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At least 721 records · Page 40Linked to original sources
PubMed2001

Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery.

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105+/-9 micrometer in length, and had a doubling time of 39+/-2 hours. Spindle-shaped clones (n=3) were larger (267+/-18 micrometer long, P<0.01) and grew slower (doubling time 65+/-4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) alpha-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca(2+) transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM alpha-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.

Actins↗
PubMedDate not supplied

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part II: preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for use as a standard challenge organism to qualify 0.1 micron rated filters.

In this article, we report on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. Filter-cloned H. pseudoflava (ATCC 700892) was easily cultured in a low nutrient broth (R2A broth) under standard laboratory conditions, reaching high titers of 10(8)-10(9) cfu/mL within 48-65 hours of incubation at 25+/-5 degrees C. Under these conditions, H. pseudoflava is a rod-shaped bacterium, averaging 0.25+/-0.03 microm by 1.65+/-0.35 microm, and appears to be smaller than Brevundimonas diminuta in width (0.31+/-0.03 microm), but somewhat longer in length (0.88+/-0.19 microm), which may partly explain the observed penetration. In total, thirty-five 0.2/0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from two different filter manufacturers were challenged with H. pseudoflava. In all cases, H. pseudoflava was shown to consistently penetrate every 0.2/0.22 microm rated filter disc tested. These tests also spanned three different challenge durations, including short-term challenges (30-40 minutes), and two different challenge fluids. The use of serial (double) 0.22 mm rated filters, which is a common industry practice to reduce the prefiltration bioburden to the final "sterilizing" filter, was also shown to be inadequate to fully retain H. pseudoflava under the challenge condition used. In contrast, two different 0.1 microm rated filter types functionally qualified with a specified high titer reduction claim for Acholeplasma laidlawii, were shown to consistently and fully retain H. pseudoflava, and retention by these two filter types was shown to be robust and independent of the challenge duration.

Acholeplasma↗
PubMed2001

Reconstruction of SEA-B7.1 double signals on human hepatocellular carcinoma cells and analysis of its immunological effect.

Since transfection of established tumors with immunostimulatory genes, such as superantigens (SAg), a family of bacterial and viral proteins with strong immunostimulatory properties, can elicit antitumor immunity, direct transfection of tumors with genes of staphylococcal enterotoxin A (SEA) could probably set up a new way of immunological pathway. In our study, human hepatocellular carcinoma (HCC) cell lines stably transduced with SEA and B7.1/SEA fused genes, HHCCSEA and HHCCBS, were obtained by using the method of retroviral mediated gene transduction. The results showed that human HCC cells could express SEA gene. Although a tiny quantity of expression was detected, a robust immune response was promoted. The cytotoxicity of CTL on HHCCBS was the same as that on HHCCSEA. But the K(m) value of the reaction of the former was lower than that of HHCCSEA. Furthermore, the activity assay of T cells by ELISPOT demonstrated that HHCCBS could elicit more CTL activity than HHCCSEA and HHCCB7.1. It suggested that the affinity of T cells to HHCCBS was higher and the maxim velocity of reaction could be attained at an early stage of the reaction. Transduced HCC cells were also analyzed for HLA expression, and it was found that a majority of the cells expressed HLA-I molecules but no HLA-DR molecules. After blocking the HLA-I molecules by HLA-I mAb, the cytotoxicity of T lymphocytes dropped remarkably. The results suggested that SEA were mainly presented by HLA-I molecules, and that B7.1 and SEA could have synergistic action at the early stage of the reaction, but the relationship between them in the consequent process needs to be clarified.

Carcinoma, Hepatocellular↗
PubMed1999

Encapsulation of rotavirus into poly(lactide-co-glycolide) microspheres.

