor formation, as well as potential approaches to therapeutic intervention against the infections.

In Phenomenology of Spirit (1806) Hegel is often held to have announced the end of history, where 'history' is to be understood as the long pursuit of ends towards which humanity had always been striving. In this, the first book in English to thoroughly critique this entrenched view, Eric Michael Dale argues that it is a misinterpretation. Dale offers a reading of his own, showing how it sits within the larger schema of Hegel's thought and makes room for an understanding of the 'end of history' as Hegel intended. Through an elegant analysis of Hegel's philosophy of history, Dale guides the reader away from the common misinterpretation of the 'end of history' to other valuable elements of Hegel's arguments which are often overlooked and deserve to endure. His book will be of great interest to scholars and advanced students of Hegel, the philosophy of history, and the history of political thought.

Adult T cell leukemia/lymphoma (ATL) is a highly aggressive CD4+/CD25+ T-cell malignancy caused by human T cell lymphotropic virus type 1 (HTLV-1). Previous studies in the MET-1 cell/NOD/SCID mouse model of ATL demonstrated that MET-1 cells are very susceptible to measles virus (MV) oncolytic therapy. To further evaluate the potential of MV therapy in ATL, the susceptibility of several HTLV-1 transformed CD4+ T cell lines (MT-1, MT-2, MT-4 and C8166-45) as well as HTLV-1 negative CD4+ T cell lines (Jurkat and CCRF-CEM) to infection with MV was tested in vitro. All cell lines were permissive to MV infection and subsequent cell death, except MT-1 and CCRF-CEM cells which were susceptible and permissive to MV infection, but resistant to cell death. The resistance to MV-mediated cell death was associated with IFNβ produced by MT-1 and CCRF-CEM cells. Inhibition of IFNβ rendered MT-1 and CCRF-CEM cells susceptible to MV-mediated cell death. Cells susceptible to MV-induced cell death did not produce nor were responsive to IFNβ. Upon infection with Newcastle Disease Virus (NDV), MT-1 and CCRF-CEM but not the susceptible cell lines up-regulated pSTAT-2. In vivo, treatment of tumors induced by MT-1 cell lines which produce IFNβ demonstrated only small increases in mean survival time, while only two treatments prolonged mean survival time in mice with MET-1 tumors deficient in type I interferon production. These results indicate that type I interferon production is closely linked with the inability of tumor cells to respond to type I interferon. Screening of tumor cells for type I interferon could be a useful strategy to select candidate patients for MV virotherapy.

The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30 II of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30 II directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30 II -mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30 II -mediated transactivation. In addition, Gal4(BD)-p30 II -mediated transactivation was competitively inhibited by the cotransfection of CMV-p30 II -HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30 II colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30 II to a highly conserved KIX region. DNA binding assays confirmed the interference of p30 II with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30 II and mediates its transcriptional effects in vivo.

The majority of patients with adult T-cell leukemia/lymphoma (ATL) resulting from human T-cell lymphotropic virus type-1 (HTLV-1) infection develop humoral hypercalcemia of malignancy (HHM). We used an animal model using severe combined immunodeficient (SCID)/beige mice to study the pathogenesis of HHM. SCID/beige mice were inoculated intraperitoneally with a human ATL line (RV-ATL) and were euthanized 20 to 32 days after inoculation. SCID/beige mice with engrafted RV-ATL cells developed lymphoma in the mesentery, liver, thymus, lungs, and spleen. The lymphomas stained positively for human CD45RO surface receptor and normal mouse lymphocytes stained negatively confirming the human origin of the tumors. The ATL cells were immunohistochemically positive for parathyroid hormone-related protein (PTHrP). In addition, PTHrP mRNA was highly expressed in lymphomas when compared to MT-2 cells (HTLV-1-positive cell line). Mice with lymphoma developed severe hypercalcemia. Plasma PTHrP concentrations were markedly increased in mice with hypercalcemia, and correlated with the increase in plasma calcium concentrations. Bone densitometry and histomorphometry in lymphoma-bearing mice revealed significant bone loss because of a marked increase in osteoclastic bone resorption. RV-ATL cells contained 1.5 HTLV-1 proviral copies of the tax gene as determined by quantitative real-time polymerase chain reaction (PCR). However, tax expression was not detected by Western blot or reverse transcriptase (RT)-PCR in RV-ATL cells, which suggests that factors other than Tax are modulators of PTHrP gene expression. The SCID/beige mouse model mimics HHM as it occurs in ATL patients, and will be useful to investigate the regulation of PTHrP expression by ATL cells in vivo.

