CD8αα+T cells exert a pro-inflammatory role in patients with psoriasis.

Background: Psoriasis is a common chronic inflammatory disease caused by excessive activation of CD4+T cells, including Th17, Th1 and Th22. The role of CD8+T cells in psoriasis pathogenesis remains poorly understood. Aim: To identify the phenotype of CD8+T cells in patients with psoriasis and to investigate its role in the formation of lesions. Methods: The phenotype of CD8+T cells in psoriatic lesions was detected by immunofluorescence staining. Flow cytometry was performed to detect their phenotype in peripheral blood. Thereafter, coculture of CD8αα+T cells with autogenous CD4+T cells was performed to investigate the function of CD8αα+T cells in patients with psoriasis. Finally, pro-inflammatory factors produced by CD8αα+T cells were examined by immunofluorescence staining and flow cytometry. Results: Compared to the CD8αß+T cells, CD8αα+T cell infiltration in psoriatic lesions markedly increased. Moreover, epidermal CD8αα+T cells exhibited tissue-resident memory T cells (TRM) phenotypes and dermal CD8αα+T cells exhibited effector memory (TEM) phenotypes in psoriatic lesions. Additionally, we found that CD8αα+T cells from patients with psoriasis did not express the markers of regulatory T cells and could promote the proliferation of CD4+T effector cells and produce interleukin-17 and interferon-γ. Conclusions: Our findings demonstrate that CD8αα+T cells contribute to the pathogenesis of psoriasis by producing pro-inflammatory factors.

Enhancing Models and Measurements of Traffic-Related Air Pollutants for Health Studies Using Dispersion Modeling and Bayesian Data Fusion.

INTRODUCTION: The adverse health effects associated with exposure to traffic-related air pollutants (TRAPs) remain a key public health issue. Often, exposure assessments have not represented the small-scale variation and elevated concentrations found near major roads and in urban settings. This research explores approaches aimed at improving exposure estimates of TRAPs that can reduce exposure measurement error when used in health studies. We consider dispersion models designed specifically for the near-road environment, as well as spatiotemporal and data fusion models. These approaches are implemented and evaluated utilizing data collected in recent modeling, monitoring, and epidemiological studies conducted in Detroit, Michigan. APPROACH: Dispersion models, which estimate near-road pollutant concentrations and individual exposures based on first principles - and in particular, high fidelity models - can provide great flexibility and theoretical strength. They can represent the spatial variability of TRAP concentrations at locations not measured by conventional and spatially sparse air quality monitoring networks. A number of enhancements to dispersion modeling and mobile on-road emissions inventories were considered, including the representation of link-based road networks and updated estimates of temporal allocation of traffic activity, emission factors, and meteorological inputs. The recently developed Research LINE-source model (RLINE), a Gaussian line-source dispersion model specifically designed for the near-road environment, was used in an operational evaluation that compared predicted concentrations of nitrogen oxides (NOx), carbon monoxide (CO), and PM2.5 (particulate matter ≤ 2.5 µm in aerodynamic diameter) with observed concentrations at air quality monitoring stations located near high-traffic roads. Spatiotemporal and data fusion models provided additional and complementary approaches for estimating TRAP exposures. We formulated both nonstationary universal kriging models that exploit the spatial correlation in the monitoring data, and data fusion models that leverage the information contained in both the monitoring data and the output of numerical models, specifically RLINE. These models were evaluated using observations of nitric oxide (NO), NOx, black carbon (BC), and PM2.5 monitored along transects crossing major roads in Detroit. We also examined model assumptions, including the appropriateness of the covariance functions, errors in RLINE outputs, and the effects of jointly modeling two pollutants and using an updated emission inventory. RESULTS: For CO and NOx, dispersion model performance was best when monitoring sites were close to major roads, during downwind conditions, during weekdays, and during certain seasons. The ability to discern local and particularly the traffic-related portion of PM2.5 was limited, a result of high background levels, the sparseness of the monitoring network, and large uncertainties for certain sources (e.g., area, fugitive) and some processes (e.g., formation of secondary aerosols). Sensitivity analyses of alternative meteorological inputs and updated emission factors showed some performance gain when using local (on-site) meteorological data and updated inventories. Overall, the operational evaluation suggested RLINE's usefulness for estimating spatially and temporally resolved exposure estimates. The application of the universal kriging models confirmed that wind speed and direction are important drivers of nonstationarity in pollutant concentrations, and that these models can predict exposure estimates that have lower prediction errors than do stationary model counterparts. The application of the Bayesian data fusion models suggested that the RLINE output had a spatially varying additive bias for NOx and PM2.5 and provided little additional information for NOx, besides what is already contained in traffic and geographical information system (GIS) covariates, but had improved estimates of PM2.5 concentrations. Results of the nonstationary Bayesian data fusion model that used RLINE output across a field spanning the measurement sites were similar to a regression-based Bayesian data fusion approach that used only RLINE output at the monitoring locations, with the latter being computationally less burdensome. Using the regression-based Bayesian data fusion model, we found that RLINE with the updated emission inventory provided results that were more useful for estimating NOx concentration at unmonitored sites, but the updated emission inventory did not improve predictions of PM2.5 concentrations. Joint modeling of NOx and PM2.5 was not useful, a result of differences in RLINE's utility in predicting PM2.5 and NOx - useful for the former, but not for the latter - and differences in the spatial dependence structures of the two pollutants. Overall, information provided by RLINE was shown to have the potential to improve spatiotemporal estimates of TRAP concentrations. CONCLUSIONS: The study results should be interpreted and generalized cautiously given the limitations of the data used. Similar analyses in other settings are recommended for confirming and extending our findings. Still, the study highlights considerations that are relevant for exposure estimates used in health studies. The ability of a dispersion model to accurately reproduce and predict a pollutant depends on the pollutant as well as on spatial and temporal factors, such as the distance and direction from the road, time-of-day, and day-of-week. The nature and source of exposure measurement errors should be taken into consideration, particularly in health studies that take advantage of time- activity information that describes where and when individuals are exposed to pollution. Efforts to refine model inputs and improve model performance can be helpful; meteorological inputs may be the most critical. For both dispersion and spatiotemporal statistical models, sufficient and high-quality monitoring data are essential for developing and evaluating these models. Our analyses using Bayesian data fusion models confirm the presence of spatially varying errors in dispersion model outputs and allow quantification of both the magnitude and the spatial nature of these errors. This valuable information can be leveraged in health studies examining air pollution exposure as well as in studies informing regulatory responses.

