Central Mechanisms Mediating Thrombospondin-4-induced Pain States.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1 Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Calcium channel α2δ1 proteins mediate trigeminal neuropathic pain states associated with aberrant excitatory synaptogenesis.

To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α2δ1 (Cavα2δ1) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Cavα2δ1 up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Cavα2δ1 with gabapentin, a ligand for the Cavα2δ1 proteins, or Cavα2δ1 antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Cavα2δ1 in orofacial pain processing. Importantly, increased Cavα2δ1 in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Cavα2δ1 up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin ß1 in primary HUVECs. The internalized integrin ß1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin ß1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin ß1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin ß1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr9²5.

Tumor-infiltrating macrophages are potential candidates for cancer immunotherapy. However, the detailed molecular mechanism underlying macrophage infiltration into tumors is poorly understood. Based on our previous finding that plasminogen activator inhibitor-1 (PAI-1) enhances vitronectin-dependent migration of macrophages, we investigated the potential role of PAI-1 in macrophage invasion into melanoma. Experimental evidence obtained from spheroid confrontation assay clearly showed that PAI-1 overexpression significantly enhanced the invasion of RAW 264.7 cells into B16F10 melanoma. We further demonstrated that PAI-1 induces phosphorylation of focal adhesion kinase (FAK) at Tyr(925), which, in turn, mediated the invasion of macrophages into the melanoma. This work further illustrates that low-density lipoprotein receptor related-protein 1 (LRP1) is essential for PAI-1-mediated FAK phosphorylation and macrophage invasion into melanoma. In conclusion, our study demonstrates a novel role of PAI-1 in macrophage invasion into melanoma and provides insights into the underlying molecular mechanism.

Thrombospondin-4 contributes to spinal sensitization and neuropathic pain states.

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Matrix metalloproteinase 9 (MMP-9)-dependent processing of ßig-h3 protein regulates cell migration, invasion, and adhesion.

Cell migration is critically involved in inflammation, cancer, and development. In this study, transforming growth factor-ß-induced protein (ßig-h3) was identified as a substrate of matrix metalloproteinase-9 (MMP-9) by site-directed mutagenesis. ßig-h3 has two cleavage sites with the consensus sequence Pro-Xaa-Xaa-Hy-(Ser/Thr) (Hy is a hydrophobic amino acid) (PGSFT beginning at amino acid 135 and PPMGT beginning at amino acid 501). Using recombinant human ßig-h3 and MMP-9, ßig-h3 from ßig-h3-transfected HEK293F cells, and MMP-9 from MMP-9-transfected HEK293F cells, human macrophages, and neutrophils, we found that MMP-9 proteolytically cleaves ßig-h3. Cleavage leads to the loss of its adhesive property and its release from extracellular matrix proteins, collagen IV, and fibronectin. Spheroids formed by increased cell-cell interactions were observed in ßig-h3-transfected HEK293F cells but not in vehicle-transfected HEK293F cells. In human glioma U87MG cells, MMP-9 constitutive overexpression resulted in endogenous ßig-h3 cleavage. ßig-h3 cleavage by MMP-9 led to increased cell invasion, and ßig-h3 knockdown also resulted in increased cell invasion. The ßig-h3 fragment cleaved by MMP-9 could bind to the surface of macrophages, and it may play a role as a peptide chemoattractant by inducing macrophage migration via focal adhesion kinase/Src-mediated signal activation. Thus, intact ßig-h3 is responsible for cell migration inhibition, cell-cell contact, and cell-extracellular matrix interaction. Experimental evidence indicates that MMP-9-cleaved ßig-h3 plays a role in MMP-9-mediated tumor cell and macrophage migration.

Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation.

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.

Prophylactic effect of a peptide vaccine targeting TM4SF5 against colon cancer in a mouse model.

Expression of transmembrane 4 superfamily member 5 protein (TM4SF5) was implicated in hepatocellular carcinoma (HCC) and colon cancer. Previously, we have shown that immunization with TM4SF5 peptide-CpG-DNA-liposome complex induces production of TM4SF5-specific antibodies and protects mice from HCC progression in an allograft model. Here, we confirmed expression of TM4SF5 in the mouse colon cancer cell line CT-26 and found that anti-TM4SF5 antibody inhibits growth of CT-26 cells. We then immunized mice with TM4SF5 peptide-CpG-DNA-liposome complex and transplanted CT-26 cells to investigate the vaccination effects. Robust production of TM4SF5-specific antibodies was induced by challenge with CT-26 cells and the tumor growth was significantly suppressed in the immunized mice. The peptide vaccine targeting TM4SF5 consequently showed a prophylactic effect against colon cancer development in a mouse model. These results suggest that the peptide vaccine can be potentially applied in humans to treat colon cancer.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvß3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

The Effects of Switching from Sevoflurane to Short-Term Desflurane prior to the End of General Anesthesia on Patient Emergence and Recovery: A Randomized Controlled Trial.

