Simultaneous determination of carbonyl compounds related to thermal treatment and oxidative stability of infant formulas by gas-diffusion microextraction and high-performance liquid chromatography with ultraviolet detection.

Infant formulae are the only possible alternative to breastfeeding during the first year of life, so it is crucial to assure their innocuousness. Infant formula undergoes heat treatments to ensure safety and shelf life. However, such processes impact health as they lead to the formation of malondialdehyde, acrolein, and α-dicarbonyl compounds, related to Maillard reaction. Thus, there is a need for improved analytical methods to ensure the safety, quality, and nutritional value of infant formulae, and also exploring the potential of specific compounds as indicators for quality control and monitoring purposes. We developed and validated a novel, efficient, and cost-effective method using gas-diffusion microextraction for the simultaneous quantification of carbonyl compounds in infant formula. Malondialdehyde, acrolein, glyoxal, methylglyoxal, and diacetyl were detected as o-phenylenediamine derivatives using HPLC with UV detection. Parameters influencing extraction efficiency were studied using an asymmetric screening design. The validated method has shown excellent linearity, sensitivity, accuracy, and precision. It was applied to analyze 26 infant formula samples, including starter, follow-up, and special formulated powdered infant formula. Methylglyoxal was found in all samples (0.201-3.153 µg mL-1), while malondialdehyde was present only in certain starter formulas (1.033-1.802 µg mL-1). Acrolein (0.510-3.246 µg mL-1), glyoxal (0.109-1.253 µg mL-1), and diacetyl (0.119-2.001 µg mL-1) were detected in various sample types. Principal components and hierarchical cluster analyses have showcased distinct sample clustering based on analyte contents. This study presents a novel methodology for the analysis of markers of thermal treatment and oxidative stability in infant formula. It contributes to the characterization of the products' composition and quality control of infant formulae, thereby enhancing their safety and nutritional adequacy. This study also presents the first reported quantification of acrolein in infant formula and introduces the application of the acrolein-o-phenylenediamine derivative for food analysis.

Experiences and reflections about behavioral pain assays in laboratory animals.

Pharmacological assays based on the measurement of nociceptive responses in laboratory animals are a fundamental tool to assess analgesic strategies. During our experience with this type of experiments, we have been repeatedly challenged by different concerns related to their interpretation or relevance. Although these subjects are frequently discussed in our lab, they do not usually find a place in research articles with original data, in which the focus on results seems mandatory. In the present manuscript we try to discuss as central issues some of these aspects that often cross transversally our research. We have gathered them in five topics inspired by the results obtained in our laboratory. The two initial sections are devoted to the influence of the behavioral method used to assess nociception on the results achieved, as well as to the possibility that data may be more easily accepted when obtained with standard methods than with alternative ones. The third topic is related to the difficulties encountered when working with a molecule that may evoke dual effects, acting as pronociceptive or antinociceptive depending on the dose. The fourth point deals with the situation in which a particular hyperalgesic reaction is related to several molecules but the single inhibition of only one of them can completely prevent it. Finally, the last issue is addressed to comment the impact in the progress of pain research of experiments performed in animal models of pathological settings.

A low pKa ligand inhibits cancer-associated pain in mice by activating peripheral mu-opioid receptors.

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC50 of NFEPP to displace bound [3H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30-100 nmol/kg) or fentanyl (17-30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

The Systemic Administration of the Chemokine CCL1 Evokes Thermal Analgesia in Mice Through the Activation of the Endocannabinoid System.

Apart from its involvement in immune functions, the chemokine CCL1 can participate in the modulation of nociceptive processing. Previous studies have demonstrated the hypernociceptive effect produced by CCL1 in the spinal cord, but its possible action on peripheral nociception has not yet been characterized. We describe here that the subcutaneous administration of CCL1 (1-10 µg/kg) produces dose-dependent and long-lasting increases in thermal withdrawal latencies measured by the unilateral hot plate test in mice. The antinociceptive nature of this effect is further supported by the reduction of spinal neurons expressing Fos protein in response to a noxious thermal stimulus observed after the administration of 10 µg/kg of CCL1. CCL1-induced antinociception was inhibited after systemic, but not spinal administration of the selective antagonist R243 (0.1-1 mg/kg), demonstrating the participation of peripheral CCR8 receptors. The absence of this analgesic effect in mice treated with a dose of cyclophosphamide that produces a drastic depletion of leukocytes suggests its dependency on white blood cells. Furthermore, whereas the antinociceptive effect of CCL1 was unaffected after the treatment with either the antagonist of opioid receptors naloxone or the cannabinoid type 1 receptor blocker AM251, it was dose-dependently inhibited after the administration of the CB2 receptor antagonist SR144528 (0.1-1 mg/kg). The detection by ELISA of an increased presence of the endocannabinoid 2-arachidonoylglycerol after the administration of an analgesic dose of CCL1 supports the notion that CCL1 can evoke thermal analgesia through the release of this endocannabinoid from circulating leukocytes.

Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers.

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

Synergistic combinations of the dual enkephalinase inhibitor PL265 given orally with various analgesic compounds acting on different targets, in a murine model of cancer-induced bone pain.

BACKGROUND: The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 (P2X3 receptor antagonist), ACEA (CB1 receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). METHODS: Experiments. Experiments were performed in 5-6 weeks old (26-33g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2×106 cells/100µL. Then 105 cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49±0.2°C. Mechanical threshold values were obtained by performing the von Frey test using the "up and down" method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. RESULTS: The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENKs, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X3 receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Nav1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. CONCLUSIONS: These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. IMPLICATIONS: This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain.

Incidence of Idiopathic Normal-Pressure Hydrocephalus in Northern Spain.

BACKGROUND: Although the incidence of idiopathic normal-pressure hydrocephalus (iNPH) can be 1.20 cases/1000 inhabitants/year in individuals ≥ 70 years old, in most series, the incidence of shunt-responsive iNPH appears to be <1/100,000 inhabitants/year. We report the results of a prospective 10-year longitudinal study of the incidence of iNPH in a northern Spanish population. METHODS: In a stable population of 590,000 inhabitants served by a single neurosurgical department, we periodically asked all primary care practitioners, neurologists, and geriatricians to refer for iNPH screening any patient with ventricular dilation who was complaining of motor disturbances, cognitive impairment, or sphincter dysfunction. RESULTS: From January 2003 to December 2012, 293 patients were referred with suspected normal-pressure hydrocephalus. In 187 patients, iNPH was diagnosed; 89 of these patients were classified as probable iNPH, and 98 were classified as possible iNPH. Cerebrospinal fluid diversion was performed in 152 patients, and 119 showed a good outcome. The incidence of iNPH was significantly greater in male patients and patients >60 years old and increased exponentially with age. After age 60, the standardized age- and sex-adjusted incidence rates for iNPH, shunt surgery for iNPH, and shunt-responsive iNPH were 13.36 cases/100,000 inhabitants/year, 10.85 cases/100,000 inhabitants/year, and 8.55 cases/100,000 inhabitants/year. No differences were detected in the response rate between probable and possible iNPH (80.52% vs. 76.00%; P = 0.497). CONCLUSIONS: Even with a protocol for patient referral in place, reported iNPH incidence was lower than predicted, reflecting a persistent problem of underdiagnosis in our population.

Epidemiology of Idiopathic Normal Pressure Hydrocephalus: A Systematic Review of the Literature.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is an important cause of gait disturbance and cognitive impairment in elderly adults. However, the epidemiology of iNPH is relatively unknown, largely as a result of the paucity of specifically designed population studies. This systematic review aims to assess the prevalence and incidence of iNPH. METHODS: A systematic literature review on the epidemiology of iNPH was conducted using MEDLINE/PubMed searching for articles published up to June 2014. RESULTS: The inclusion criteria were met by 21 studies. Of the studies, 12 were specifically designed for detecting cases of iNPH; however, only 4 were prospective. In people >65 years old, pooled prevalence obtained from specific population studies was 1.3%, which was almost 50-fold higher than that inferred from door-to-door surveys of dementia or parkinsonism. Prevalence may be higher in assisted-living and extended-care residents, with 11.6% of patients fulfilling the criteria for suspected iNPH and 2.0% of patients showing permanent improvement after cerebrospinal fluid diversion. The only prospective population-based survey that reported iNPH incidence estimated 1.20 cases/1000 inhabitants/year, 15-fold higher than estimates obtained from studies based on hospital catchment areas. The incidence of shunt surgery for iNPH and shunt-responsive iNPH obtained from incident cases of hospital catchment areas appears to be <2 cases/100,000 inhabitants/year and 1 case/100,000 inhabitants/year, respectively. No population-based study reporting the real values for these 2 parameters could be found. CONCLUSIONS: iNPH appears to be extremely underdiagnosed. Properly designed and adequately powered population-based studies are required to characterize the epidemiology of this disease accurately.