[Study on diffusion kurtosis imaging in hyperacute phase of mild traumatic brain injury].

Objective: To explore the changes of cerebral white matter in the hyperacute period (<24 h) patients with mild traumatic brain injury (mTBI) by diffusion kurtosis imaging(DKI) technique. Methods: A total of 52 patients with mTBI were included in this study, collected in Emergency Department of Affiliated Hospital of Hangzhou Normal University from May 2016 to June 2017. Twenty-one healthy controls were recruited at the mean time(gender, age and years of education were matched with the patients). DKI data were acquired with 3.0 T scanners.The FSL software was used to preprocess the DKI data, and the white matter abnormalities were detected by tract-based spatial statistics (TBSS). Results: There were no statistical differences in gender, age and years of education between mTBI patients and healthy controls (P=0.427, P=0.235, P=0.165). The values of MK of the body of corpus callosum (BCC), the genu of corpus callosum (GCC), the splenium of corpus callosum (SCC), the bilateral anterior limb of interbal capsule (ALIP), the right posterior limb of internal capsule (PLIC_R), the bilateral anterior corona radiate (ACR), the bilateral posterior corona radiate (PCR), the bilateral superior corona radiate (SCR), the left inferior fronto-occipital fasciculus (IFOF_L)and the bilateral superior longitudinal fasciculus (SLF) were lower in mTBI patients((1.095±0.080), (1.130±0.066), (1.160±0.080), (1.135±0.077), (1.108±0.076), (1.203±0.069), (1.073±0.056), (1.052±0.055), (1.170±0.055), (1.149±0.050), (1.028±0.056), (1.051±0.059), (0.868±0.060), (1.194±0.048), (1.183±0.054) mm(2)/s) than those in healthy controls((1.153±0.054), (1.184±0.057), (1.215±0.068), (1.181±0.053), (1.163±0.062), (1.258±0.041), (1.115±0.037), (1.096±0.049), (1.210±0.040), (1.190±0.049), (1.063±0.042), (1.087±0.057), (0.913±0.063), (1.236±0.047), (1.214±0.038) mm(2)/s)(P<0.01). However, there were no statistical differences in fractional anisotropy (FA) between groups (P>0.01). Conclusion: DKI technology is more sensitive in detecting cerebral white matter abnormalities in patients with hyperacute mTBI which routine MRI findings was normal.

[Efficacy of endoscopic approach to reconstruct the medial orbital fracture with perpendicular plate of ethmoid].

Objective:To study the efficacy of endoscopic sinus approach in the repair of medial orbital fracture with perpendicular plate of ethmoid.Method:Retrospective chart was reviewed in 10 cases receiving endoscopic approach to reconstruct the medial orbital fracture with perpendicular plate of ethmoid.We observed the improvement of the symptom such as diplopia, eye movement,and enophthalmos of the preoperative and postoperative.Result:After postoperative follow-up of 4 months to 23 months, all the patients had no graft loss or displacement, infection and other complications, and except for 1 patient with mild diplopia, other patients recovered completely, including eye movement disorder, diplopia,and enophthalmos.Conclusion:Endoscopic approach is a safe and effective treatment in the repair of medial orbital fracture with perpendicular plate of ethmoid.

Increased miR-155-5p expression in dermal mesenchymal stem cells of psoriatic patients: comparing the microRNA expression profile by microarray.

Mesenchymal stem cells (MSCs) have pleiotropic immuno-modulatory effects and pro-angiogenic ability, leading to the presumption that MSCs may be involved in the pathogenesis of many inflammatory or autoimmune disorders, including psoriasis. In a previous study, we reported the specific gene expression profile of dermal MSCs from psoriasis. Inflammation- and angiogenesis-related genes, such as lipopolysaccharide-induced tumor necrosis factor-alpha transcription factor (LITAF), dual-specificity protein phosphatase 1 (DUSP1), vascular endothelial growth factor α (VEGFα), and insulin-like growth factor-binding protein-5 (IGFBP5), are abnormally expressed in psoriatic dermal MSCs. As a key regulator of gene expression, miRNA are involved in a wide variety of biological processes; in fact, several miRNAs have been implicated in the development and progression of inflammatory or autoimmune disorders. In this study, we compared the miRNA expression profiles of dermal MSCs from patients with psoriasis to those in MSCs from normal individuals by microarray, and found that the pro-inflammatory miRNA miR-155 was significantly overexpressed in psoriatic MSCs (2.44 fold, P < 0.001). Additionally, the expression of miR-155 target gene TAB2 (8.47 ± 1.55 vs 6.38 ± 2.10, P < 0.01,) and the downstream gene iNOS (5.26 ± 2.58 vs 3.73 ± 1.89, P < 0.05) was found to be inhibited in psoriatic dermal MSCs by real-time PCR. Therefore, we speculated that the elevation in miR-155 levels may be an indicator of, or a key regulatory pathway in, the pathogenesis of psoriasis, resulting in functionally impaired dermal MSCs.

LITAF, HHEX, and DUSP1 expression in mesenchymal stem cells from patients with psoriasis.

Psoriasis is a common chronic relapsing inflammatory skin disease, in which mesenchymal stem cells (MSCs) have been hypothesized to play an important role in abnormal localized inflammation and vascular proliferation observed in skin lesions. Previous studies have revealed abnormal gene expression patterns, DNA methylation status, and cytokine secretion of MSCs in psoriatic skin lesions, as well as some gene expression abnormalities related to inflammation and angiogenesis. We further verified the gene and protein expressions of inflammation-related lipopolysaccharide-induced tumor necrosis factor-alpha transcription factor (LITAF), dual-specificity protein phosphatase 1 (DUSP1), and angiogenesis-related hematopoietically expressed homeobox (HHEX) in MSCs derived from the skin lesions of psoriasis patients. The gene expression of LITAF, DUSP1, and HHEX in dermal MSCs was measured at the mRNA level using reverse transcription-polymerase chain reaction and the corresponding protein expression levels were analyzed by western blotting analysis. The gene and protein expression levels of LITAF, HHEX, and DUSP1 in dermal MSCs were significantly lower in psoriasis patients compared to controls. Amplification and western blotting results were consistent with our previously reported gene chip data. Our results suggest that dermal MSCs in psoriatic skin lesions may be involved in the development, progression, and regulation of localized inflammatory abnormalities by reducing the expression of LITAF, HHEX, and DUSP1, which are related to inflammation and angiogenesis.

Impact of BMMSCs from different sources on proliferation of CD34⁺ cells.

There are significant differences on the biological characteristics of bone marrow mesenchymal stem cells (BMMSCs), immunological response, and antigen-presenting functions between patients with psoriasis and normal subjects, but there are no significant differences in aborted fetuses. We examined the differences in BMMSCs between aborted fetuses and patients with psoriasis in this study. Bone marrow from normal subjects, aborted fetuses, and patients with psoriasis were obtained using a MidiMACS machine. Density gradient centrifugation method was used to isolate the bone marrow mononuclear cells of patients with psoriasis and aborted fetus and the cells were cultivated. Bone marrow CD34(+) cells from normal subjects were isolated. MTT colorimetric detection was used to test the proliferation activity of bone marrow CD34(+) cells. The purity of bone marrow CD34(+) cells and BMMSCs was determined by flow cytometry. The BMMSC culture supernatant fluid of patients with psoriasis and aborted fetuses showed no statistically significant difference with bone marrow CD34(+) cell proliferation in normal subjects (P > 0.05).

Research Note Mesenchymal stem cells from skin lesions of psoriasis patients promote proliferation and inhibit apoptosis of HaCaT cells.

Psoriasis is an inflammatory skin disease characterized by excessive proliferation and abnormal differentiation and apoptosis of keratinocytes (KCs). Mesenchymal stem cells (MSCs) from skin lesions of psoriasis patients demonstrate abnormal cytokine secretion, which may affect KC proliferation and apoptosis. Here, we explored how MSCs from skin lesions of psoriasis patients affect HaCaT cell proliferation and apoptosis. First, flow cytometry and multipotent differentiation methods were used to identify skin MSCs, which were then co-cultured with HaCaT cells. HaCaT cell proliferation was analyzed in real-time, and cell cycle progression and apoptosis were assessed by flow cytometry. Cell morphologies and multipotencies of skin MSCs were similar between the psoriasis group and healthy control group, with high levels of CD29, CD44, CD73, CD90, and CD105 and limited expression of CD34, CD45, and HLA-DR. MSCs from skin lesions of psoriasis patients promote KC proliferation more potently and are less capable of inducing KC apoptosis. This may underlie KC proliferation and abnormal apoptosis in psoriasis skin lesions, which results in abnormal thickening of the epidermis.

CXCL12 G801A polymorphism and susceptibility to glioma: a case­control study.

Previous studies have demonstrated that the CXCL12 G801A polymorphism is closely correlated with tumor susceptibility. In addition, the CXCL12/CXCR4 pathway is closely related to proliferation, metastasis, and invasion of glioma. However, the genetic effects of the CXCL12 G801A polymorphism on glioma risk in Chinese populations remain unknown. In this study, we investigated the potential associations between the CXCL12 G801A polymorphism with glioma susceptibility and its clinicopathological characteristics. Frequencies of CXCL12 G801A polymorphic variants between glioma patients (N = 750) and healthy controls (N = 750) were assessed using restriction length fragment polymorphism analysis. The association among the CXCL12 G801A polymorphism, glioma grade (WHO classification), and histological type was also evaluated. Our results showed that patients with glioma had significantly higher frequency of the CXCL12-3' A/A genotypes (P = 0.039) as compared with healthy controls. When stratified by the glioma histology, high-grade glioma patients had significantly higher frequency of the CXCL12-3' A/A genotypes (P = 0.019) as compared with low-grade glioma patients. When stratified by the WHO grade, significantly higher frequency of the CXCL12-3' A/A genotype was observed in stage IV glioma patients (P = 0.037). We conclude that the CXCL12 G801A polymorphism is a risk factor that increases susceptibility to gliomas in a subset of the general Han Chinese population.

Carbohydrate accumulation may be the proximate trigger of anthocyanin biosynthesis under autumn conditions in Begonia semperflorens.

Many plant leaves appear red in the autumn, and many papers have focused on the environmental factors and role of anthocyanin in this process. However few papers have examined the substances that are induced during this process. We hypothesised that excess sugar accumulation directly induces anthocyanin accumulation under autumn conditions. Using two methods (restricting phloem movement and exogenous sucrose feeding), we found that both surplus photosynthate and exogenous sucrose could induce anthocyanin biosynthesis, corresponding to up-regulation of several enzymes involved in anthocyanin biosynthesis (phenylalanine ammonia lyase, chalcone isomerase, dihydroflavonol 4-reductase and flavonoid 3-O-glucosyl transferase) and in transport (glutathione S-transferase). Our results suggest that excess carbohydrate may be the proximate trigger for induction of anthocyanin biosynthesis in autumn, but only when carbohydrates are accumulated for storage.

Assessment of modeled mercury dry deposition over the Great Lakes region.

Three sets of model predicted values for speciated mercury concentrations and dry deposition fluxes over the Great Lakes region were assessed using field measurements and model intercomparisons. The model predicted values were produced by the Community Multiscale Air Quality Modeling System for the year 2002 (CMAQ2002) and for the year 2005 (CMAQ2005) and by the Global/Regional Atmospheric Heavy Metals Model for the year 2005 (GRAHM2005). Median values of the surface layer ambient concentration of gaseous elemental mercury (GEM) from all three models were generally within 30% of measurements. However, all three models overpredicted surface-layer concentrations of gaseous oxidized mercury (GOM) and particulate bound mercury (PBM) by a factor of 2-10 at the majority of the 15 monitoring locations. For dry deposition of GOM plus PBM, CMAQ2005 showed a clear gradient with the highest deposition in Pennsylvania and its surrounding areas while GRAHM2005 showed no such gradient in this region; however, GRAHM2005 had more hot spots than those of CMAQ2005. Predicted dry deposition of GOM plus PBM from these models should be treated as upper-end estimates over some land surfaces in this region based on the tendencies of all the models to overpredict GOM and PBM concentrations when compared to field measurements. Model predicted GEM dry deposition was found to be as important as GOM plus PBM dry deposition as a contributor to total dry deposition. Predicted total annual mercury dry deposition were mostly lower than 5 µg m(-2) to the surface of the Great lakes, between 5 and 15 µg m(-2) to the land surface north of the US/Canada border, and between 5 and 40 µg m(-2) to the land surface south of the US/Canada border. Predicted dry deposition from different models differed from each other by as much as a factor of 2 at regional scales and by a greater extent at local scales.

Orphanin FQ produces gender-specific modulation of trigeminal nociception: behavioral and electrophysiological observations.

The present study aimed to determine if orphanin FQ, an endogenous ligand for the opioid receptor like-1 receptor, produces gender-specific effects in the modulation of N-methyl-D-aspartate (NMDA)-evoked responses of trigeminal nociceptive neurons, and in the NMDA-induced nociceptive behavior. Single-unit extracellular recordings were made from nociceptive-specific and wide dynamic range neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in anesthetized (1.5 g/kg urethane) rats. In the proestrous female, orphanin FQ applied microiontophoretically produced facilitation of the NMDA-evoked responses in 50% (16/32) of nociceptive neurons, inhibition in 31% (10/32), and biphasic effects in 19% (6/32). In contrast, in the male, it inhibited the responses in 86% (18/21), and facilitated the responses in 14% (4/21). In ovariectomized animals, orphanin FQ inhibited the responses in 75% (9/12) of nociceptive neurons, facilitated the responses in 17% (2/12) and produced biphasic effects in 8% (1/12). In contrast, in estradiol-treated ovariectomized rats, it facilitated the responses in 46% (5/11), inhibited the responses in 36% (4/11) and produced biphasic effects in 18% (2/11). For behavioral studies, NMDA-induced scratching behavior was used to assess the effects of orphanin FQ. Twenty-eight male, ovariectomized and estradiol-treated ovariectomized rats were microinjected with NMDA (2 nmol in 10 microl) alone through a cannula implanted in the medullary region, while another 27 rats were microinjected with orphanin FQ (10 nmol in 10 microl) 10 min prior to giving NMDA. Orphanin FQ reduced the NMDA-induced nociceptive scratching behavior by 92% in the male, and by 96% in ovariectomized rats. In contrast, in estradiol-treated ovariectomized animals, orphanin FQ facilitated the NMDA-induced scratching behavior by 210%. We conclude from these studies that orphanin FQ is primarily pronociceptive in the female and primarily antinociceptive in the male. Furthermore, we suggest that estrogen is involved in generating the gender-specific effects of orphanin FQ.