While sevoflurane and desflurane have been regarded as inhalation agents providing rapid induction and emergence, previous studies demonstrated the superiority of desflurane-anesthesia compared to sevoflurane-anesthesia in the postoperative recovery in obese and geriatric patients. We investigated whether a short-term switch of sevoflurane to desflurane at the end of sevoflurane-anesthesia enhances patient postoperative recovery profile in non-obese patients. We randomly divide patients undergoing elective surgery (n = 60) into two groups: sevoflurane-anesthesia group (Group-S, n = 30) and sevoflurane-desflurane group (Group-SD, n = 30). In Group-S, patients received only sevoflurane-anesthesia until the end of surgery (for >2 hours). In Group-SD, sevoflurane was stopped and switched to desflurane-anesthesia before the completion of sevoflurane-anesthesia (for approximately 30 minutes). We assessed the intergroup differences in the times to get eye-opening, extubation, and a bispectral index of 80 (BIS-80). Group-SD showed significantly shorter times to get eye-opening (438 ± 101 vs. 295 ± 45 s; mean difference, 143 s; 95% confidence interval [CI], 101-183; p < 0.001), extubation (476 ± 108 vs. 312 ± 42 s; mean difference, 164 s; 95% CI, 116-220; p < 0.001), and BIS-80 (378 ± 124 vs. 265 ± 49 minutes; mean difference, 113 s; 95% CI, 58-168 p < 0.001) compared to Group-S. There was no between-group difference in postoperative nausea, vomiting, and hypoxia incidences. Our results suggested that the short-term (approximately 30 minutes) switch of sevoflurane to desflurane at the end of sevoflurane-anesthesia can facilitate the speed of postoperative patient recovery.

Thrombin-dependent MMP-2 activity is regulated by heparan sulfate.

Like most metalloproteases, matrix metalloprotease 2 (MMP-2) is synthesized as a zymogen. MMP-2 propeptide plays a role in inhibition of catalytic activity through a cysteine-zinc ion pairing, disruption of which results in full enzyme activation. A variety of proteases have been shown to be involved in the activation of pro-MMP-2, including metalloproteases and serine proteases. In the previous study we showed that MMP-2 activation occurred via specific cleavages of the propeptide by thrombin followed by intermolecular autoproteolytic processing for full enzymatic activity. Thrombin also degraded MMP-2, but this degradation was reduced greatly under cell-associated conditions with a concomitant increase in activation, prompting us to elucidate the molecular mechanisms underlying thrombin-mediated MMP-2 activation. In the present study we demonstrate that heparan sulfate is essential for thrombin-mediated activation of pro-MMP-2. Binding of heparan sulfate to thrombin is primarily responsible for this activation process, presumably through conformational changes at the active site. Furthermore, interaction of MMP-2 with exosites 1 and 2 of thrombin is crucial for thrombin-mediated MMP-2 degradation, and inhibition of this interaction by heparan sulfate or hirudin fragment results in a decrease in MMP-2 degradation. Finally, we demonstrated interaction between exosite 1 and hemopexin-like domain of MMP-2, suggesting a regulatory role of hemopexin-like domain in MMP-2 degradation. Taken together, our experimental data suggest a novel regulatory mechanism of thrombin-dependent MMP-2 enzymatic activity by heparan sulfate proteoglycans.

Production of antibodies with peptide-CpG-DNA-liposome complex without carriers.

BACKGROUND: The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. RESULTS: We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. CONCLUSIONS: Our overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

Application of pulsed radiofrequency currents to rat dorsal root ganglia modulates nerve injury-induced tactile allodynia.