Two small-scale double emulsion techniques for incorporation of formaldehyde-inactivated rotavirus particles (FRRV) into poly(lactide-co-glycolide) (PLG) microspheres were developed and optimised. The effects of high-speed homogenisation versus vortex mixing on the double emulsion stability, microsphere size, entrapment efficiency and in vitro release of FRRV in the second emulsification step were studied. A stable double emulsion was verified only when using vortex mixing in this step. Slow removal of the organic phase allowed measurement of the size of the emulsion droplets and subsequent prediction of the size of the resulting microspheres. Microspheres in the size range of 1-10 microm were prepared using both techniques. The homogenisation technique was sensitive to changes in the operating time, the emulsification energy and the volume of the outer aqueous phase, while the vortex technique was more robust. Rotavirus was released in vitro in a triphasic manner with both techniques. The more robust vortex technique was selected for preparation of PLG microspheres containing rotavirus for in vivo studies. After immunisation of mice with a single intramuscular injection, the PLG-FRRV microspheres elicited an IgG antibody response in serum detected by ELISA equally high as that elicited with FRRV alone. These results indicate that the antigenicity of FFRV was retained after incorporation into PLG microspheres using the vortex technique.

Animals↗
PubMed2002

Analysis of gene function in somatic mammalian cells using small interfering RNAs.

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.

Animals↗
PubMed1999

Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex.

We report here evidence for mutations that break domain-domain functional communication in a synthetase-tRNA complex. Each synthetase is roughly divided into two major domains that are paralleled by the two arms of the L-shaped tRNA structure. The active-site-containing domain interacts with the acceptor arm of the tRNA. The second domain frequently interacts with the anticodon-containing arm. By an induced-fit mechanism, contacts with the anticodon can activate formation of a robust transition state at a site over 70 A away. This induced-fit-based activation is thought to occur through domain-domain signaling and is seen by the enhancement of aminoacylation of the anticodon-containing full tRNA versus a substrate based on the acceptor arm alone. Here we describe a rationally designed mutant methionyl-tRNA synthetase containing two point substitutions at sites that potentially link an anticodon-binding motif to the catalytic domain. The double mutation had no effect on interactions with either the isolated acceptor arm or the anticodon stem-loop. In contrast to interactions with the separate pieces, the mutant enzyme was severely impaired for binding the native tRNA and lost much of its ability to enhance the rate of charging of the full tRNA over that of a substrate based on the acceptor arm alone. We propose that these residues are part of a network for facilitating domain-domain communication for formation of an active synthetase-tRNA complex by induced fit.

Acylation↗
PubMed2004

Experimental pain models reveal no sex differences in pentazocine analgesia in humans.

BACKGROUND: Accumulating evidence suggests that there are sex differences in analgesic responses to opioid agonists. Several studies using an oral surgery pain model have reported more robust analgesia to kappa-agonist-antagonists (e.g., pentazocine, nalbuphine, butorphanol) among women than among men. However, evidence of sex differences in kappa-agonist-antagonist effects from studies of experimentally induced pain in humans is lacking. METHODS: Therefore, the analgesic effects of intravenous pentazocine (0.5 mg/kg) were determined in healthy women (n = 41) and men (n = 38) using three experimental pain models: heat pain, pressure pain, and ischemic pain. Each pain procedure was conducted before and after double-blind administration of both pentazocine and saline, which occurred on separate days in counterbalanced order. RESULTS: Compared with saline, pentazocine produced significant analgesic responses for all pain stimuli. However, no sex differences in pentazocine analgesia emerged. Effect sizes for the sex differences were computed; the magnitude of effects was small, and an equal number of measures showed greater analgesia in men than in women. Also, analgesic responses were not highly correlated across pain modalities, suggesting that different mechanisms may underlie analgesia for disparate types of pain. CONCLUSIONS: These findings indicate significant analgesic responses to pentazocine in both men and women across multiple experimental pain assays, and the absence of sex differences contrasts with previous data from the oral surgery model. The most likely explanation for the discrepancy in results is that of differences in the pain assays. These findings are important because they suggest that sex differences in opioid analgesia may be specific to certain types of pain.

Adolescent↗
PubMed2002

Time-resolved, undersampled projection reconstruction imaging for high-resolution CE-MRA of the distal runoff vessels.