Endomorphin-1 and endomorphin-2 modulate responses of trigeminal neurons evoked by N-methyl-D-aspartic acid and somatosensory stimuli.

The present study investigated the modulation of N-methyl-D-aspartate (NMDA)-evoked and peripheral cutaneous stimulus-evoked responses of trigeminal neurons by endomorphins, endogenous ligands for the mu-opioid receptor. Effects of endomorphins, administered microiontophoretically, were tested on the responses of nociceptive neurons recorded in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in anesthetized rats. Endomorphin-1 and endomorphin-2 predominantly reduced the NMDA-evoked responses, producing an inhibitory effect of 54.1 +/- 2.96% (mean +/- SE; n = 34, P < 0.001) in 92% (34/37) of neurons and 63.6 +/- 3.61% (n = 32, P < 0.001) in 91% (32/35) of neurons, respectively. The inhibitory effect of endomorphins was modality specific; noxious stimulus-evoked responses were reduced more than nonnoxious stimulus-evoked responses. Naloxone applied at iontophoretic current that blocked the inhibitory effect of [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin, reduced the peak inhibitory effect of endomorphins on the NMDA- and natural stimulus-evoked responses. We suggest that endomorphins by acting at micro-opioid receptor selectively modulate noxious stimulus-evoked responses in the medullary dorsal horn.

Orphanin FQ (nociceptin) modulates responses of trigeminal neurons evoked by excitatory amino acids and somatosensory stimuli, and blocks the substance P-induced facilitation of N-methyl-D-aspartate-evoked responses.

The present investigation details the modulation of medullary dorsal horn neuron responses to excitatory amino acids and peripheral cutaneous stimuli by orphanin FQ (nociceptin), an endogenous ligand for the opioid receptor-like, receptor. Effects of orphanin FQ, administered microiontophoretically or given intracerebroventricularly, were tested on the responses of nociceptive-specific, wide dynamic range and low threshold neurons recorded in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in anesthetized (urethane or pentobarbital) male rats. Microiontophoretic application of orphanin FQ reduced the N-methyl-D-aspartate-evoked responses in 86% (71/82) of neurons, and the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-evoked responses in 86% (30/35) of neurons. However, orphanin FQ produced a longer lasting inhibitory effect on the N-methyl-D-aspartate-evoked responses relative to the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-evoked responses. The inhibitory effect of orphanin FQ was not modality-specific, responses evoked by noxious as well as non-noxious stimuli were reduced in 22/23 neurons. However, the inhibitory effect was more pronounced on noxious stimulus-evoked responses. Naloxone applied at currents that antagonized the inhibitory effects of selective agonists at mu and kappa opioid receptors failed to inhibit the effects of orphanin FQ. Microiontophoretic co-application of substance P with N-methyl-D-aspartate facilitated the N-methyl-D-aspartate-evoked responses in 52% (26/50) of nociceptive neurons. Orphanin FQ blocked or reduced the substance P-induced facilitation by 86+/-24.4% (n = 14). In order to compare electrophysiological data with previous behavioral observations, effects of orphanin FQ administered intracerebroventricularly were tested on the excitatory amino acid-evoked responses. Orphanin FQ reduced the N-methyl-D-aspartate-evoked responses in 85% (11/13) of neurons whereas the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-evoked responses were facilitated in 69% (9/13) of neurons. We suggest that orphanin FQ produces a predominantly inhibitory effect on, (i) noxious stimuli evoked responses, (ii) excitatory amino acid receptor-mediated transmission and, (iii) the substance P-induced facilitation of the N-methyl-D-aspartate-evoked responses. We conclude that orphanin FQ primarily produced an antinociceptive action at the level of the dorsal horn of the medulla.