BACKGROUND: Application of pulsed radiofrequency (PRF) currents to the dorsal root ganglia (DRG) has been reported to produce relief from certain pain states without causing thermal ablation. In this study, we examined the direct correlation between PRF application to DRG associated with spinal nerve injury and reversal of injury-induced behavioral hypersensitivity in a rat neuropathic pain model. METHODS: Neuropathic lesioning was performed via left L5 spinal nerve ligation on male adult Sprague-Dawley rats. Once the injured rats had developed tactile allodynia, one group was then assigned to PRF treatment of the L5 DRG and another group was assigned to the sham treatment to the DRG. Behavioral testing was performed on both the control and treated paws using the von Frey filament test before the surgery and at indicated days. The resulting data were analyzed using a linear mixed model to assess the overall difference between the treatment groups and the overall difference among the study days. Cohen's d statistic was computed from paired difference-from-baseline scores for each of the 14 study days after treatment and these measures of effect size were then used to descriptively compare the recovery patterns over time for each study group. RESULTS: Spinal nerve injury resulted in the development of behavioral hypersensitivity to von Frey filament stimulation (allodynia) in the hindpaw of the left (injury) side. Mixed linear modeling showed a significant difference between the treatment groups (P = 0.0079) and a significant change of paw withdrawal threshold means over time (P = 0.0006) for all 12 animals. Evaluation of Cohen's d (effect size) revealed that the PRF-treated animals exhibited better recovery and recorded larger effect sizes than the sham-treated animals on 10 of the 14 post-PRF treatment days and exhibited moderate-to-strong effects posttreatment at days 8 to 10 and at and beyond day 32. CONCLUSIONS: Findings from this study support that PRF of the DRG causes reversal of nerve injury (spinal nerve ligation)-induced tactile allodynia in rats. This allodynia reversal indicates that nonablative PRF acting via modulation of the DRG can speed recovery in nerve injury-induced pain.

ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function.

BACKGROUND: The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9). RESULTS: We found that a variant of ASB9 that lacks the SOCS box (ASB9DeltaSOCS) was naturally detected in human cell lines but not in peripheral blood mononuclear cells or normal hepatocytes. We also identified ubiquitous mitochondrial creatine kinase (uMtCK) as a new target of ASB9 in human embryonic kidney 293 (HEK293) cells. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of uMtCK in a SOCS box-independent manner. The overexpression of ASB9, but not ASB9DeltaSOCS, induces ubiquitination of uMtCK. ASB9 and ASB9DeltaSOCS can interact and colocalise with uMtCK in the mitochondria. However, only expression of ASB9 induced abnormal mitochondrial structure and a decrease of mitochondrial membrane potential. Furthermore, the creatine kinase activities and cell growth were significantly reduced by ASB9 but not by ASB9DeltaSOCS. CONCLUSIONS: ASB9 interacts with the creatine kinase system and negatively regulates cell growth. The differential expression and function of ASB9 and ASB9DeltaSOCS may be a key factor in the growth of human cell lines and primary cells.

Treatment of phalangeal osteomyelitis caused by a cat bite.

In the case of a cat bite patient, even if the external wound is small, prophylactic antibiotics should be used early and a closed observation is needed. If symptoms persist, deep infection should be considered.

Treatment with phosphodiester CpG-ODN ameliorates atopic dermatitis by enhancing TGF-ß signaling.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG phosphorothioate (PS CpG-ODN) are known to decrease IgE synthesis in Th2 allergy responses. Nonetheless, the therapeutic role of PS CpG-ODN is limited due to cytotoxicity. Therefore, we developed a phosphodiester (PO) form of CpG-ODN (46O) with reduced toxicity but effective against allergies. In this study, we first compared the toxicity of 46O with CpG-ODNs containing a PS backbone (1826S). We also investigated the therapeutic efficacy and mechanism of 46O injected intravenously in a mouse model of ovalbumin (OVA)-induced atopic dermatitis (AD). To elucidate the mechanism of 46O underlying the inhibition of IgE production, IgE- and TGF-ô€…-associated molecules were evaluated in CD40/IL-4- or LPS/IL-4-stimulated B cells. Our data showed that the treatment with 46O was associated with a lower hematological toxicity compared with 1826S. In addition, injection with 46O reduced erythema, epidermal thickness, and suppressed IgE and IL-4 synthesis in mice with OVA-induced AD. Additionally, 46O induced TGF-ß production in LPS/IL-4-stimulated B cells via inhibition of Smad7, which suppressed IgE synthesis via interaction between Id2 and E2A. These findings suggest that enhanced TGF-ß signaling is an effective treatment for IgE-mediated allergic conditions, and 46O may be safe and effective for treating allergic diseases such as AD and asthma. [BMB Reports 2021; 54(2): 142-147].

