Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions. This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters

CytoNorm : a normalization algorithm for cytometry data

High-dimensional flow cytometry has matured to a level that enables deep phenotyping of cellular systems at a clinical scale. The resulting high-content data sets allow characterizing the human immune system at unprecedented single cell resolution. However, the results are highly dependent on sample preparation and measurements might drift over time. While various controls exist for assessment and improvement of data quality in a single sample, the challenges of cross-sample normalization attempts have been limited to aligning marker distributions across subjects. These approaches, inspired by bulk genomics and proteomics assays, ignore the single-cell nature of the data and risk the removal of biologically relevant signals. This work proposes CytoNorm, a normalization algorithm to ensure internal consistency between clinical samples based on shared controls across various study batches. Data from the shared controls is used to learn the appropriate transformations for each batch (e.g., each analysis day). Importantly, some sources of technical variation are strongly influenced by the amount of protein expressed on specific cell types, requiring several population-specific transformations to normalize cells from a heterogeneous sample. To address this, our approach first identifies the overall cellular distribution using a clustering step, and calculates subset-specific transformations on the control samples by computing their quantile distributions and aligning them with splines. These transformations are then applied to all other clinical samples in the batch to remove the batch-specific variations. We evaluated the algorithm on a customized data set with two shared controls across batches. One control sample was used for calculation of the normalization transformations and the second control was used as a blinded test set and evaluated with Earth Mover's distance. Additional results are provided using two real-world clinical data sets. Overall, our method compared favorably to standard normalization procedures

Growth methods of c-axis oriented MgB2 thin films by pulsed laser deposition

High quality MgB2 thin films have been obtained by pulsed laser deposition both on MgO and on Al2O3 substrates using different methods. In the standard two-step procedure, an amorphous precursor layer is deposited at room temperature starting both from stoichiometric target and from boron target: after this first step, it is annealed in magnesium atmosphere in order to crystallize the superconducting phase. The so obtained films show a strong c-axis orientation, evidenced by XRD analysis, a critical temperature up to 38 K and very high critical fields along the basal planes, up to 22T at 15K. Also an in situ one step technique for the realization of superconducting MgB2 thin films has been developed. In this case, the presence of an argon buffer gas during deposition is crucial and we observe a strong dependence of the quality of the deposited film on the background gas pressure. The influence of the Ar atmosphere has been confirmed by time and space-resolved spectroscopy measurements on the emission spectrum of the plume. The Ar pressure modifies strongly the plasma kinetics by promoting excitation and ionization of the plume species, especially of the most volatile Mg atoms, increasing their internal energy.Comment: Paper presented at Boromag Workshop, Genoa 17-19 June 2002, in press on SUS

Laser-sintered thin films of doped SiGe nanoparticles

We present a study of the morphology and the thermoelectric properties of short-pulse laser-sintered (LS) nanoparticle (NP) thin films, consisting of SiGe alloy NPs or composites of Si and Ge NPs. Laser-sintering of spin-coated NP films in vacuum results in a macroporous percolating network with a typical thickness of 300 nm. The Seebeck coefficient is independent of the sintering process and typical for degenerate doping. The electrical conductivity of LS films rises with increasing temperature, best described by a power-law and influenced by two-dimensional percolation effects.Comment: 4 pages, 4 figure

Selective nociceptor activation in volunteers by infrared diode laser

<p>Abstract</p> <p>Background</p> <p>Two main classes of peripheral sensory neurons contribute to thermal pain sensitivity: the unmyelinated C fibers and thinly myelinated Aδ fibers. These two fiber types may differentially underlie different clinical pain states and distinctions in the efficacy of analgesic treatments. Methods of differentially testing C and Aδ thermal pain are widely used in animal experimentation, but these methods are not optimal for human volunteer and patient use. Thus, this project aimed to provide psychophysical and electrophysiological evidence that whether different protocols of infrared diode laser stimulation, which allows for direct activation of nociceptive terminals deep in the skin, could differentially activate Aδ or C fiber thermonociceptors in volunteers.</p> <p>Results</p> <p>Short (60 ms), high intensity laser pulses (SP) evoked monomodal "pricking" pain which was not enhanced by topical capsaicin, whereas longer, lower power pulses (LP) evoked monomodal "burning" pain which was enhanced by topical capsaicin. SP also produced cortical evoked EEG potentials consistent with Aδ mediation, the amplitude of which was directly correlated with pain intensity but was not affected by topical capsaicin. LP also produced a distinct evoked potential pattern the amplitude of which was also correlated with pain intensity, which was enhanced by topical capsaicin, and the latency of which could be used to estimate the conduction velocity of the mediating nociceptive fibers.</p> <p>Conclusions</p> <p>Psychophysical and electrophysiological data were consistent with the ability of short high intensity infrared laser pulses to selectively produce Aδ mediated pain and of longer pulses to selectively produce C fiber mediated thermal pain. Thus, the use of these or similar protocols may be useful in developing and testing novel therapeutics based on the differential molecular mechanisms underlying activation of the two fiber types (e.g., TRPV1, TRPV2, etc). In addition, these protocol may be useful in determining the fiber mediation of different clinical pain types which may, in turn be useful in treatment choice.</p

