The safety and effectiveness of a long-acting transdermal fentanyl solution compared with oxymorphone for the control of postoperative pain in dogs: a randomized, multicentered clinical study.

A prospective, double-blinded, positive-controlled, multicenter, noninferiority study was conducted to evaluate the safety and effectiveness of transdermal fentanyl solution (TFS) compared with oxymorphone for the control of postoperative pain in dogs. Five hundred and two (502) client-owned dogs were assigned to a single dose of TFS (2.7 mg/kg) applied 2-4 h prior to surgery or oxymorphone hydrochloride (0.22 mg/kg) administered subcutaneously 2-4 h prior to surgery and q6h through 90 h. Pain was evaluated over 4 days by blinded observers using a modified Glasgow composite pain scale, and the a priori criteria for treatment failure was a pain score ≥ 8 or adverse event necessitating withdrawal. Four TFS- and eight oxymorphone-treated dogs were withdrawn due to lack of pain control. Eighteen oxymorphone-treated, but no TFS-treated dogs were withdrawn due to severe adverse events. The one-sided upper 95% confidence interval of the difference between TFS and oxymorphone treatment failure rates was -5.3%. Adverse events associated with oxymorphone were greater in number and severity compared with TFS. It was concluded that a single administration of TFS was safe and noninferior to repeated injections of oxymorphone for the control of postoperative pain over 4 days at the dose rates of both formulations used in this study.

Pharmacokinetics and dose selection of a novel, long-acting transdermal fentanyl solution in healthy laboratory Beagles.

A novel, transdermal fentanyl solution (TFS) was developed that delivers sustained concentrations of fentanyl for days following a single application. The pharmacokinetics following a single topical dose was examined. Eighteen adult Beagle dogs were divided into three groups of six dogs (3M, 3F). Each group was administered a single dose of 1.3 (25), 2.6 (50), or 5.2 mg/kg (100 µL/kg) of TFS. The dose was applied to the clipped, ventral abdominal skin using a 1-mL tuberculin syringe. Immediately following dosing, collars were placed on each dog through 72 h to prevent direct licking of the application site. Serial jugular venous blood samples were collected at 0 (predosing), 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 144, 168, 240, 336, 408, and 504 h after dosing and assayed for plasma fentanyl concentration. Fentanyl was rapidly detected following application with a mean absorption lag time (t(lag) ) of 0.333 h in the 1.3 mg/kg group and 0 in the other two groups. The mean C(max) increased with dose and were 2.28, 2.67, and 4.71 ng/mL in the 1.3, 2.6 and 5.2 mg/kg dose groups, respectively. Mean terminal half-lives were 53.7, 69.6, and 103 h in the 1.3, 2.6, and 5.2 mg/kg dose groups, respectively. The mean AUC(0-LLOQ) from lowest to highest dose groups were 157, 268, and 645 ng·h/mL and were dose proportional with a R(2) value of 0.9818. Adverse reactions were limited to the highest dose group and included sedation (four of six dogs) and decreased food and water intake (one dog). A dose of 2.6 mg/kg (50 µL/kg) is proposed for further development studies based on the lack of adverse events that were observed compared to the 5.2 mg/kg group and a more rapid onset of action and longer duration of action compared to the 1.3 mg/kg group.

Pharmacokinetics and the effect of application site on a novel, long-acting transdermal fentanyl solution in healthy laboratory Beagles.