The ability of a peptide vaccine derived from the human T-lymphotropic virus type 1 (HTLV-1) surface envelope glycoprotein protein (gp46) to mimic the native protein and elicit a protective immune response has been examined. This peptide construct, designated MVFMF2, comprises amino acids (aa) 175-218 of gp46 linked by a four residue turn (GPSL) to a promiscuous T-cell epitope from the measles virus fusion protein (MVF, aa 288-302). The peptide was structurally characterized by circular dichroism (CD) spectroscopy and was found to contain alpha-helical secondary structure. The immunogenicity of MVFMF2 in rabbits and mice was evaluated by direct ELISA and competitive ELISA using peptide constructs and the recombinant protein ACH-RE3 (aa 165-306). This peptide, when administered with adjuvant (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine, nor-MDP) was immunogenic in an outbred population of both rabbits and mice. Furthermore, the peptide construct was encapsulated in biodegradable microspheres of poly(D,L-lactide-co-glycolide) to eliminate booster immunization and to examine adjuvant requirements. The data indicate that MVFMF2 shows enhanced immunogenicity when encapsulated in biodegradable microspheres. Inoculation of the encapsulated peptide produced a similar humoral response to that of the free peptide, but did not require the use of adjuvant. Elicited anti-rabbit and anti-mouse antibodies recognized whole viral preparations and the recombinant protein ACH-RE3 in ELISA assays. Additionally, inoculated rabbits exhibited enhanced reactivity to viral antigens by western blot compared to non-vaccinated controls. Although anti-rabbit and anti-mouse antibodies were capable of inhibiting syncytium formation at low dilutions, rabbits were not protected from cell-associated viral challenge. Future development of vaccines to HTLV-1 may need to incorporate the ability to elicit cell-mediated immune responses in order to protect against cell-associated viral infection.

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containing regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases associated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12, (I) that contains four SH3 binding motifs (PXXP) that localizes to cellular endomembranes when overexpressed in cultured cells. Differential splicing of pX ORF II results in the production of two nuclear proteins, p13(II) and p30(II). p13(II) also localizes to mitochondria. p30(II) shares homology with the POU family of transcription factors. We have identified functional roles of pX ORF I and ORF II in establishment and maintenance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for its ability to infect quiescent peripheral blood mononuclear cells (PBMC). Our data indicate that T cells infected with the wild-type clone of HTLV-1 (ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in the activation of quiescent lymphocytes, a prerequisite for effective viral replication in vivo. To test the ability of p30(II) to function as a transcription factor we have constructed p30(II) as a Gal4-fusion protein. When transfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusion protein induces transcriptional activity up to 50-fold in both 293 and HeLa-Tat cells. These systems will be useful to identify molecular mechanisms that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. Emerging evidence indicates an important role for the pX ORF I-encoded accessory protein p12Iin viral replication, but its contribution to viral pathogenesis remains to be defined. p12Iis a conserved, membrane-associated protein containing four SH3-binding motifs (PXXP). Its interaction with the interleukin-2 (IL-2) receptor β- and γ-chains implies an involvement of p12Iin intracellular signaling pathways. In addition, we have demonstrated that expression of pX ORF I p12Iis essential for persistent infection in rabbits. In contrast, standard in vitro systems have thus far failed to demonstrate a contribution of p12Ito viral infectivity and ultimately cellular transformation. In this study we developed multiple in vitro coculture assays to evaluate the role of p12Iin viral infectivity in quiescent peripheral blood mononuclear cells to more accurately reflect the virus-cell interactions as they occur in vivo. Using these assays, we demonstrate a dramatic reduction in viral infectivity in quiescent T lymphocytes for a p12 mutant viral clone (ACH.p12) in comparison to the wild-type clone ACH. Moreover, addition of IL-2 and phytohemagglutinin during the infection completely rescued the ability of ACH.p12 to infect primary lymphocytes. When newly infected primary lymphocytes are used to passage virus, ACH.p12 also exhibited a reduced ability to productively infect activated lymphocytes. Our data are the first to demonstrate a functional role for pX ORF I in the infection of primary lymphocytes and suggest a role for p12Iin activation of host cells during early stages of infection.