Hypernociceptive responses following the intratibial inoculation of RM1 prostate cancer cells in mice.

BACKGROUND: Pain due to bone metastases of prostatic origin is a relevant clinical issue. We study here the nociceptive responses obtained in mice receiving the intratibial inoculation of RM1 prostate cancer cells. METHODS: 10(2) -10(5) RM1 cells were inoculated to C57BL/6 mice and tumor development was analysed histologically and with luciferase-expressing RM1 cells. Spinal astroglial (GFAP) or microglial (Iba-1) expression was assessed with immunohistochemical methods and hypernociception was measured by the unilateral hot plate, the paw pressure and the von Frey tests. The analgesic effect of morphine, zoledronic acid or the CCR2 antagonist RS504393 was measured. Levels of the chemokines CCL2, CCL3, and CCL5 were determined by ELISA. RESULTS: The inoculation of 10(3) RM1 cells induced tumoral growth in bone with a mixed osteoclastic/osteoblastic pattern and evoked astroglial, but not microglial, activation in the spinal cord. Hyperalgesia and allodynia were already established four days after inoculation and dose-dependently inhibited by the s.c. administration of morphine (1-5 mg/kg) or zoledronic acid (1-3 mg/kg). CCL2 and CCL5, but not CCL3, were released by RM1 cells in culture whereas only an increased presence of CCL2 was found in bone tumor homogenates. The administration of the CCR2 antagonist RS504393 (0.3-3 mg/kg) inhibited RM1 induced thermal hyperalgesia without modifying mechanical allodynia. CONCLUSION: The intratibial inoculation of RM1 cells in immunocompetent mice induces hypernociceptive responses and can be useful to perform studies of bone cancer induced pain related to androgen-independent prostate cancer. The antinociceptive role derived from the blockade of the CCR2 chemokine receptors is further envisaged.

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.

The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.

The chemokine CCL5 induces CCR1-mediated hyperalgesia in mice inoculated with NCTC 2472 tumoral cells.

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10-30µg) but not spinal (3-5µg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863. The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium. The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

Involvement of spinal chemokine CCL2 in the hyperalgesia evoked by bone cancer in mice: a role for astroglia and microglia.

The hypernociceptive role played by the chemokine CCL2, and its main receptor, CCR2, in pathological settings is being increasingly recognized. We aimed to characterize the involvement of spinal CCL2 in the hyperalgesia due to the intratibial inoculation of fibrosarcoma NCTC 2472 cells in mice. The intrathecal (i.t.) administration of the CCR2 antagonist RS 504393 (1­3 µg) or an anti-CCL2 antibody inhibited tumoral hyperalgesia. No change in the expression of spinal CCR2 was detected by western blot, whereas immunohistochemical experiments demonstrated increased CCL2 staining at the superficial laminae of the spinal cord ipsilateral to the tumor. This spinal CCL2 does not seem to be released from nociceptors since CCL2 mRNA and CCL2 levels in DRGs, as measured by RT-PCR and ELISA, remain unmodified in tumor-bearing mice. In contrast, immunohistochemical assays demonstrated the spinal up-regulations of GFAP and Iba-1, respective markers of astroglia and microglia, and the expression of CCL2 in both types of glial cells at the superficial laminae of the spinal cord of tumor-bearing mice. Finally, since CCL2 could induce astroglial or microglial activation, we studied whether the blockade of CCR2 could inhibit the increased spinal glial expression. GFAP, but not Iba-1, up-regulation was reduced in tumor-bearing mice treated for 3 days with i.t. RS 504393, indicating that spinal CCL2 acts as an astroglial activator in this setting. The participation at spinal level of CCL2/CCR2 in tumoral hypernociception, together with its previously described involvement at periphery, makes attractive the modulation of this system for the alleviation of neoplastic pain.

[Reference values and variability study of thyroid hormones in pregnant women from El Bierzo]. / Valores de referencia y estudio de la variabilidad de hormonas tiroideas en gestantes de El Bierzo.