Imaging of the blood vessels below the knee using contrast-enhanced (CE) MRI is challenging due to the need to coordinate image acquisition and arrival of the contrast in the targeted vessels. Time-resolved acquisitions have been successful in consistently capturing images of the arterial phase of the bolus of contrast agent in the distal extremities. Although time-resolved exams are robust in this respect, higher spatial resolution for the depiction of tight stenoses and the small vessels in the lower leg is desirable. A modification to a high-spatial-resolution T(1)-weighted pulse sequence (projection reconstruction-time resolved imaging of contrast kinetics (PR-TRICKS)) that improves the through-plane spatial resolution by a factor of 2 and maintains a high frame rate is presented. The undersampled PR-TRICKS pulse sequence has been modified to double the spatial resolution in the slice direction by acquiring high-spatial-frequency slice data only after first pass of the bolus of contrast agent. The acquisition reported in the present work (PR-hyperTRICKS) has been used to image healthy volunteers and patients with known vascular disease. The temporal resolution was found to be beneficial in capturing arterial phase images in the presence of asymmetric filling of vessels.

Contrast Media↗
PubMed2006

Bis(n-octylamino)perylene-3,4:9,10-bis(dicarboximide)s and their radical cations: synthesis, electrochemistry, and ENDOR spectroscopy.

1,6- and 1,7-bis(n-octylamino)perylene-3,4:9,10-bis(dicarboximide) were synthesized by reaction of n-octylamine with the corresponding dibromo compounds. These compounds display intense charge-transfer optical transitions in the visible spectrum (approximately 550-750 nm) and fluoresce weakly (Phi(F) < 0.06). Cyclic voltammetry reveals that each chromophore undergoes facile and reversible oxidation and reduction. Spectroelectrochemical studies show that the radical cations of these chromophores are stable and show no signs of deprotonation of the secondary amines. Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) studies of the chemically generated radical cations of these chromophores corroborate the spectroelectrochemical data by showing that the radical cations persist for days at room temperature in methylene chloride solution. These experiments and complementary density functional theory (DFT) calculations provide a comprehensive picture of the molecular orbitals, spin density distributions, and geometries of the radical cations. The redox properties and stability of these alkylamino-functionalized perylene compounds make them a valuable addition to the family of robust perylene-based chromophores that can be used to develop new photoactive charge transport materials.

Journal Article↗
PubMed1999

Reproducibility of measurements of potential doubling time of tumour cells in the multicentre National Cancer Institute protocol T92-0045.

We compared the flow cytometric measurement and analysis of the potential doubling time (Tpot) between three centres involved in the National Cancer Institute (NCI) protocol T92-0045. The primary purpose was to understand and minimize the variation within the measurement. A total of 102 specimens were selected at random from patients entered into the trial. Samples were prepared, stained, run and analysed in each centre and a single set of data analysed by all three centres. Analysis of the disc data set revealed that the measurement of labelling index (LI) was robust and reproducible. The estimation of duration of S-phase (T(S)) was subject to errors of profile interpretation, particularly DNA ploidy status, and analysis. The LI dominated the variation in Tpot such that the level of final agreement, after removal of outliers and ploidy agreement, reached correlation coefficients of 0.9. The sample data showed poor agreement within each of the components of the measurement. There was some improvement when ploidy was in agreement, but correlation coefficients failed to exceed values of 0.5 for Tpot. The data suggest that observer-associated analysis of T(S) and tissue processing and tumour heterogeneity were the major causes of variability in the Tpot measurement. The first two aspects can be standardized and minimized, but heterogeneity will remain a problem with biopsy techniques.

Aneuploidy↗
PubMed1997

[Limits of perceptual robustness in perspective distortion].

