Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia

Resiniferatoxin (RTX) is an ultrapotent capsaicin analog that binds to the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). There is a large body of evidence supporting a role for TRPV1 in noxious-mediated and inflammatory hyperalgesic responses. In this study, we evaluated low, graded, doses of perineural RTX as a method for regional pain control. We hypothesized that this approach can provide long-term, but reversible, blockade of a portion of nociceptive afferent fibers within peripheral nerves when given at a site remote from the neuronal perikarya in the dorsal root ganglia. Following perineural RTX application to the sciatic nerve, we demonstrated a significant inhibition of inflammatory nociception that was dose- and time-dependent. At the same time, treated animals maintained normal proprioceptive sensations and motor control, and other nociceptive responses were largely unaffected. Using a range of mechanical and thermal algesic tests, we found that the most sensitive measure following perineural RTX administration was inhibition of inflammatory hyperalgesia. Recovery studies showed that physiologic sensory function could return as early as two weeks post-RTX treatment, however, immunohistochemical examination of the DRG revealed a partial, but significant reduction in the number of the TRPV1-positive neurons. We propose that this method could represent a beneficial treatment for a range of chronic pain problems, including neuropathic and inflammatory pain not responding to other therapies

Constrained TRPV1 agonists synthesized via silver-mediated intramolecular azo-methine ylide cycloaddition of α-iminoamides

As part of an effort to identify agonists of TRPV1, a peripheral sensory nerve ion channel, high throughput screening of the NIH Small Molecule Repository (SMR) collection identified MLS002174161, a pentacyclic benzodiazepine. A synthesis effort was initiated that ultimately afforded racemic seco analogs 12 of the SMR compound via a silver mediated intramolecular [3+2] cycloaddition of an azo-methine ylide generated from α-iminoamides 11. The cycloaddition set four contiguous stereocenters and, in some cases, also spontaneously afforded imides 13 from 12. The synthesis of compounds 12, the features that facilitated the conversion of 12–13, and their partial agonist activity against TRPV1 are discussed

Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies

<p>Abstract</p> <p>Background</p> <p>Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.</p> <p>Methods</p> <p>Here we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as <it>Renilla </it>luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA.</p> <p>Results</p> <p>Analysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100–1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (<it>r</it>_{<it>s </it>}= 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3–4 of the CMV antigens.</p> <p>Conclusion</p> <p>These results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.</p

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates

<p>Abstract</p> <p>Background</p> <p>Ablation of TRPV1-expressing nociceptive fibers with the potent capsaicin analog resiniferatoxin (RTX) results in long lasting pain relief. RTX is particularly adaptable to focal application, and the induced chemical axonopathy leads to analgesia with a duration that is influenced by dose, route of administration, and the rate of fiber regeneration. TRPV1 is expressed in a subpopulation of unmyelinated C- and lightly myelinated Adelta fibers that detect changes in skin temperature at low and high rates of noxious heating, respectively. Here we investigate fiber-type specific behaviors, their time course of recovery and molecular correlates of axon damage and nociception using infrared laser stimuli following an RTX-induced peripheral axonopathy.</p> <p>Results</p> <p>RTX was injected into rat hind paws (mid-plantar) to produce thermal hypoalgesia. An infrared diode laser was used to stimulate Adelta fibers in the paw with a small-diameter (1.6 mm), high-energy, 100 msec pulse, or C-fibers with a wide-diameter (5 mm), long-duration, low-energy pulse. We monitored behavioral responses to indicate loss and regeneration of fibers. At the site of injection, responses to C-fiber stimuli were significantly attenuated for two weeks after 5 or 50 ng RTX. Responses to Adelta stimuli were significantly attenuated for two weeks at the highest intensity stimulus, and for 5 weeks to a less intense Adelta stimulus. Stimulation on the toe, a site distal to the injection, showed significant attenuation of Adelta responses for 7- 8 weeks after 5 ng, or 9-10 weeks after 50 ng RTX. In contrast, responses to C-fiber stimuli exhibited basically normal responses at 5 weeks after RTX. During the period of fiber loss and recovery, molecular markers for nerve regeneration (ATF3 and galanin) are upregulated in the dorsal root ganglia (DRG) when behavior is maximally attenuated, but markers of nociceptive activity (c-Fos in spinal cord and MCP-1 in DRG), although induced immediately after RTX treatment, returned to normal.</p> <p>Conclusion</p> <p>Behavioral recovery following peripheral RTX treatment is linked to regeneration of TRPV1-expressing Adelta and C-fibers and sustained expression of molecular markers. Infrared laser stimulation is a potentially valuable tool for evaluating the behavioral role of Adelta fibers in pain and pain control.</p