Salbutamol and the conditioning of latissimus dorsi for cardiomyoplasty.

BACKGROUND: Cardiomyoplasty is a new surgical alternative therapy for CHF. Although conditioning of muscle for cardiomyoplasty has a positive effect on fatigue resistance it also produces negative effects. In this study we assessed the effect of salbutamol, a beta2-agonist, on both the positive and the negative effects of conditioning. METHODS: In a control group of six animals one latissimus dorsi was subject to chronic, 1 Hz, low-frequency stimulation (CLFS) while the other served as a control. The experimental group of seven dogs received a continuous SC infusion of salbutamol and one latissimus dorsi was subjected to CLFS. The other muscle demonstrated the effects of salbutamol per se. After 42 days the animals were anesthetized and fatigue resistance, muscle mass, and mechanical properties of the muscles were evaluated. RESULTS: Salbutamol increased muscle mass, tetanic tension, and rate of rise and fall of tetanic tension. It diminished fatigue resistance and had no effect on shortening velocity. Chronic stimulation decreased muscle mass, tetanic tension, rate of rise and fall of tetanic tension, and muscle shortening velocity in both groups of dogs. Salbutamol diminished the declines in muscle mass, rate of tension development, and rate of muscle shortening due to CLFS, but did not change the effects of CLFS on tetanic tension and the rate of fall of tetanic tension. Salbutamol did not alter the increase in fatigue resistance induced by CLFS. CONCLUSIONS: The favorable effect of CLFS on fatigue resistance was unaffected by salbutamol. The unfavorable effects of CLFS on loss of muscle mass, rate of tension development, and decline in shortening velocity were partially blocked by salbutamol, improving the ability of the latissimus dorsi to augment cardiac systole.

alpha2-adrenoceptors modulate NMDA-evoked responses of neurons in superficial and deeper dorsal horn of the medulla.

Extracellular single unit recordings were made from neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in 21 male rats anesthetized with urethan. NMDA produced an antagonist-reversible excitation of 46 nociceptive as well as nonnociceptive neurons. Microiontophoretic application of a preferential alpha2-adrenoceptor (alpha2AR) agonist, (2-[2, 6-dichloroaniline]-2-imidazoline) hydrochloride (clonidine), reduced the NMDA-evoked responses of 86% (6/7) of nociceptive-specific (NS) neurons, 82% (9/11) of wide dynamic range (WDR) neurons, and 67% (4/6) of low-threshold (LT) neurons in the superficial dorsal horn. In the deeper dorsal horn, clonidine inhibited the NMDA-evoked responses of 94% (16/17) of NS and WDR neurons and 60% (3/5) of LT neurons. Clonidine facilitated the NMDA-evoked responses in 14% (1/17) of NS, 9% (1/11) of WDR, and 33% (2/6) of LT neurons in the superficial dorsal horn. Idazoxan, an alpha2AR antagonist, reversed the inhibitory effect of clonidine in 90% (9/10) of neurons, whereas prazosin, an alpha1-adrenoceptor antagonist with affinity for alpha2BAR, and alpha2CAR, were ineffective. We suggest that activation of alpha2ARs produces a predominantly inhibitory modulation of the NMDA-evoked responses of nociceptive neurons in the medullary dorsal horn.

The beta2-agonist salbutamol affects the expression of phospholamban and both isoforms of SERCA in canine skeletal muscle and blocks changes in these induced by neuromuscular stimulation.

Chronic administration of salbutamol induced expression of hybrid fibers in canine skeletal muscles. Fast-twitch fibers expressed SERCA2a (the slow-twitch isoform of sarcoplasmic reticulum Ca2+-ATPase) and slow-twitch fibers expressed SERCA1 (the fast-twitch isoform of the Ca2+-ATPase). The proportion of fibers that became hybrid increased from a small percentage in the control muscles to 30% in the predominantly fast-twitch latissimus dorsi and to 45% in the predominantly slow-twitch vastus intermedius. In contrast to this response by the SERCA genes the phospholamban gene response was muscle specific. The fraction of fibers that expressed phospholamban decreased slightly in the latissimus dorsi while increasing moderately in the vastus intermedius. The effects of chronic neurostimulation of the latissimus dorsi on SERCA1, SERCA2a and phospholamban levels were mostly blocked by salbutamol. While 100% of fibers from neurostimulated muscles expressed phospholamban, only 51% of the fibers from the neurostimulated and salbutamol-treated muscles expressed it. In the neurostimulated muscle, very few muscle fibers expressed SERCA1a while 61% of the fibers that received salbutamol expressed it, albeit as hybrid fibers. The levels of SERCA2a in response to these interventions were just the opposite. In the neurostimulated muscle 37.5% of fibers were hybrid and 62.5% expressed SERCA2a only. With co-administration of neurostimulation and salbutamol, 61.3% of fibers were hybrid and 38.7% expressed SERCA2a only.