Walleye dermal sarcoma (WDS) is a common disease of walleye fish in the United States and Canada. These proliferative lesions are present autumn through winter and regress in the spring. Walleye dermal sarcoma virus (WDSV), a retrovirus distantly related to other members of the family Retroviridae , has been etiologically linked to the development of WDS. We have reported that the D-cyclin homologue [retroviral (rv) cyclin] encoded by WDSV rescues yeast conditionally deficient for cyclin synthesis from growth arrest and that WDSV-cyclin mRNA is present in developing tumors. These data strongly suggest that the rv-cyclin plays a central role in the development of WDS. To test the ability of the WDSV rv-cyclin to induce cell proliferation, we have generated transgenic mice expressing the rv-cyclin in squamous epithelia from the bovine keratin-5 promoter. The transgenic animals were smaller than littermates, had reduced numbers of hair follicles, and transgenic females did not lactate properly. Following injury the transgenic animals developed severe squamous epithelial hyperplasia and dysplasia with ultrastructural characteristics of neoplastic squamous epithelium. Immunocytochemistry studies demonstrated that the hyperplastic epithelium stained positive for cytokeratin and were abnormally differentiated. Furthermore, the rv-cyclin protein was detected in the thickened basal cell layers of the proliferating lesions. These data are the first to indicate that the highly divergent WDSV rv-cyclin is a very potent stimulator of eukaryotic cell proliferation and to demonstrate the potential of a cyclin homologue encoded by a retrovirus to induce hyperplastic skin lesions.

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia and is characterized by long periods of clinical latency with low levels of viral production. Transcription of HTLV-1 is controlled through sequences in the promoter and enhancer regions of the long terminal repeat of the integrated provirus. Important among these sequences are three 21 bp imperfect repeats responsive to the viral oncogenic protein Tax (TRE). Members of the CREB/ATF-1/CREM family of transcription factors bind to TRE-1 and are critical for HTLV-1 transcription. Other less studied family members include the inducible cAMP early repressor (ICER) proteins. ICER proteins lack phosphorylation and activation domains and are potent inhibitors of transcription. The ability of ICER to bind TRE-1 and its effects on HTLV-1 Tax mediated transcription have not been studied in the natural cell targets of the virus, peripheral blood mononuclear cells (PBMC). We show that ICER mRNA levels are low in quiescent PBMC, but rise and remain elevated for up to 18 hr after mitogenic stimulation of these cells. Electrophoretic mobility shift assays using recombinant Tax and ICER demonstrate that ICER binds TRE-1 and that binding is increased in the presence of Tax. Furthermore, over expression of ICER IIgamma suppressed Tax-mediated transcription whereas an anti-sense ICER II plasmid designed to block endogenous ICER enhanced Tax-mediated transcription in activated PBMC. Together our data indicate that ICER inhibits Tax-mediated transcription in activated PBMC and suggest a role for ICER in maintenance of HTLV-1 persistence.

Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12 I has a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (α, β, or γ c ) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.