Nitric oxide induction of IRE1-alpha-dependent CREB phosphorylation in human glioma cells.

In this study, the function of nitric oxide (NO) in endoplasmic reticulum (ER)-related cell death in human glioma cells was investigated. Treatment of human CRT-MG cells with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and thapsigargin, an ER stress inducer, increased cytosolic Ca(2+) and caused apoptosis in a dose-dependent manner. Expression of the ER-associated molecules inositol-requiring enzyme 1 (IRE1)-alpha, p-eIF, and Ero1-alpha were also elevated in thapsigargin- or NO donor-treated cells. Furthermore, thapsigargin and SNAP treatment increased IRE1-alpha nuclease activity, induced IRE1-alpha/TRAF2 complex formation, and increased p-JNK1/2 levels, suggesting that NO activates the IRE1-alpha/TRAF2/JNK pathway in the ER. Expression of IRE1-alpha increased concomitantly with cAMP responsive element binding protein (CREB) phosphorylation. siRNA knock down of IRE1-alpha reduced phospho-CREB levels and abolished its nuclear translocation. The levels of phospho-CREB and IRE1-alpha increased with NO donor concentration, which resulted in cell death. IRE1-alpha and phospho-CREB levels in glioblastoma U87MG cells were higher than those in normal astrocytes in response to NO. In addition, treatment with the intracellular cytokine interleukin-1beta induced cell death associated with NO and increased IRE1-alpha and p-CREB levels. These data reveal that intracellular NO affects IRE1-alpha-dependent CREB phosphorylation in human glioma cells. Therefore, an IRE1-alpha-dependent phospho-CREB signaling pathway responsive to NO/Ca(2+) may play an important role in regulating ER-related cell death in glioma.

Enhanced pre-synaptic glutamate release in deep-dorsal horn contributes to calcium channel alpha-2-delta-1 protein-mediated spinal sensitization and behavioral hypersensitivity.

Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavalpha2delta1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavalpha2delta1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavalpha2delta1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavalpha2delta1 transgenic mice. Our data indicated that overexpression of Cavalpha2delta1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavalpha2delta1-mediated spinal sensitization in which elevated Cavalpha2delta1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavalpha2delta1 expression.

Immunostimulation and anti-DNA antibody production by backbone modified CpG-DNA.

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-DNA) have gained attention as potentially useful therapeutics. However, the phosphorothioate-modified CpG-DNAs (PS-ODN) can induce backbone-related side effects. Here, we compared the immunostimulatory activity of natural phosphodiester CpG-DNA (PO-ODN) from Mycobacterium bovis and PS-ODN in mice. Both PO-ODN and PS-ODN induced production of IL-12. PS-ODN increased spleen weights, spleen cell numbers, and the migration of macrophages into the peritoneal cavity in the mice in a CG sequence-dependent manner. PS-ODN induced anti-PS-ODN antibody production in the mice, and the PS-ODN-specific IgM was cross-reactive with other PS-ODNs in a CG sequence-independent manner. In contrast, PO-ODN did not affect on spleen weights, cell numbers, or IgM production. These results may provide an explanation for the side effects in immunotherapeutic application of PS-ODN. They also suggest that PO-ODN may be more optimal than PS-ODN to enhance innate immune responses without severe side effects.

2,4-Dinitrofluorobenzene modifies cellular proteins and induces macrophage inflammatory protein-2 gene expression via reactive oxygen species production in RAW 264.7 cells.

The skin sensitizer 2,4-dinitrofluorobenzene (DNFB) provokes delayed hypersensitivity responses as a result of topical application to the skin. Here, we demonstrate that DNFB modifies proteins in RAW 264.7 cells and skin tissues in NC/Nga mice; we also show the functional involvement of DNFB-induced modification of cellular proteins in the DNFB-induced macrophage inflammatory protein (MIP)-2 gene expression in RAW 264.7 cells. In addition, we demonstrate that DNFB strongly induces reactive oxygen species (ROS) production. Our RT-PCR analysis and reporter gene assays reveal that the DNFB-induced intracellular ROS production is necessary for MIP-2 gene expression by DNFB. We observed that the vitamin C and chemical oxidant scavenger N-acetyl-cysteine have an inhibitory effect on the generation of ROS, the activation of MAP kinase pathways, and the MIP-2 gene expression in DNFB-treated RAW 264.7 cells. These results provide insight into the mechanisms involved in DNFB-induced contact hypersensitivity.