Pictures often do not appear distorted even when viewed at oblique angles. Three hypotheses have been put forth to explain this robustness of virtual space toward affine transformations. First, array specificity holds that the perception of depicted space is fully specified by the information available at the point of observation. Second, the notion of a compensatory mechanism involves an unconscious recreation of the scene according to the original viewpoint. Third, the indiscrimination hypothesis denies the ability of the visual system to resolve or detect affine transformations up to a certain degree. Three experiments were conducted to investigate these claims. Using a double projection technique devised by Cutting (1987), Experiment I showed that observers are able to discriminate and compensate, to some degree, for affine transformations if information about the projection surface is available. However, observers relied on relative image velocities rather than reconstructing the object. In Experiment 2 additional observer motion was simulated. In single and double projection trials that required more difficult judgments of object rotation, compensation was poor and observers seemed to rely on local cues. Finally, real and simulated rotation of the projection surface revealed that observers are able to compensate for only one primary projection surface slant. The results reject the indiscrimination hypothesis and support the notion of array specificity.

Adult↗
PubMed1998

Recombinant human glial growth factor 2 (rhGGF2) improves functional recovery of crushed peripheral nerve (a double-blind study).

This in vivo double-blind study evaluated the effect of recombinant human glial growth factor 2 (rhGGF2), a Schwann cell mitogen, on the recovery of motor function of rat sciatic nerve following crush injury. Seventy three rats were divided into three groups. Group I (n=5), sham operated; Groups II (n=34) and III (n=34) received a 100 g crush load for 2 h over a 5 mm segment of the sciatic nerve. Group III was treated with 1 mg/kg rhGGF2, via subcutaneous injection one day before nerve crush and daily for the following four days. Group II received an equivalent volume of saline as a control. Motor functional recovery was assessed by calculating the sciatic functional index (SFI) and the recovery rate of tetanic contractile force of the extensor digitorum longus (EDL) muscle. Recovery of nerve function was evident at day 11 after crush in the rhGGF2-treated animals, whereas the nerves in controls were still paralyzed. The rhGGF2-treated animals showed a significant improvement of the SFI between days 11-21 postoperatively when compared to controls. The isometric tetanic contractile force was stronger in the rhGGF2-treated group than in controls, with a significant difference at 40 to 70 Hz stimulus frequencies on day 4. Correlation analysis showed that tetanic contractile force had a linear correlation with the SFI. Histologic assessment indicated that the rhGGF2-treated animals showed less severe degeneration and earlier robust remyelination of axons than controls. The results suggest that treatment with rhGGF2 is effective in promoting nerve regeneration as seen in measurements of functional recovery and qualitative assessment of nerve morphology. The mechanism of GGF's protective effect may be related to its direct action on Schwann cells, stimulating their mitosis as well as inducing neurotrophic factors essential to neuronal maintenance and repair.

Animals↗
PubMed1995

Electrical consequences of spine dimensions in a model of a cortical spiny stellate cell completely reconstructed from serial thin sections.

We built a passive compartmental model of a cortical spiny stellate cell from the barrel cortex of the mouse that had been reconstructed in its entirety from electron microscopic analysis of serial thin sections (White and Rock, 1980). Morphological data included dimensions of soma and all five dendrites, neck lengths and head diameters of all 380 spines (a uniform neck diameter of 0.1 micron was assumed), locations of all symmetrical and asymmetrical (axo-spinous) synapses, and locations of all 43 thalamocortical (TC) synapses (as identified from the consequences of a prior thalamic lesion). In the model, unitary excitatory synaptic inputs had a peak conductance change of 0.5 nS at 0.2 msec; conclusions were robust over a wide range of assumed passive-membrane parameters. When recorded at the soma, all unitary EPSPs, which were initiated at the spine heads, were relatively iso-efficient; each produced about 1 mV somatic depolarization regardless of spine location or geometry. However, in the spine heads there was a twentyfold variation in EPSP amplitudes, largely reflecting the variation in spine neck lengths. Synchronous activation of the TC synapses produced a somatic depolarization probably sufficient to fire the neuron; doubling or halving the TC spine neck diameters had only minimal effect on the amplitude of the composite TC-EPSP. As have others, we also conclude that from a somato-centric viewpoint, changes in spine geometry would have relatively little direct influence on amplitudes of EPSPs recorded at the soma, especially for a distributed, synchronously activated input such as the TC pathway. However, consideration of the detailed morphology of an entire neuron indicates that, from a dendro-centric point of view, changes in spine dimension can have a very significant electrical impact on local processing near the sites of input.