Application of transdermal drugs to different anatomical sites can result in different absorption characteristics. The pharmacokinetics (PKs) and bioequivalence of a single 2.6 mg/kg (50 µL/kg) dose of a novel, long-acting transdermal fentanyl solution were determined when applied topically to the ventral abdominal or dorsal interscapular skin of 40 healthy laboratory Beagles. The PKs were differentiated by a more rapid initial absorption of fentanyl from the dorsal application site. Mean plasma fentanyl concentrations remained above 0.6 ng/mL from 4 to 96 h in the dorsal application group and from 8 to 144 h in the ventral application group. Bioequivalence analysis demonstrated that the sites were not equivalent; the 90% confidence intervals of the ratio of the geometric means for both the maximum concentration (C(max)) and the area under the curve (AUC) were not contained within the 80-125% interval. The C(max) was 2.34 ± 1.29 (mean ± standard deviation) and 2.02 ± 0.84 ng/mL for the ventral and dorsal application groups, respectively. The terminal elimination half-lives (t(1/2)) for both groups were similar with values of 137 ± 58.9 and 117 ± 59.6 h for the ventral and dorsal application site groups, respectively. A mean absorption rate of ≥ 2 µg · kg/h was maintained from 2 to 144 h following dorsal application and from 2 to 264 h following ventral application. These results suggest that transdermal fentanyl solution could be applied as a single dose to the dorsal scapular area 2-4 h prior to surgery with analgesia lasting a minimum of 4 days.

Naloxone reversal of an overdose of a novel, long-acting transdermal fentanyl solution in laboratory Beagles.

Opioid overdose in dogs is manifested by clinical signs such as excessive sedation, bradycardia, and hypothermia. The ability of two different intramuscular (i.m.) naloxone reversal regimens to reverse the opioid-induced effects of a fivefold overdose of long-acting transdermal fentanyl solution was evaluated in dogs. Twenty-four healthy Beagles were administered a single 13 mg/kg dose (fivefold overdose) of transdermal fentanyl solution and randomized to two naloxone reversal regimen treatment groups, hourly administration for 8 h of 40 (n = 8) or 160 µg/kg i.m. (n = 16). All dogs were sedated and had reduced body temperatures and heart rates (HRs) prior to naloxone administration. Both dosage regimens significantly reduced sedation (P < 0.001), and the 160 µg/kg naloxone regimen resulted in a nearly threefold lower odds of sedation than that of the 40 µg/kg i.m. naloxone regimen (P < 0.05). Additionally, naloxone significantly increased the mean body temperatures and HR (P < 0.001), although the 160 µg/kg regimen increased body temperature and HR more (P < 0.05). However, the narcotic side effects of fentanyl returned within 1-3 h following termination of the naloxone dosage regimens. The opioid-induced effects of an overdose of transdermal fentanyl solution can be safely and effectively reversed by either 40 or 160 µg/kg i.m. naloxone administered at hourly intervals.

The margin of safety of a single application of transdermal fentanyl solution when administered at multiples of the therapeutic dose to laboratory dogs.

Previous studies have demonstrated that a single, topical application of a novel, long-acting transdermal fentanyl solution provides analgesic fentanyl concentrations for at least 4 days. The objective of this study was to describe the margin of safety following application at multiples of the therapeutic dose. Twenty-four laboratory dogs were administered a single placebo or 1×, 3×, or 5× multiple of the dose of 2.6 mg/kg (50 µL/kg) to the ventral abdominal skin and observed for 14 days. Plasma fentanyl concentrations increased in proportion to dose. Adverse reactions in the 1× group were transient and included a low prevalence (≤ 33%) of mild sedation, reduced food intake, modest weight loss, and minimal reductions in heart rate and rectal temperature. Moderate to severe sedation emerged in the 3× and 5× groups, which was associated with a dose-limiting reduction in food and water intake, necessitating maintenance fluid replacement for the first 2 days following application. Also observed in the higher-dose groups were an increased prevalence of abnormal stools and transient lens opacities. All abnormal health observations were completely resolved prior to necropsy on day 14, and there were no histological abnormalities identified. These data support the safe use of the 1× dose and describe the outcome of an overdose of up to 5× dose in the absence of opioid reversal.

Population pharmacokinetics of transdermal fentanyl solution following a single dose administered prior to soft tissue and orthopedic surgery in dogs.