Comparison of Radioimmunoprecipitation With Luciferase Immunoprecipitation for Autoantibodies to GAD65 and IA-2β

OBJECTIVE - To compare the sensitivity and specificity of luciferase immunoprecipitation (LIPS) with radioimmunoprecipitation (RIP) for the measurement of autoantibodies to the type 1 diabetes autoantigens glutamic acid decarboxylase 65 (GAD65) and insulinoma-associated protein (IA)-2 beta. RESEARCH DESIGN AND METHODS - Sera from 49 type 1 diabetic patients and 100 nondiabetic control subjects from Diabetes Antibody Standardization Program 2007 were used to screen for autoantibodies to GAD65. An additional 200 type 1 diabetic patients and 200 nondiabetic control subjects were used to validate the GAD65 results and screen for autoantibodies to IA-2 beta. RESULTS - LIPS showed equal sensitivity and specificity to RIP for detecting autoantibodies to GAD65 and IA-2 beta. Receiver-operating characteristic analysis revealed that the detection of autoantibodies to GAD65 and IA-2 beta by LIPS and RIP were not statistically different. CONCLUSIONS - The LIPS assay does not require the use of radioisotopes or in vitro transcription/translation and is a practical alternative at the clinical level for the RIP assay

Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation

BACKGROUND: Spinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury. RESULTS: Lumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation. CONCLUSION: These data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease

Anti-HTLV antibody profiling reveals an antibody signature for HTLV-I-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP)

<p>Abstract</p> <p>Background</p> <p>HTLV-I is the causal agent of adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Biomarkers are needed to diagnose and/or predict patients who are at risk for HAM/TSP or ATLL. Therefore, we investigated using luciferase immunoprecipitation technology (LIPS) antibody responses to seven HTLV-I proteins in non-infected controls, asymptomatic HTLV-I-carriers, ATLL and HAM/TSP sera samples. Antibody profiles were correlated with viral load and examined in longitudinal samples.</p> <p>Results</p> <p>Anti-GAG antibody titers detected by LIPS differentiated HTLV-infected subjects from uninfected controls with 100% sensitivity and 100% specificity, but did not differ between HTLV-I infected subgroups. However, anti-Env antibody titers were over 4-fold higher in HAM/TSP compared to both asymptomatic HTLV-I (<it>P </it>< 0.0001) and ATLL patients (<it>P </it>< 0.0005). Anti-Env antibody titers above 100,000 LU had 75% positive predictive value and 79% negative predictive value for identifying the HAM/TSP sub-type. Anti-Tax antibody titers were also higher (<it>P </it>< 0.0005) in the HAM/TSP compared to the asymptomatic HTLV-I carriers. Proviral load correlated with anti-Env antibodies in asymptomatic carriers (<it>R </it>= 0.76), but not in HAM/TSP.</p> <p>Conclusion</p> <p>These studies indicate that anti-HTLV-I antibody responses detected by LIPS are useful for diagnosis and suggest that elevated anti-Env antibodies are a common feature found in HAM/TSP patients.</p

A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy- or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene-related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber-mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch