Antisense oligodeoxynucleotide to δ opioid receptors attenuates morphine dependence in mice

The effect of intracerebroventricular (i.c.v.) treatment with antisense oligodeoxynucleotide (A-oligo) to δ opioid receptor mRNA on the morphine-induced place preference and naloxone-precipitated jumping was examined in morphine-dependent mice. Morphine (5 mg/kg, s.c.) produced a significant place preference. I.c.v. pretreatment with A-oligo (0.01–1 μg/mouse) dose-dependently attenuated this morphine (5 mg/kg, s.c.)-induced place preference, while mismatched oligodeoxynucleotide (M-oligo; 1 μg/mouse, i.c.v.) was ineffective. Naloxone (3 mg/kg, s.c.) precipitated jumping in morphine-dependent mice. I.c.v. pretreatment with A-oligo (1 μg/mouse) attenuated this naloxone (3 mg/kg, s.c.)-precipitated jumping in morphine-dependent mice, while M-oligo (1 μg/mouse, i.c.v.) was ineffective. These data demonstrate that the selective reduction in supraspinal δ opioid receptor function caused by pretreatment with A-oligo attenuated the morphine-induced place preference and naloxone-precipitated jumping in morphine-dependent mice, suggesting that the rewarding effect of and physical dependence on morphine may be modulated by central δ opioid receptors.     

Human cancer-associated mutations in the Aα subunit of protein phosphatase 2A increase lung cancer incidence in Aα knock-in and knockout mice

Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding Aα subunit, one of several regulatory B subunits, and the catalytic Cα subunit. Aα mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant Aα subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated Aα point mutants defective in binding B' subunits, one knockout mouse expressing truncated Aα defective in B and C subunit binding, and a floxed mouse for generating conditional Aα knockouts. We found that the cancer-associated Aα mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of Aα mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the Aα mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.     

α-Subunit G-protein antisense oligodeoxynucleotide effects on supraspinal (i.c.v.) α2-adrenoceptor antinociception in mice

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) α₂-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tailflick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18–24 h later. G_i3α antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. G_i2α antisense produced differential effects on the three agonists. G_i1α and G_sα antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. μ-opioid antinociception is mediated via G_i2α, the present results suggest that different receptors may mediate antinociception via different G-protein subunits and, hence, that specific subunits might offer novel targets for drug discovery.     

Endogeneous morphine and codeine in mice — effect of muramyl dipeptide

Administration of morphine exerts many effects on the immune system. On the other hand little attention has been paid to the fact, that endogenous morphine and codeine exists in mammals, including man. This raises the question, whether or not endogenous opiate alkaloids play some role in immunoregulation. In addition muramyl-dipeptide (MDP), product of baterial cell wall degradation and a potent immunomdulatory agent exhibits a broad spectrum of effects including effects on CNS functions. The present study investigated whether or not the endogenous levels of morphine and codeine are affected by administration of MDP in mice. Marked variation was found in spleen, brain, small intestine and heart in morphine and codeine concentrations. The intraperitoneal administration of MDP produced a significant increase in tissue morphine levels 30 minutes after injection.     

Targeting of the Gi2α Gene in es cells with Replacement and Insertion Vectors

Abstract

Five replacement vectors (RV) and one insertion vector (IV) were constructed in which ca. 10 kb of genomic G_i2α sequence, flanked on one (IV) or both (RV) sides by a thymidine kinase (TK) marker, were disrupted by a Neo marker inserted into the Ncol site of exon 3. G418^RFIAU^R clones corresponding to ca. 4×10⁸ ES cells electroporated with replacement vectors were analyzed and revealed no targeting event. The insertion vector, however, was integrated by a single reciprocal recombination resulting in a duplication of homology (Hit step; G418^RFIAUS^S, which was lost-together with the plasmid and the TK sequences - by intrachromosomal recombination (Run step; G418^RFIAU^R). Thus, the Hit and Run strategy can be used with a selectable marker disrupting the targeted gene, giving rise to the same targeted product that would have been expected to arise from a double crossover with a replacement vector.     

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice: Evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal μ opioid-mediated antinociception in mice is mediated via the μ₁ subtype. The purpose of the present study was to determine if the highly μ-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain μ₁, but not μ₂, opioid receptors. CXBK or normal Crl:CD-1 ^®(ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55°C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by β-FNA (demonstrating a μ opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of δ opioid receptors). These findings suggest that μ₂ opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.     

The effect of NF-κB antisense oligonucleotide on transdifferentiation of fibroblast in lung tissue of mice injured by bleomycin

To investigate the influence of NF-κB antisense oligonucleotide on transdifferentiation of fibroblast in the pathological process of bleomycin-induced pulmonary fibrosis in mice. 6 h before molding of C57BL/6 model of pulmonary fibrosis in mice, NF-κB antisense oligonucleotide was injected from caudal vein. Then the lung tissue was collected for primary culture as well as model group and control group. Cultured cells were used for immunocytochemical staining of p65, IκB-α and α-SMA proteins as well as in situ hybridization staining of p65 and IκB-α. Then image analysis was carried out. The expressions of all the indicators were expressed as mean optical density. Compared with the control group, the expressions of p65 protein, IκB-α protein and α-SMA protein of model group were increased, as well as the expressions of p65 mRNA and IκB-α mRNA (P < 0.05). Compared with model group, the expressions of all indicators of intervention group were decreased (P < 0.05). P65 protein and p65 mRNA were positively correlated with the expression of α-SMA protein respectively. p65 protein and p65 mRNA were positively correlated with the expressions of IκB-α protein and IκB-α mRNA respectively. NF-κB antisense oligonucleotide can inhibit the transdifferentiation of fibroblast towards myofibroblast in the pathological process of bleomycin-induced pulmonary fibrosis in mice.     

μ- and δ-opioid receptor antagonists precipitate similar withdrawal phenomena in butorphanol and morphine dependence

The relative involvement of μ- and δ-opioid receptors in the mediation of butorphanol-, as compared to morphine-, dependence was examined with the use of highly selective antagonists at μ- and δ-opioid receptors. Extracellular fluid levels of glutamate (Glu) and aspartate (Asp) were measured within the pontine locus coeruleus following precipitation of withdrawal from dependence on either butorphanol or morphine in conscious Sprague-Dawley rats. Dependence was induced by intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/μl/h), morphine (26 nmol/μl/h) or saline vehicle (1 μl/h) for 3 days by means of an osmotic minipump. Microdialysis probes (2 mm tip) were inserted into the locus coeruleus 24 h before precipitation of withdrawal by i.c.v. injection of either the μ-opioid receptor antagonist,d-Pen-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; 4.8 nmol/5 μl or 48 nmol/5 μl), or the δ-opioid receptor antagonist, naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2′3′-indolmorphinan hydrochloride; 48 nmol/5 μl or 100 nmol/5 μl). Baseline levels of Glu ranged from 9.59±1.27 to 12.84 ±3.01 μM in the various treatment groups. Level of Asp were similar. Precipitation of withdrawal by CTOP elicited significant increases of Glu and Asp in both morphine- and butorphanol-dependent rats. Maximal increases in Glu of 425% and 258% above baseline levels were elicited in the first 15 min microdialysis sample following i.c.v. injection of CTOP in morphine- and butorphanol-dependent rats, respectively. Behavioral signs of withdrawal were greater in morphine than butorphanol-dependent groups. The i.c.v. treatment with naltrindole elicited increases in Glu and Asp that were similar, although less marked, than those precipitated by CTOP treatment. Administration of naltrindole produced equivalent signs of withdrawal in both morphine- and butorphanol-dependent rats. Withdrawal from dependence on both morphine and butorphanol is characterized by elevations in coerulear levels of excitatory amino acids. Responses elicited following the use of selective μ- and δ-opioid receptor antagonists to precipitate withdrawal suggest that the role played by these receptors in mediation of the signs and symptoms of withdrawal do not differ greatly between butorphanol- and morphine-dependent rats.     