HTLV-I is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with tropical spastic paraparesis/HTLV-I-associated myelopathy. Following integration into the host cell genome, HTLV-I replication is regulated by both host and viral mechanisms that control transcription. Low levels of viral transcription (basal transcription) occur before expression of the virally encoded Tax protein (Tax-mediated transcription). Members of the cyclic adenosine monophosphate (cAMP) response element binding (CREB)/activating transcription factor 1 (ATF-1) family of transcription factors bind three 21-bp repeats (Tax-responsive element-1, or TRE-1) within the viral promoter and are important for basal and Tax-mediated transcription. Using mitogen stimulated and quiescent peripheral blood mononuclear cells (PBMC) and Jurkat cells, we compared differences in basal transcription and amounts and binding of transcription factors with TRE-1. We demonstrate that amounts of transcriptionally active phosphorylated CREB protein (P-CREB) differ between activated PBMC and Jurkat cells. Following stimulation, P-CREB levels remain elevated in PBMC for up to 24 hours whereas CREB is dephosphorylated in Jurkat cells within 4 hours following stimulation. The differences in P-CREB levels between PBMC and Jurkat cells were directly correlated with basal transcription of HTLV-I in the two cell types. Using electrophoretic mobility shift assays, we determined that the pattern of band migration differed between the two cell types. These data demonstrate that PBMC differentially regulate basal HTLV-I transcription compared with Jurkat T cells, and this differential regulation is due, in part to differential phosphorylation and binding of CREB/ATF-1 to TRE-1 in the HTLV-I promoter. We demonstrate the utility of using primary lymphocyte models to study HTLV-I transcription in the context of cell signaling and suggest that activated PBMC maintain elevated levels of P-CREB, which promote basal HTLV-I transcription and enhance viral persistence in vivo.

The polymerase chain reaction (PCR) has been established as a highly sensitive technique for detection of viral DNA or RNA. However, due to inherent limitations of PCR the amount of amplified product often does not correlate with the initial amount of template DNA. This is particularly true for PCR detection of viral infections that are characterized by low in vivo viral copy numbers in certain stages of the infection, such as human T-cell lymphotropic virus type 1 (HTLV-1) and simian T-cell lymphotropic virus type 1 (STLV-1). Therefore, we developed a quantitative competitive polymerase chain reaction (qcPCR) for detection of HTLV-1 and STLV-1 proviral DNA. The assay was optimized using an infectious HTLV-1 clone, ACH, HTLV-1 infected cell lines, MT-2.6 and HUT-102 and STLV-1 infected lines Kia and Matsu. Applicability of this system was demonstrated by determining HTLV-1 proviral load in peripheral blood mononuclear cells (PBMC) of human subjects with HTLV-1 associated diseases and an asymptomatic carrier as well as rabbits infected experimentally. This qcPCR method, the first designed specifically for HTLV-1 and STLV-1, will provide an important tool for pathogenesis studies of HTLV-1 and for evaluating the efficacy of antiviral drugs and vaccines against the viral infection using animal models.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and HTLV-1-associated myelopathy. Novel, yet conserved RNA transcripts encoded from open reading frames (ORFs) I and II of the viral pX region are expressed both in vitro and in infected individuals. The ORF I mRNA encodes the protein p12I, which has been shown to localize to cellular endomembranes, cooperate with bovine papillomavirus E5 in transformation, as well as bind to the IL-2 receptor β and γ chains and the H+ vacuolar ATPase. It is unknown what role p12I plays in the viral life cycle. Using an infectious molecular clone of HTLV-1 (ACH) and a derivative clone, ACH.p12I, which fails to produce the p12Imessage, we investigated the importance of p12I in infected primary cells and in a rabbit model of the infection. ACH.p12I was infectious in vitro as shown by viral passage in culture and no qualitative or quantitative differences were noted between ACH and ACH.p12I in posttransfection viral antigen production. However, in contrast to ACH, ACH.p12I failed to establish persistent infection in vivo as indicated by reduced anti-HTLV-1 antibody responses, failure to demonstrate viral p19 antigen production in peripheral blood mononuclear cell (PBMC) cultures, and only transient detection of provirus by polymerase chain reaction in PBMC from ACH.p12I-inoculated rabbits. These results are the first to show the essential role of HTLV-1 p12I in the establishment of persistent viral infection in vivo and suggest potential new targets in antiviral strategies to prevent HTLV-1 infection.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.