Neck pain and anxiety do not always go together

Chronic pain and psychosocial distress are generally thought to be associated in chronic musculoskeletal disorders such as non-specific neck pain. However, it is unclear whether a raised level of anxiety is necessarily a feature of longstanding, intense pain amongst patient and general population sub-groups. In a cohort of 70 self-selected female, non-specific neck pain sufferers, we observed relatively high levels of self-reported pain of 4.46 (measured on the 11 point numerical pain rating scale (NRS-101)) and a longstanding duration of symptoms (156 days/year). However, the mean anxiety scores observed (5.49), fell well below the clinically relevant threshold of 21 required by the Beck Anxiety Inventory. The cohort was stratified to further distinguish individuals with higher pain intensity (NRS>6) and longer symptom duration (>90 days). Although a highly statistically significant difference (p = 0.000) was subsequently observed with respect to pain intensity, in the resulting sub-groups, none such a difference was noted with respect to anxiety levels. Our results indicate that chronic, intense pain and anxiety do not always appear to be related. Explanations for these findings may include that anxiety is not triggered in socially functional individuals, that individual coping strategies have come into play or in some instances that a psychological disorder like alexithymia could be a confounder. More studies are needed to clarify the specific role of anxiety in chronic non-specific musculoskeletal pain before general evidence-driven clinical extrapolations can be made

Multiomic immune clockworks of pregnancy

Preterm birth is the leading cause of mortality in children under the age of five worldwide. Despite major efforts, we still lack the ability to accurately predict and effectively prevent preterm birth. While multiple factors contribute to preterm labor, dysregulations of immunological adaptations required for the maintenance of a healthy pregnancy is at its pathophysiological core. Consequently, a precise understanding of these chronologically paced immune adaptations and of the biological pacemakers that synchronize the pregnancy “immune clock” is a critical first step towards identifying deviations that are hallmarks of peterm birth. Here, we will review key elements of the fetal, placental, and maternal pacemakers that program the immune clock of pregnancy. We will then emphasize multiomic studies that enable a more integrated view of pregnancy-related immune adaptations. Such multiomic assessments can strengthen the biological plausibility of immunological findings and increase the power of biological signatures predictive of preterm birth

Single-cell Analysis of the Neonatal Immune System Across the Gestational Age Continuum

Although most causes of death and morbidity in premature infants are related to immune maladaptation, the premature immune system remains poorly understood. We provide a comprehensive single-cell depiction of the neonatal immune system at birth across the spectrum of viable gestational age (GA), ranging from 25 weeks to term. A mass cytometry immunoassay interrogated all major immune cell subsets, including signaling activity and responsiveness to stimulation. An elastic net model described the relationship between GA and immunome (R=0.85, p=8.75e-14), and unsupervised clustering highlighted previously unrecognized GA-dependent immune dynamics, including decreasing basal MAP-kinase/NFkB signaling in antigen presenting cells; increasing responsiveness of cytotoxic lymphocytes to interferon-a; and decreasing frequency of regulatory and invariant T cells, including NKT cells and MAIT cells. Knowledge gained from the analysis of the neonatal immune landscape across GA provides a mechanistic framework to understand the unique susceptibility of preterm infants to both hyper-inflammatory diseases and infections

Multiomics modeling of the immunome, transcriptome, microbiome, proteome and metabolome adaptations during human pregnancy.

MotivationMultiple biological clocks govern a healthy pregnancy. These biological mechanisms produce immunologic, metabolomic, proteomic, genomic and microbiomic adaptations during the course of pregnancy. Modeling the chronology of these adaptations during full-term pregnancy provides the frameworks for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia.ResultsWe performed a multiomics analysis of 51 samples from 17 pregnant women, delivering at term. The datasets included measurements from the immunome, transcriptome, microbiome, proteome and metabolome of samples obtained simultaneously from the same patients. Multivariate predictive modeling using the Elastic Net (EN) algorithm was used to measure the ability of each dataset to predict gestational age. Using stacked generalization, these datasets were combined into a single model. This model not only significantly increased predictive power by combining all datasets, but also revealed novel interactions between different biological modalities. Future work includes expansion of the cohort to preterm-enriched populations and in vivo analysis of immune-modulating interventions based on the mechanisms identified.Availability and implementationDatasets and scripts for reproduction of results are available through: https://nalab.stanford.edu/multiomics-pregnancy/.Supplementary informationSupplementary data are available at Bioinformatics online