A novel, long-acting transdermal fentanyl solution (TFS) that delivers sustained plasma fentanyl concentrations following a single application for the control of postoperative pain has recently been approved for use in dogs. The pharmacokinetics (PKs) of this formulation have been evaluated in healthy laboratory dogs, but they have not been reported in a clinical population of dogs for which it is indicated. Plasma fentanyl concentrations were determined from 215 dogs following a single, small-volume (∼50 µL/kg) dose of TFS administered 2-4 h prior to orthopedic or soft tissue surgery. A population PK model was fit, and a 1-compartment open PK model with first-order absorption and an absorption lag-time best described the data. No tested clinical covariates had a significant effect on the PKs. The final model adequately described the population PKs and gave results consistent with laboratory PK studies in healthy dogs. The PKs were primarily characterized by a rapid initial increase in plasma fentanyl concentrations and a long terminal half-life of 74.0 (95% C.I. [54.7-113]) h governed by flip-flop kinetics for the typical subject. The plasma fentanyl concentrations were sustained over days in the range considered to be analgesic for postoperative pain in dogs.

The effectiveness of a long-acting transdermal fentanyl solution compared to buprenorphine for the control of postoperative pain in dogs in a randomized, multicentered clinical study.

A prospective, double-blinded, positive-controlled, multicenter, noninferiority clinical study was conducted to evaluate the safety and effectiveness of a long-acting transdermal fentanyl solution (TFS) for the control of postoperative pain. Four hundred forty-five client-owned dogs of various breeds were randomly assigned to receive a single dose of TFS (2.6 mg/kg [∼50 µL/kg]) (N = 223) applied 2-4 h prior to surgery or buprenorphine (20 µg/kg) (N = 222) administered intramuscularly 2-4 h prior to surgery and every 6 h through 90 h. There were 159 (35.7%) males and 286 (64.3%) females ranging from 0.5 to 16 years of age and 3 to 98.5 kg enrolled. Pain was scored using the modified Glasgow Composite Pain Scale with an a priori dropout criteria of ≥ 8 (20 maximum score). The one-sided upper 95% confidence interval of the mean difference between fentanyl and buprenorphine treatment failures was 5.6%, which was not greater than the a priori selected margin difference of 15%. Adverse events attributed to either treatment were minimal in impact and were approximately equal between groups. Sustained plasma fentanyl concentrations provided by a single pre-emptive dose of TFS are safe and effective and are noninferior to repeated injections of buprenorphine in controlling postoperative pain over 4 days. This long-acting fentanyl formulation provides veterinarians with a novel, registered option for the control of postoperative pain in dogs that improves dosing compliance and potentially mitigates the disadvantages of oral, parenteral, and patch delivered opioids.

Comparison of gene transfer efficiencies and gene expression levels achieved with equine infectious anemia virus- and human immunodeficiency virus type 1-derived lentivirus vectors.

This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.

Endoplasmic reticulum and cis-Golgi localization of human T-lymphotropic virus type 1 p12(I): association with calreticulin and calnexin.

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. We have demonstrated an important role of pX ORF I expression, which encodes p12(I), in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12(I) may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12(I) in cell activation, we characterized the subcellular localization of p12(I) in transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12(I) accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The location of p12(I) was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12(I) with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12(I) directly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12(I) in the establishment of HTLV-1 infection by activation of host cells.

Rhesus monkey model for fetal gene transfer: studies with retroviral- based vector systems.

Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human. Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days). Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer). PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth. Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.

Analysis of gene transfer efficiency of retrovirus producer cell transplantation for in situ gene transfer to hematopoietic cells.

OBJECTIVE: The aim of this study was to assess the gene transfer efficiency of an in situ administration protocol for hematopoietic stem/progenitor cells in the rhesus macaque (Macaca mulatta) animal model. MATERIALS AND METHODS: Moloney murine leukemia virus amphotropic vector producer cells (1--2 x 10(8) cells/animal) were transplanted into the femoral bone marrow cavities of six macaques. To determine if the levels of gene transfer could be increased, a second injection at the same dose of producer cells was performed into the iliac crest in three of the six macaques. RESULTS: We demonstrated that 0.02-0.1% of peripheral blood mononuclear cells contained the vector transgene for up to 12 months following the initial administration of producer cells. Hematopoietic progenitor cell assays indicated that the neomycin phosphotransferase gene was detected in 10--30% of progenitor cell colonies. A humoral immune response directed toward viral particles was demonstrated in all animals. Additionally, we demonstrated that an increase in the levels of transduced cells, up to 1% of circulating peripheral blood mononuclear cells and granulocytes, contain the transgene following producer cell readministration. CONCLUSIONS: These data demonstrate the successful in situ gene transfer to hematopoietic stem/progenitor cells and circulating peripheral blood mononuclear cells that persists as long as 12 months postinjection, in the absence of any preconditioning.