Viral protein expression is postulated to play a critical role in the pathogenesis of human T-cell lymphotropic virus type 1 (HTLV-1)-associated diseases. Therefore, knowledge of the cellular events which initiate or enhance viral gene expression is important in understanding the mechanism of HTLV-1-induced disease. In this report, we examined the modulation of transcription of the HTLV-1 long terminal repeat (LTR) following induction of the cellular stress response. We demonstrate by both in vitro transcription assays and transient transfections that induction of the stress response increases basal transcription from the LTR. Transient cotransfection assays indicate that stress induction of viral transcription is Tax independent. In addition, we provide evidence that the sequences responsible for the enhanced transcription are -52 through +157 of the U3/R region of the HTLV-1 LTR. Finally, our data suggest that the increase in transcription is mediated through an intermediate polymerase II/polymerase III transcriptional complex, demonstrated by the inability to abolish the effect with low concentrations of alpha-amanitin.

The learning from incidents and subsequent improvements made for the protection of people and integrity of offshore installations over the last 25 years has no doubt saved numerous lives. Places of refuge, blast protection, rapid isolation processes, offshore survival training and improved evacuation systems have all proved themselves in the prevention of harm either in actual or potential major accidents offshore. Today, when emergency situations arise offshore we consistently witness immediate and efficient activation of tested incident response processes to ensure the safety of personnel through rapid shut down and evacuation. But what about after all personnel offshore are ‘safe’? When the safety of personnel is addressed during a serious ongoing release of oil and gas, operators can be faced with a longer-term, large-scale environmental response. What can we learn from previous incidents to respond effectively to incidents that potentially threaten the environment? It is well known that environmental impacts from the oil & gas industry readily capture public interest, with environmental images remaining at the forefront of media and public attention for many months. This paper examines the environmental response to the G4 well control incident which commenced on 25th March 2012 at the Elgin installation well head platform in the Central North Sea. This is one of the largest North Sea gas/condensate releases in recent history and continued for 51 days until the leak was stopped on 15 May 2012. In the first 24 hours of the incident, all 238 personnel on board the Elgin platform and Rowan Viking jack-up drilling rig which was working over the Elgin well head platform were safely evacuated without any injury. Within the first 24 hours also, TOTAL’s Environment group was activated to implement immediate actions to address and monitor the significant ongoing condensate spill to sea. The presentation of this paper will discuss: Spill combat options mobilisedConcurrent implementation of both medium and long-term response strategiesEnvironmental monitoring approaches and resultsStakeholder interactions (including the regulator, media and other interested parties)Learning and recommendations to industry

We transfected human and rabbit peripheral blood mononuclear cells (PBMC) with the ACH molecular clone of human T-cell lymphotropic virus type 1 (HTLV-1) to study its in vitro and in vivo properties. PBMC transfected with ACH were shown to transfer infection to naive PBMC. ACH transformed rabbit PBMC, as indicated by interleukin-2-independent proliferation of a transfectant culture. This transformant culture was shown by flow cytometric analysis to be a CD4+ CD25+ T-lymphocyte population containing, as determined by Southern blot analysis, at least three integrated HTLV-1 proviral copies. HTLV-1 infection was produced in rabbits inoculated with ACH-transfected, irradiated PBMC. Inoculated rabbits seroconverted to positivity for antibodies against HTLV-1 and had steady or rising HTLV-1 enzyme-linked immunosorbent assay antibody titers. Western blot (immunoblot) analysis revealed sustained seroconversion of rabbits to positivity for antibodies against all major viral antigenic determinants. Infection of rabbits was further demonstrated by antigen capture assay of p24 in PBMC and lymph node cultures and PCR amplification of proviral sequences from PBMC. These data suggest that ACH, like wild-type HTLV-1, infects and transforms primary CD4+ T lymphocytes and is infectious in vivo. This clone will facilitate investigations into the role of viral genes on biological properties of HTLV-1 in vitro and in vivo.