BACKGROUND AND OBJECTIVES: El Bierzo area is characterized by low urinary iodine levels in the pregnant population. Guidelines recommend that local reference values are established for the diagnosis of thyroid dysfunction in pregnancy. Our objectives were to establish reference values for thyroid-stimulating hormone (TSH), free thyroxine (FT4) and free triiodothyronine (FT3) in women in the first trimester of pregnancy and to explore the factors influencing variability in these hormones. PATIENTS AND METHODS: A retrospective study of 412 women in the first trimester of pregnancy who were measured serum levels of TSH, FT4, and FT3; 163 women with conditions with a potential influence on thyroid function were excluded. Thirty smoking pregnant women were also excluded from the study of reference values. Factors examined in the variability study included age, body mass index (BMI), and smoking. A multifactorial analysis of covariance was performed. RESULTS: Reference values in first-trimester pregnant women were: TSH: 0.497-3.595 mIU/L; FT4: 0.90-1.42 ng/dL; FT3: 2.49-3.56 pg/mL. TSH levels depended on mother age and on interaction between age and smoking. FT3 levels depended on the mother's BMI and smoking, and there was also an interaction between both factors. CONCLUSION: The reference values found may be used to assess thyroid dysfunction in pregnant women from El Bierzo. TSH and FT3 levels are influenced by age and BMI of the mother and by smoking, in addition to the interaction of these factors.

Spinal CCL2 and microglial activation are involved in paclitaxel-evoked cold hyperalgesia.

The antineoplastic paclitaxel induces a sensory neuropathy that involves the spinal release of neuroinflammatory mediators and activation of glial cells. Although the chemokine CCL2 can evoke glial activation and its participation in neuropathic pain has been demonstrated in other models, its involvement in paclitaxel-evoked neuropathy has not been previously explored. Paclitaxel-evoked cold hypernociception was assessed in mice by the unilateral cold plate test and the effects on cold hyperalgesia of the CCR2 antagonist RS 504393, the CCR1 antagonist J113863, the microglial inhibitor minocycline or an anti-CCL2 antibody were tested. Furthermore, ELISA measurements of CCL2 concentration and immunohistochemical assays of Iba-1 and GFAP, markers of microglial and astroglial cells respectively, were performed in the lumbar spinal cord. Cold hypernociception measured 3 days after the administration of paclitaxel (10mg/kg) was inhibited by the s.c. (0.3-3mg/kg) or i.t. (1-10 µg) administration of RS 504393 but not of J113863 (3-30 mg/kg). CCL2 levels measured by ELISA in the lumbar spinal cord were augmented in mice treated with paclitaxel and the i.t. administration of an anti-CCL2 antibody completely suppressed paclitaxel-evoked cold hyperalgesia, strongly suggesting that CCL2 is involved in the hypernociception evoked by this taxane. Besides, the implication of microglial activation is supported by the increase in the immunolabelling of Iba-1, but not GFAP, in the spinal cord of paclitaxel-treated mice and by the inhibition of cold hyperalgesia produced by the i.t. administration of the microglial inhibitor minocycline (1-10 nmol). Finally, the neutralization of spinal CCL2 by the i.t. administration of a selective antibody for 3 days almost totally inhibited paclitaxel-evoked microglial activation. In conclusion, our results indicate that paclitaxel-evoked cold hypernociception depends on the activation of CCR2 due to the spinal release of CCL2 and the subsequent microglial activation.

CCL2 released at tumoral level contributes to the hyperalgesia evoked by intratibial inoculation of NCTC 2472 but not B16-F10 cells in mice.