Animals↗
PubMed2000

A cost-benefit analysis using a willingness-to-pay questionnaire of intranasal budesonide for seasonal allergic rhinitis. Rhinocort Study Group.

BACKGROUND: The cost-benefit of intranasal steroids for the treatment of seasonal allergic rhinitis is unknown. OBJECTIVE: To determine the cost-benefit of intranasal budesonide for seasonal allergic rhinitis. METHODS: Subjects who were symptomatic for a baseline period of 7 to 10 days were randomized to receive intranasal budesonide by Turbuhaler (400 microg) (n = 121) or aqueous spray (256 microg) (n = 121) once daily for 4 weeks. A willingness-to-pay questionnaire that measured benefits of treatment was administered before and at study completion. Costs were collected and compared with benefits. RESULTS: Subjects were willing to spend on average $15.89/wk (range $1 to $75) to alleviate the problems of seasonal ragweed rhinitis. Eighty percent of subjects felt that, with treatment, rhinitis had less of an impact on their lives, compared with previous years. The mean willingness-to-pay for the drug used during another ragweed season was $12.95/wk. This was 92% (95% CI, 85% to 100%) of the pre-treatment estimate. There was no relationship between an indirect assessment of income and willingness-to-pay estimates. The benefit was greater than the cost by a mean of $5.80/wk (95% CI, $3.52 to 8.08), P < .0001. There was no difference in costs, willingness-to-pay, or cost-benefit comparing delivery modes. A sensitivity analysis revealed the conclusions were robust. CONCLUSIONS: Intranasal budesonide is cost-beneficial in the treatment of seasonal allergic rhinitis and a willingness-to-pay questionnaire may provide a useful method to assess a therapy's benefit.

Administration, Intranasal↗
PubMed2005

Electrochemical impedance sensing of DNA hybridization on conducting polymer film-modified diamond.

The impedimetric sensing of DNA hybridization on polyaniline/polyacrylate (PANI/PAA)-modified boron-doped diamond (BDD) electrode has been investigated. An ultrathin film of PANI-PAA copolymer was electropolymerized onto the diamond surfaces to provide carboxylic groups for tethering to DNA sensing probes. The electrochemical impedance and the intrinsic electroactivity of the polymer-diamond interface were analyzed after the hybridization reaction with target and non-target DNA. The impedance measurement shows changes in the impedance modulus as well as electron-transfer resistance at the stage of probe DNA immobilization (single-strand), as well as after hybridization with target DNA (double-strand). DNA hybridization increases the capacitance of the polymer-DNA layer and reduces the overall impedance of the DNA-polymer-diamond stack significantly. The polymer-modified BDD electrode shows no detectable nonspecific adsorption, with good selectivity between the complementary DNA targets and the one-base mismatch targets. The detection limit was measured to be 2 x 10(-8) M at 1000 Hz. Denaturing test on the hybridized probe and subsequent reuse of the probe indicates chemical robustness of the sensor. Our results suggest that electropolymerization followed by the immobilization of biomolecules is a simple and effective way of creating a functional biomolecular scaffold on the diamond surface. In addition, label-free electrochemical impedance method can provide direct and noninvasive sensing of DNA hybridization on BDD.

Boron↗
PubMed2002

Achieving remission from depression with venlafaxine and venlafaxine extended release: a literature review of comparative studies with selective serotonin reuptake inhibitors.