Lymphangiosarcoma was diagnosed from biopsy material obtained from an 8-week-old puppy with a progressively enlarging subcutaneous inguinal swelling. Histologically, the tumor was composed of endothelial cells immediately adjacent to large collagen bundles. Tumor cells formed irregular vascular channels which extended along the connective tissue investments of small vessels and nerves of the subcutis and deep dermis. Similar neoplastic tissue extensively infiltrated an inguinal lymph node. Neoplastic cells were immunohistochemically stained for factor 8-related antigen and were weakly positive when compared with several hemangiomas and hemangiosarcomas. Transmission electron microscopy revealed numerous micropinocytotic vesicles and a continuous basal lamina. The puppy was euthanatized at 8 months of age due to severe septic polyarthritis. Lymphangiosarcoma was documented at the site of the original tumor as well as in the axillary lymph node at necropsy.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with a variety of immunoregulatory disorders. HTLV-1 has been shown to bind to and infect a variety of hematopoietic and nonhematopoietic cells. However, both in vivo and in vitro, the provirus is mostly detected in and preferentially transforms CD4+ T cells. The molecular mechanism that determines the CD4+ T-cell tropism of HTLV-1 has not been determined. Using cocultures of purified CD4+ and CD8+ T cells with an HTLV-1 producing cell line, we measured viral transcription by using Northern (RNA) blot analysis, protein production by using a p24 antigen capture assay and flow cytometric analysis for viral envelope, and proviral integration by using DNA slot blot analysis. We further measured HTLV-1 long terminal repeat-directed transcription in purified CD4+ and CD8+ T cells by using transient transfection assays and in vitro transcription. We demonstrate a higher rate of viral transcription in primary CD4+ T cells than in CD8+ T cells. HTLV-1 protein production was 5- to 25-fold greater in CD4+ cocultures and mRNA levels were 5-fold greater in these cultures than in the CD8+ cocultures. Transient transfection and in vitro transcription indicated a modest increase in basal transcription in CD4+ T cells, whereas there was a 20-fold increase in reporter gene activity in CD4+ T cells cotransfected with tax. These data suggest that unique or activated transcription factors, particularly Tax-responsive factors in CD4+ T cells, recognize regulatory sequences within the HTLV-1 long terminal repeat, and this mediates the observed enhanced viral transcription and ultimately the cell tropism and leukemogenic potential of the virus.

Peripheral cellular immunity of ten dogs with keratoconjunctivitis sicca (KCS) that had not been treated with topical corticosteroids or cyclosporine was evaluated (by use of in vitro lymphocyte proliferation assays and CD4+/CD8+ lymphocyte subset analysis) before and after 1 and 3 months of treatment with topical ocular 2% cyclosporine (CsA). In vitro lymphocyte proliferation and CD4+/CD8+ lymphocyte subset analysis was done in eight normal dogs at the 0, 1 and 3 month time periods to use for comparison. There was no significant difference in lymphocyte proliferation or numbers of CD4+ or CD8+ lymphocytes in dogs with KCS and normal dogs prior to CsA treatment. However, by 1 month's time, lymphocyte proliferation had decreased in the CsA-treated Dogs with KCS, and by 3 months there was a significant difference (P0.0001) from the normal dogs. These results suggest that dogs with KCS may not have altered peripheral cellular immunity and that use of topical 2% cyclosporine for treatment of KCS causes a suppression of lymphocyte proliferation after 1 to 3 months of use.

