A quantitative real time PCR method to analyze T cell receptor Vβ subgroup expansion by staphylococcal superantigens

<p>Abstract</p> <p>Background</p> <p>Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by <it>Staphylococcus aureus</it>, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor β chain (Vβ). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vβ subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vβ nucleotide sequences and SYBR Green I reporter has not been applied to assess Vβ-dependent expansion of T cells by SAgs.</p> <p>Methods</p> <p>Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vβ subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vβ nucleotide sequences.</p> <p>Results</p> <p>qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R²of standard curves were -3.3764 ± 0.0245 and 0.99856 ± 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vβ specificities observed in this study were similar to those reported in previous studies.</p> <p>Conclusions</p> <p>The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vβ-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.</p

Two opiod forms of stress analgesia: Studies of tolerance and cross-tolerance

We have previously reported that stress analgesia sensitive to and insensitive to opiate antagonists can be differentially produced in rats by varying the severity or temporal pattern of inescapable footshock. In these studies, we give further evidence for the opioid and non-opioid bases of these paradigms of stress analgesia. We find that naloxone-sensitive analgesia demonstrates tolerance with repeated stress and cross-tolerance with morphine, whereas naloxone-insensitive analgesia demonstrates neither of these characteristics. Moreover, different forms of opioid, but not non-opioid, stress analgesia manifest cross-tolerance with each other. These data are discussed in terms of the similarities and differences between two forms of opioid stress analgesia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26230/1/0000310.pd

The ACTTION-APS-AAPM Pain Taxonomy (AAAPT) Multidimensional Approach to Classifying Acute Pain Conditions.

Objective: With the increasing societal awareness of the prevalence and impact of acute pain, there is a need to develop an acute pain classification system that both reflects contemporary mechanistic insights and helps guide future research and treatment. Existing classifications of acute pain conditions are limiting, with a predominant focus on the sensory experience (e.g., pain intensity) and pharmacologic consumption. Consequently, there is a need to more broadly characterize and classify the multidimensional experience of acute pain. Setting: Consensus report following expert panel involving the Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION), American Pain Society (APS), and American Academy of Pain Medicine (AAPM). Methods: As a complement to a taxonomy recently developed for chronic pain, the ACTTION public-private partnership with the US Food and Drug Administration, the APS, and the AAPM convened a consensus meeting of experts to develop an acute pain taxonomy using prevailing evidence. Key issues pertaining to the distinct nature of acute pain are presented followed by the agreed-upon taxonomy. The ACTTION-APS-AAPM Acute Pain Taxonomy will include the following dimensions: 1) core criteria, 2) common features, 3) modulating factors, 4) impact/functional consequences, and 5) putative pathophysiologic pain mechanisms. Future efforts will consist of working groups utilizing this taxonomy to develop diagnostic criteria for a comprehensive set of acute pain conditions. Perspective: The ACTTION-APS-AAPM Acute Pain Taxonomy (AAAPT) is a multidimensional acute pain classification system designed to classify acute pain along the following dimensions: 1) core criteria, 2) common features, 3) modulating factors, 4) impact/functional consequences, and 5) putative pathophysiologic pain mechanisms. Conclusions: Significant numbers of patients still suffer from significant acute pain, despite the advent of modern multimodal analgesic strategies. Mismanaged acute pain has a broad societal impact as significant numbers of patients may progress to suffer from chronic pain. An acute pain taxonomy provides a much-needed standardization of clinical diagnostic criteria, which benefits clinical care, research, education, and public policy. For the purposes of the present taxonomy, acute pain is considered to last up to seven days, with prolongation to 30 days being common. The current understanding of acute pain mechanisms poorly differentiates between acute and chronic pain and is often insufficient to distinguish among many types of acute pain conditions. Given the usefulness of the AAPT multidimensional framework, the AAAPT undertook a similar approach to organizing various acute pain conditions

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

Self-help interventions for depressive disorders and depressive symptoms: a systematic review

<p>Abstract</p> <p>Background</p> <p>Research suggests that depressive disorders exist on a continuum, with subthreshold symptoms causing considerable population burden and increasing individual risk of developing major depressive disorder. An alternative strategy to professional treatment of subthreshold depression is population promotion of effective self-help interventions that can be easily applied by an individual without professional guidance. The evidence for self-help interventions for depressive symptoms is reviewed in the present work, with the aim of identifying promising interventions that could inform future health promotion campaigns or stimulate further research.</p> <p>Methods</p> <p>A literature search for randomised controlled trials investigating self-help interventions for depressive disorders or depressive symptoms was performed using PubMed, PsycINFO and the Cochrane Database of Systematic Reviews. Reference lists and citations of included studies were also checked. Studies were grouped into those involving participants with depressive disorders or a high level of depressive symptoms, or non-clinically depressed participants not selected for depression. A number of exclusion criteria were applied, including trials with small sample sizes and where the intervention was adjunctive to antidepressants or psychotherapy.</p> <p>Results</p> <p>The majority of interventions searched had no relevant evidence to review. Of the 38 interventions reviewed, the ones with the best evidence of efficacy in depressive disorders were S-adenosylmethionine, St John's wort, bibliotherapy, computerised interventions, distraction, relaxation training, exercise, pleasant activities, sleep deprivation, and light therapy. A number of other interventions showed promise but had received less research attention. Research in non-clinical samples indicated immediate beneficial effects on depressed mood for distraction, exercise, humour, music, negative air ionisation, and singing; while potential for helpful longer-term effects was found for autogenic training, light therapy, omega 3 fatty acids, pets, and prayer. Many of the trials were poor quality and may not generalise to self-help without professional guidance.</p> <p>Conclusion</p> <p>A number of self-help interventions have promising evidence for reducing subthreshold depressive symptoms. Other forms of evidence such as expert consensus may be more appropriate for interventions that are not feasible to evaluate in randomised controlled trials. There needs to be evaluation of whether promotion to the public of effective self-help strategies for subthreshold depressive symptoms could delay or prevent onset of depressive illness, reduce functional impairment, and prevent progression to other undesirable outcomes such as harmful use of substances.</p