Repression of tax-mediated human t-lymphotropic virus type 1 transcription by inducible cAMP early repressor (ICER) protein in peripheral blood mononuclear cells.

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.

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.

Comparison of HTLV-I basal transcription and expression of CREB/ATF-1/CREM family members in peripheral blood mononuclear cells and Jurkat T cells.

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.

Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction.

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.

Selective ablation of human T-cell lymphotropic virus type 1 p12I reduces viral infectivity in vivo.

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 p12(I), 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 beta and gamma chains and the H+ vacuolar ATPase. It is unknown what role p12(I) plays in the viral life cycle. Using an infectious molecular clone of HTLV-1 (ACH) and a derivative clone, ACH.p12(I), which fails to produce the p12(I) message, we investigated the importance of p12(I) in infected primary cells and in a rabbit model of the infection. ACH.p12(I) was infectious in vitro as shown by viral passage in culture and no qualitative or quantitative differences were noted between ACH and ACH.p12(I) in posttransfection viral antigen production. However, in contrast to ACH, ACH.p12(I) 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.p12(I)-inoculated rabbits. These results are the first to show the essential role of HTLV-1 p12(I) in the establishment of persistent viral infection in vivo and suggest potential new targets in antiviral strategies to prevent HTLV-1 infection.

Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB.

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.

CD2 signalling induces phosphorylation of CREB in primary lymphocytes.

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP-sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact).

Signaling via the CD2 receptor enhances HTLV-1 replication in T lymphocytes.

Human T lymphotropic virus type 1 (HTLV-1) is considered the etiologic agent of adult T cell leukemia/lymphoma and several chronic progressive immune-mediated diseases. Approximately 1-4% of infected individuals develop disease, generally decades following infection. Increased proviral transcription, mediated by the viral 40-kDa trans-activating protein, Tax, has been implicated in the pathogenesis of HTLV-1-associated diseases. Since the HTLV-1 promoter contains sequences responsive to cyclic AMP and protein kinase C, we hypothesized that lymphocyte activation signals initiated through the TCR/CD3 complex or CD2 receptor promote viral replication in HTLV-1-infected lymphocytes. We demonstrate that mAbs directed against the CD2, but not the CD3 receptor increase viral p24 capsid protein 1.5- to 5.7-fold in CD2/CD3+ HTLV-1-infected cell culture supernatants. Northern blot analysis demonstrated a 2.5- to 4-fold increase in all species of viral mRNA following CD2 cross-linking of OSP2/4 cells, an immortalized HTLV-1 cell line. Consistent with transcriptional regulation, reporter gene activity increased approximately 11-fold in CD2-stimulated Jurkat T cells cotransfected with a Tax-expressing plasmid and a CAT reporter gene construct under control of the HTLV-1 promoter. These data suggest a possible physiologic mechanism, whereby CD2-mediated cell adhesion and lymphocyte activation may promote viral transcription in infected lymphocytes.

Transcriptional modulation of viral reporter gene constructs following induction of the cellular stress response.

In this study, we report that commonly used methods of transient transfection induce the cellular stress response and a recovery period is required following transfection when analyzing cellular stress responsive genes. Four transfection methods were examined for their ability to induce the stress response by measuring the expression of heat shock protein (hsp) 72. We demonstrate that electroporation increases expression of hsp 72 in HUT 78 cells. Additionally, DEAE-dextran and liposome-mediated transfection resulted in increased hsp 72 expression in an adherent cell line (HeLa). Liposome-mediated transfection differentially induced cell stress, dependent on the transfection time in serum-free culture conditions. The stress responsiveness of two viral promoters, the HTLV-1 long terminal repeat and CMV immediate early transcriptional unit were examined. We found the maximal stress-mediated enhancement of transcription with both promoters did not occur until the cells recovered for 24 h following transfection.