The participation of the chemokine CCL2 (monocyte chemoattractant protein-1) in inflammatory and neuropathic pain is well established. Furthermore, the release of CCL2 from a NCTC 2472 cells-evoked tumor and its involvement in the upregulation of calcium channel α2δ1 subunit of nociceptors was demonstrated. In the present experiments, we have tried to determine whether the increase in CCL2 levels is a common property of painful tumors and, in consequence, the administration of a chemokine receptor type 2 (CCR2) antagonist can inhibit tumoral hypernociception. CCL2 levels were measured by ELISA in the tumoral region of mice intratibially inoculated with NCTC 2472 or B16-F10 cells, and the antihyperalgesic and antiallodynic effects evoked by the administration of the selective CCR2 antagonist RS 504393 were assessed. Cultured NCTC 2472 cells release CCL2 and their intratibial inoculation evokes the development of a tumor in which CCL2 levels are increased. Moreover, the systemic or peritumoral administration of RS 504393 inhibited thermal and mechanical hyperalgesia, but not mechanical allodynia evoked after the inoculation of these cells. Thermal hyperalgesia was also inhibited by the peritumoral administration of a neutralizing CCL2 antibody. In contrast, no change in CCL2 levels was observed in mice inoculated with B16-F10 cells, and RS 504393 did not inhibit the hypernociceptive reactions evoked by their intratibial inoculation. The peripheral release of CCL2 is involved in the development of thermal and mechanical hyperalgesia, but not mechanical allodynia evoked by the inoculation of NCTC 2472 cells, whereas this chemokine seems unrelated to the hypernociception induced by B16-F10 cells.

An inhibitor of neuronal exocytosis (DD04107) displays long-lasting in vivo activity against chronic inflammatory and neuropathic pain.

Small peptides patterned after the N terminus of the synaptosomal protein of 25 kDa, a member of the protein complex implicated in Ca(2+)-dependent neuronal exocytosis, inhibit in vitro the release of neuromodulators involved in pain signaling, suggesting an in vivo analgesic activity. Here, we report that compound DD04107 (palmitoyl-EEMQRR-NH(2)), a 6-mer palmitoylated peptide that blocks the inflammatory recruitment of ion channels to the plasma membrane of nociceptors and the release of calcitonin gene-related peptide from primary sensory neurons, displays potent and long-lasting in vivo antihyperalgesia and antiallodynia in chronic models of inflammatory and neuropathic pain, such as the complete Freund's adjuvant, osteosarcoma, chemotherapy, and diabetic neuropathic models. Subcutaneous administration of the peptide produced a dose-dependent antihyperalgesic and antiallodynic activity that lasted ≥24 h. The compound showed a systemic distribution, characterized by a bicompartmental pharmacokinetic profile. Safety pharmacology studies indicated that the peptide is largely devoid of side effects and substantiated that the in vivo activity is not caused by locomotor impairment. Therefore, DD04107 is a potent and long-lasting antinociceptive compound that displays a safe pharmacological profile. These findings support the notion that neuronal exocytosis of receptors and neuronal algogens pivotally contribute to chronic inflammatory and neuropathic pain and imply a central role of peptidergic nociceptor sensitization to the pathogenesis of pain.

Potentiation of acute morphine-induced analgesia measured by a thermal test in bone cancer-bearing mice.

Agonists of µ-opioid receptors are currently used in the management of cancer pain. However, several data suggest that the analgesic effect of morphine can diminish during the development of experimental tumors. By using a thermal test, we have studied whether the analgesic effect evoked by morphine is altered in mice bearing two painful bone tumors. The analgesic effect evoked by systemic morphine remained unaltered after the intratibial inoculation of B16-F10 melanoma cells and was potentiated after the inoculation of NCTC 2472 osteosarcoma cells. Although the number of spinal µ-opioid receptors measured by western blot studies was not augmented in osteosarcoma-bearing mice, the analgesia evoked by intrathecal (i.t.) morphine was also enhanced. The analgesic response produced by the spinal administration of the Gi/o protein activator mastoparan was amplified, whereas the analgesic response evoked by the i.t. administration of the N-type calcium channel blocker ω-conotoxin remained unaltered. The efficacy of the GIRK channel blocker tertiapin-Q to antagonize the analgesic effect produced by a maximal dose of morphine was also increased in osteosarcoma-bearing mice. Our results seem to indicate that the analgesic effect of morphine on thermal nociception can be enhanced in response to the development of particular bone tumors in mice, being this potentiation probably related to a greater efficacy of the transduction system driven by Gi/o proteins and GIRK channels.

Antinociceptive effects induced through the stimulation of spinal cannabinoid type 2 receptors in chronically inflamed mice.