Isolated and identified more than 30 years ago, Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL), an aggressive lymphoproliferative disease of activated CD4+ T cells, and other inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A variety of animal models have contributed to the fundamental knowledge of HTLV-1 transmission, pathogenesis and to the design of novel therapies to treat HTLV-1 associated diseases. Small animal models (rabbits, rats, mice) as well as large animal models (monkeys) have been utilized to significantly advance characterization of the viral proteins and of virus-infected cells in the early steps of infection, as well as in the development of leukemogenic and immunopathogenic processes. Over the past two decades, the creation of new immuno-compromised mouse strains that are robustly reconstituted with a functional human immune system (HIS) after being transplanted with human tissues or progenitor cells has revolutionized the in vivo investigation of viral infection and pathogenesis. Recent observations obtained in HTLV-1-infected humanized HIS mice that develop lymphomas provide the opportunity to study the evolution of the proviral clonality in human T cells present in different lymphoid organs. Current progress in the improvement of those humanized models will favor the testing of drugs and the development of targeted therapies against HTLV-1-associated diseases.

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15-20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3-5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma, or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as p30, p12, p13 and the antisense-encoded HTLV-1 basic leucine zipper factor (HBZ). While progress has been made in knowledge of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full-length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1.

Human T lymphotropic virus type 1 (HTLV-1) and HTLV-2 are related but pathogenically distinct viruses. HTLV-1 mainly causes adult T cell leukemia, while HTLV-2 is not associated with leukemia. In vitro , HTLV-1 and HTLV-2 predominantly transform CD4 + and CD8 + T cells, respectively: the genetic determinant maps to the viral envelope. Herein, we investigate whether this transformation tropism occurs during initial infection or subsequently during the cellular transformation process. Since most individuals are chronically infected at the time of detection, we utilized an established rabbit model to longitudinally measure the early HTLV-1 and HTLV-2 infection and replication kinetics in purified CD4 + and CD8 + T cells. HTLV-1 and HTLV-2 were detected in both CD4 + and CD8 + T cells within 1 week postinoculation. In HTLV-1-infected rabbit CD4 + T cells, proviral burden and tax / rex mRNA expression peaked early, and expression levels were directly proportional to each other. The late expression of the antisense transcript ( Hbz or Aph-2 ) correlated directly with a late proviral burden peak in HTLV-1- or HTLV-2-infected rabbit CD8 + T cells, respectively. This study provides the first in vivo evidence that these viruses do not exhibit cellular preference during initial infection. We further evaluated the transformation tropism of HTLV-1 and HTLV-2 over a 9-week period using in vitro cell growth/immortalization assays. At the early weeks, both HTLV-1 and HTLV-2 showed proportionate growth of CD4 + and CD8 + T cells. However, beyond week 5, the predominance of one particular T cell type emerged, supporting the conclusion that transformation tropism is a postinfection event due to selective clonal expansion over time.

INTRODUCTION AND OBJECTIVES: Heat shock proteins (HSPs) are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system. Furthermore, several HSPs are implicated with the prognosis of specific cancers. HSP60 and HSP90 have been proposed as prognostic factors for bladder cancer (BC). Since HSPs proteins are among the most immunogenic reported molecules and BCG therapy is immune dependent, the role of HSPs in patients with BC treated with BCG warrants investigation. Previously, our group showed that in primary T1 BC treated with BCG, FGRF3 mutation and protein overexpression were associated with a decreased risk of tumor progression. Here, we evaluated HSP70 expression levels and its relationship to pathological and clinical parameters in the same group of previously untreated primary T1 BC treated with BCG. We chose this patient specific BC population to minimize the influence of other factors such as previous treatments. METHODS: 69 patients diagnosed with primary T1 BC (confirmed by pathological review) treated at the University Health Network, Toronto were included in the study. Microarrays were built and HSP70 protein expression was determined by standard immunohistochemistry (HSP70 Antibody, StressMarq Biosciences Inc, Victoria, BC, Canada). Slides were co-reviewed with an experienced uro-pathologist with staining scores dependent on the expression and intensity of the marker. HSPs expression was correlated with pathological, clinical outcomes and with the expression of FGFR3. FGFR3 mutation status was examined by multiplex PCR-SNaPshot analysis. Kaplan-Meier method and multivariate Cox-regression analysis were used for data analysis. RESULTS: Mean age of patients was 71.1 years ( 8.5). HSP70 was found to be expressed in 29/53 (55%) high-grade tumors and in 9/14 (64%) low-grade tumors. Kaplan-Meier survival analysis demonstrated that the lack of HSP70 expression was a significant predictor for disease recurrence (p 0.05) but did not affect progression. In a multivariate model adjusting for grade, size and concomitant CIS, lack of HSP70 expression remained a significant predictor for recurrence (HR of 1.952, 95% CI 1.02-3.75; p 0.045). HSP70 was shown to correlate with FGFR3 expression and mutation (p 0.05). CONCLUSIONS: HSP70 is a promising marker in T1 BC treated with BCG. Both HSP70 and FGFR3 may play an important prognostic role in T1 BC identifying a group at lower risk of recurrence.