OBJECTIVE: To evaluate data supporting the ability of venlafaxine, an antidepressant with a dual mechanism of action, to produce remission from depression. METHOD: Review of multicentre, double-blind, randomized studies comparing venlafaxine or venlafaxine extended release (XR) with a selective serotonin reuptake inhibitor (SSRI), using Hamilton Depression Rating Scale total scores in the range of < or = 7 and < 10 as the final outcome measure, to evaluate the ability of venlafaxine/venlafaxine XR to produce full remission from depression. RESULTS: Venlafaxine/venlafaxine XR demonstrated higher rates of remission than did the SSRIs and placebo. CONCLUSION: With full remission rather than response as the measure of outcome, venlafaxine/venlafaxine XR demonstrated more robust antidepressant efficacy than the SSRIs and placebo. This finding suggests that venlafaxine/venlafaxine XR are appropriate standard-of-care therapies for the treatment of patients with major depressive disorder.

Antidepressive Agents, Second-Generation↗
PubMed1995

[Analgesia with intra-articular morphine following knee joint arthroscopy? A double-blind, randomized study with patient-controlled analgesia].

UNLABELLED: Previous studies investigating the peripheral action of locally instilled morphine after arthroscopic knee surgery found evidence for an analgesic effect. Follow-up studies have lead to conflicting results. We used patient-controlled analgesia (PCA) to test the analgesic potency of intraarticular morphine. METHODS. Patients undergoing arthroscopic knee surgery under general anaesthesia received, after written informed consent and in double-blind and randomised manner, 1 mg morphine diluted in 10 ml saline either intraarticularly or intravenously at the end of the surgical procedure. A control injection of 10 ml saline was given at the other site. The pain intensity on a visual analogue scale (VAS) and the cumulative morphine consumption were recorded at 1, 2, 3, 4, 6, 8 and 24 h after the end of general anaesthesia. STATISTICS: Wilcoxon rank sum test with P < 0.05. RESULTS. A total of 59 patients were included in the study; 29 received morphine intraarticularly (verum group), 30 intravenously (control group). There was no difference in gender, age, duration of arthroscopy or anaesthesia. There were more than 60% diagnostic arthroscopies in both groups; other types of surgery were comparable, with the exception of cruciate band repair procedures only in the control group. We found no difference in morphine consumption or pain intensity between the two groups throughout the study period. Median overall consumption of morphine after 24 h was 14 mg in the verum group and 15 mg in the control group, with wide interindividual variation. Pain intensities were remarkably low. The peak pain intensity of both groups was found at 1 h postoperatively, with median 16/100 on the VAS in both groups. Blinding was robust. CONCLUSION. We found no reduction in postoperative morphine supplementation after 1 mg morphine intraarticularly compared to 1 mg intravenously given at the end of knee arthroscopies. There were also no differences in pain intensities on a VAS. We conclude that titration of postoperative pain with a morphine-filled PCA pump was unable to show a difference in analgesic potency between intraarticular and intravenous morphine.

Adolescent↗
PubMed2003

A novel multivalent human CTL peptide construct elicits robust cellular immune responses in HLA-A*0201 transgenic mice: implications for HTLV-1 vaccine design.

Cytotoxic T-lymphocytes are critical in the clearance of chronic viral infections such as HTLV-1. Peptide-based vaccines may have potential application in invoking antiviral CTL responses. In the development of vaccination strategies, it is becoming increasingly important to elicit a broad immune response against several epitopes simultaneously that may provide large population coverage. In the present study, we addressed this issue, namely the processing and presentation of multiple CTL epitopes simultaneously for the generation of multispecific CTL responses. We designed a novel multivalent peptide consisting of three HLA-A(*)0201 restricted CTL epitopes, with intervening double arginine residues in tandem. These epitopes were derived from the HTLV-1 regulatory protein Tax, which is an attractive target for vaccine development against HTLV-1. Arginine residues were included to provide cleavage sites for proteasomes, to generate the intended MHC Class I ligands. Proteasomal digestion studies and mass spectrometry analysis showed cleavage of the multivalent construct to generate the individual epitopes. Immunization of HLA-A(*)0201 transgenic mice with this construct efficiently elicited cellular responses to each intended epitope in vivo, further validating the applicability of this approach. These data may have potential in the development of immunotherapeutic strategies for the treatment of HTLV-1 disease and in the future design of multivalent subunit peptide vaccines.

Animals↗