The stimulation of spinal cannabinoid type 2 (CB(2)) receptors is a suitable strategy for the alleviation of experimental pain symptoms. Several reports have described the up-regulation of spinal cannabinoid CB(2) receptors in neuropathic settings together with the analgesic effects derived from their activation. Besides, we have recently reported in two murine bone cancer models that the intrathecal administration of cannabinoid CB(2) receptor agonists completely abolishes hyperalgesia and allodynia, whereas spinal cannabinoid CB(2) receptor expression remains unaltered. The present experiments were designed to measure the expression of spinal cannabinoid CB(2) receptors as well as the analgesic efficacy derived from their stimulation in mice chronically inflamed by the intraplantar injection of complete Freund's adjuvant 1 week before. Both spinal cannabinoid CB(2) receptors mRNA measured by real-time PCR and cannabinoid CB(2) receptor protein levels measured by western blot remained unaltered in inflamed mice. Besides, the intrathecal (i.t.) administration of the cannabinoid CB(2) receptor agonists AM1241, (R,S)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole, (0.03-1 µg) and JWH 133, (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran, (3-30 µg) dose-dependently blocked inflammatory thermal hyperalgesia and mechanical allodynia. The analgesic effects induced by both agonists were counteracted by the coadministration of the selective cannabinoid CB(2) receptor antagonist SR144528, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide, (5 µg) but not by the cannabinoid CB(1) receptor antagonist AM251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, (10 µg). The effects induced by AM1241 were also inhibited by the coadministration of the opioid receptor antagonist, naloxone (1 µg). These results demonstrate that effective analgesia can be achieved in chronic inflammatory settings through the stimulation of spinal cannabinoid CB(2) receptors even if this receptor population is not up-regulated.

Involvement of Gi/o proteins and GIRK channels in the potentiation of morphine-induced spinal analgesia in acutely inflamed mice.

The analgesic efficacy of opiates can be enhanced in inflammatory states due to peripheral and spinal alterations. We describe here that the analgesic effect induced by intrathecal (i.t.) morphine assessed by measuring thermal withdrawal latencies is enhanced in carrageenan-inflamed mice. The spinal micro-opioid receptor (MOR) population is not up-regulated as demonstrated by Western blot assays. In contrast, behavioural experiments show the involvement of changes in transduction mechanisms activated by spinal opioid receptors. The i.t. administration of the nitric oxide (NO) synthase inhibitor L-NMMA (3-30 microg) antagonised with a similar potency and efficacy morphine-induced analgesia in inflamed and non-inflamed mice, discarding that an increase in NO release could be responsible of the enhancement of morphine-induced analgesia. The analgesic effects evoked by the i.t. administration of the direct G(i/o) protein activator mastoparan (0.03-10 microg), but not those induced by the N-type calcium channel blocker omega-conotoxin GVIA (3-30 ng), were potentiated in inflamed mice, suggesting that postsynaptic and not presynaptic mechanisms could be involved. Furthermore, the inhibitory effects on morphine-induced analgesia produced by the G(i/o) protein inhibitor pertussis toxin (0.1-17 ng) or the G-coupled inwardly rectifying potassium (GIRK) channels inhibitor tertiapin-Q (0.75-750 ng) were greatly enhanced in inflamed mice. These results suggest that differences in the transduction mechanism activated by MOR at postsynaptic level, probably related with GIRK channels activity, could participate in the potentiation of morphine-induced spinal analgesia in acutely inflamed mice.

Metalloproteinase MT5-MMP is an essential modulator of neuro-immune interactions in thermal pain stimulation.

Peripheral interactions between nociceptive fibers and mast cells contribute to inflammatory pain, but little is known about mechanisms mediating neuro-immune communication. Here we show that metalloproteinase MT5-MMP (MMP-24) is an essential mediator of peripheral thermal nociception and inflammatory hyperalgesia. We report that MT5-MMP is expressed by CGRP-containing peptidergic nociceptors in dorsal root ganglia and that Mmp24-deficient mice display enhanced sensitivity to noxious thermal stimuli under basal conditions. Consistently, mutant peptidergic sensory neurons hyperinnervate the skin, a phenotype that correlates with changes in the regulated cleavage of the cell-cell adhesion molecule N-cadherin. In contrast to basal nociception, Mmp24(-/-) mice do not develop thermal hyperalgesia during inflammation, a phenotype that appears associated with alterations in N-cadherin-mediated cell-cell interactions between mast cells and sensory fibers. Collectively, our findings demonstrate an essential role of MT5-MMP in the development of dermal neuro-immune synapses and suggest that this metalloproteinase may be a target for pain control.