Transcription rates of SERCA and phospholamban genes change in response to chronic stimulation of skeletal muscle.

Chronic low frequency stimulation of predominantly fast-twitch skeletal muscles decrease the levels of SERCA1 (fast-twitch sarco(endo)plasmic reticulum Ca2+-ATPase) mRNA, and increase the levels of SERCA2 (slow-twitch sarco(endo)plasmic reticulum Ca2+-ATPase) and phospholamban mRNAs. To assess the role of transcription in these changes in mRNA levels, nuclei were isolated from chronically stimulated canine latissimus dorsi muscles and transcription rates were estimated by nuclear run-on assays. Decreases in the rates of SERCA1 gene transcription matched the fall in its mRNA level and increases in the rates of SERCA2 and phospholamban gene transcription matched the increases in their mRNAs.

Correlations between MyoD, myogenin, SERCA1, SERCA2 and phospholamban transcripts during transformation of type-II to type-I skeletal muscle fibers.

Canine latissimus dorsi, composed predominantly of fast-twitch muscle fibers, were subjected to chronic 1 Hz neuromuscular stimulation for periods up to 42 days to induce changes in gene expression. This produced down regulation of SERCA1 (fast-twitch isoform of sarco(endo)plasmic reticulum Ca2+-ATPase), a gene product of fast-twitch muscle, and up regulation fo SERCA2 (slow-twitch isoform of sarco(endo)plasmic reticulum Ca2+-ATPase) and phospholamban, products of genes expressed by slow-twitch muscles. To assess the involvement of MyoD and myogenin in the regulation of the expression of these genes their levels were measured during the stimulation period. The prompt, at 7 days, fall in SERCA1 mRNA preceded the fall in MyoD by about 7 days, suggesting that the decline in MyoD was not causally related to the decline in SERCA1. The prompt rise in SERCA2 mRNA at 7 days preceded the rise in myogenin by 14 days. The rise in myogenin at 21 days did correlate with the similar rise in phospholamban mRNA.

Fast- and slow-twitch isoforms (SERCA1 and SERCA2a) of sarcoplasmic reticulum Ca-ATPase are expressed simultaneously in chronically stimulated muscle fibers.

Using an immunohistochemical double-labeling technique, we observed that different isoforms of sarcoplasmic reticulum Ca-ATPase are co-expressed in single fibers of canine fast-twitch skeletal muscles stimulated chronically at low frequency. By 7 days of neuromuscular stimulation, the population of hybrid fibers expressing both SERCA1 and SERCA2a [fast- and slow-twitch isoforms of sarco(endo)plasmic reticulum Ca(2+)-ATPase] had increased from 1.5% to 9.2% of fibers. By 14 days of stimulation 90% of the pure fast-twitch fibers (expressing only SERCA1) were replaced by hybrid fibers. An additional 28 days of stimulation caused all fast-twitch fibers to express SERCA2a at the same level as found in nonstimulated slow-twitch fibers (expressing only SERCA2a). At this time, one-half of the previously hybrid fibers had become pure-slow-twitch fibers. The remaining one-half of the hybrid fibers expressed SERCA1 at a very low level. Extending stimulation to 70 days did not further change the percentage of fibers that were slow-twitch or hybrid. Immunoblot studies at the whole-muscle level confirmed that changes in SERCA expression at 42 days of neuromuscular stimulation were complete. Immunohistochemical analysis of longitudinal sections of muscle showed that the changes in SERCA protein were uniform along the length of the muscle fiber, indicating that nuclei along its length responded equally to chronic stimulation.