nodal stage, they are associated with inferior disease outcomes. Detection of even 1 CTC/7.5 mL blood in UCB patients prior to RC is an independent predictor for disease recurrence and cancer-related death. These findings are very important for future investigations, as they are in contradiction to theories supporting a cut-off value of 5 or more CTC needed for accurate outcome prediction. Therefore, CTC may represent a feasible biomarker for monitoring response to neoadjuvant and adjuvant chemotherapy.

A group of nursing, social work, education, and English faculty worked together for a year to explore how literature experiences designed for medical education might enhance professional preparation in their fields and address their common dilemmas of caregiving. The resulting insights reveal the ways in which adaptations of narrative medicine models offer benefits for students in these “caring professions.” They also indicate the promise of interdisciplinary reading experiences among students from these fields and suggest how these frameworks might address their common challenges of burnout and erosion of empathy in early clinical experience. This “open letter” to future students who will participate in an interdisciplinary reading group describes the challenges facing the professions of nursing, social work, and education, and explores the ways that doing narrative work together will prepare students to meet them.

Our students are busy, busy taking classes, busy working part-time (sometimes full-time) jobs, busy talking/texting on their cell phones, busy surfing the Internet searching for information to complete the most recently assigned paper or project. All of this busy-ness occurs within the contexts of environments filled with the visual and auditory noise of twenty-first-century American life inside and outside of classrooms and schools. The activities and the environment are the taken-for-granted milieu in the lives of our students. In our own lives as well if we are honest with ourselves. We do not, like Jakob in Anne Michael’s novel Fugitive Pieces (1997), know how “to seek by way of silence” (111). It is important to point out that the silence I reveal in this chapter is of a particular kind, because as Paul Woodruff argues, “Excessive noise and [a particular species] of silence both fall away from the ideal of reverence in the classroom” (2001, 193).

Whole blood lysis and fixation methods for flow cytometric (FCM) analysis were tested for their ability to reduce the infectivity of human T-lymphotropic virus type 1 (HTLV-I). Our goals were to: (1) determine the effects of 1.0 and 2.0% paraformaldehyde (PF) fixation on HTLV-I infected cell lines and (2) assess the infectivity of blood samples containing HTLV-I-infected cells following processing with 5 commercially available products (Immuno-lyse, ImmunoPrep/Q-Prep, FACS lysis solution, GenTrak lyse and fix reagent, and Ortho-mune lysing reagent) compared to ammonium chloride lysis with either 0.1 or 1.0% PF fixation. Infectivity was determined by monitoring HTLV-I p24 antigen production in cocultures of treated leukocytes with uninfected peripheral blood mononuclear cells (PBMC). Each method effectively reduced the viability of treated leukocytes. Commercial lysis/fixation methods significantly reduced HTLV-I infectivity compared to prepared ammonium chloride/PF-based methods. For all preparations, increasing the time of fixation (e.g., 60 min) effectively reduced viral infectivity. Taken together, these data suggest that commercially available fixatives greatly reduce, but do not eliminate the risk of HTLV-I infection during processing of viral-infected cells for FCM analysis.