Changes in HIF-1α protein, pyruvate dehydrogenase phosphorylation, and activity with exercise in acute and chronic hypoxia

Exercise under acute hypoxia elicits a large increase in blood lactate concentration ([La](b)) compared with normoxic exercise. However, several studies in humans show that with the transition to chronic hypoxia, exercise [La](b) returns to normoxic levels. Although extensively examined over the last decades, the muscle-specific mechanisms responsible for this phenomenon remain unknown. To assess the changes in skeletal muscle associated with a transition from acute to chronic hypoxia, CD-1 mice were exposed for 24 h (24H), 1 wk (1WH), or 4 wk (4WH) to hypobaric hypoxia (equivalent to 4,300 m), exercised under 12% O(2), and compared with normoxic mice (N) at 21% O(2). Since the enzyme pyruvate dehydrogenase (PDH) plays a major role in the metabolic fate of pyruvate (oxidation vs. lactate production), we assessed the changes in its activity and regulation. Here we report that when run under hypoxia, 24H mice exhibited the highest blood and intramuscular lactate of all groups, while the 1WH group approached N group values. Concomitantly, the 24H group exhibited the lowest PDH activity, associated with a higher phosphorylation (inactive) state of the Ser(232) residue of PDH, a site specific to PDH kinase-1 (PDK1). Furthermore, protein levels of PDK1 and its regulator, the hypoxia inducible factor-1α (HIF-1α), were both elevated in the 24H group compared with N and 1WH groups. Overall, our results point to a novel mechanism in muscle where the HIF-1α pathway is desensitized in the transition from acute to chronic hypoxia, leading to a reestablishment of PDH activity and a reduction in lactate production by the exercising muscles.     

A comparison of two subarachnoid α2-agonists,xylazine and clonidine,with respect to duration of antinociception,and hemodynamic effects in goats

Subarachnoid administration of clonidine and xylazine produces antinociception in several species and in humans. The present study compares these two drugs administered by the subarachnoid route to goats. Goats (n=6) were randomly assigned to three treatment groups. All animals of each group received 0.06 mg kg⁻¹ clonidine (CLO), 0.1 mg kg⁻¹ xylazine (XIL) and 0.9% saline solution (SS), with a minimum interval of 1 week between treatments. All injections were made into the subarachnoid space between the last lumbar vertebra and the first sacral vertebra. Analgesia, ataxia, sedation, cardiovascular and respiratory effects, and rectal temperature were evaluated at predefined regular time intervals before drug administration (baseline) and after administration. The onset of analgesia by clonidine and xylazine was observed in 6.8±1.8 and 9.5±2.6 min (mean±S.D.), respectively. Both α₂-agonists produced analgesia of dorsocaudal rib areas, flanks, hind limbs, perineum and tail, sedation and ataxia. The duration of antinociception after clonidine administration was 118.8±24 min and after xylazine 88.3±15 min (mean±S.D.). Clonidine and xylazine subarachnoidally administered induced a significant (P<0.05) decrease in heart and respiratory rates and hypothermia in relation to the basal value. Neither drug significantly altered blood pressure. Both α₂-agonist drugs induced frequent diuresis and an increase in salivation. We conclude that subarachnoid clonidine produces longer antinociception with less ataxia than xylazine in goats. However, the drug induced bradycardia, a decrease in the respiratory rate and hypothermia, with a small compromise in the blood pressures at the doses studied. Further studies should be done to determine whether this analgesia is sufficient for surgical procedures.     

Disruption of the Gi2α locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair

We have used an insertion vector-based approach to target the G_i2 gene in AB-1 embryonic stem cells. 105 bp located 0.8–0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineeredNot I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained (hit step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy, 2-fluoro--d-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination (run step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified hit and run strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated G_i2 allele is also demonstrated.     

Ric-8A and Giα Recruit LGN,NuMA, and Dynein to the Cell Cortex To Help Orient the Mitotic Spindle

In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein α (Gα) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and Gα_i function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to Gα_i, thus preventing its GEF activity for Gα_i. Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased Gα_i expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.The cortical capture of astral microtubules is essential to generate the forces needed for mitotic spindle positioning for both symmetric and asymmetric cell divisions (23, 29). Failure to either capture astral microtubules or the inappropriate application of pulling forces adversely affects mitotic spindle orientation, and can impede embryogenesis and alter cell fate decisions. Studies examining mitotic spindle orientation in Drosophila embryonic and larval neuroblasts have identified two critical pathways, the Gα/Pins/Mud pathway and the Pins/Dlg/Khc73 pathway (29). The heterotrimeric G-protein α subunit (Gα), Pins (Partner-of-Inscuteable), and Mud (Mushroom body defect) are members of an evolutionarily conserved noncanonical G-protein signaling pathway, which form a tripartite protein complex linked to the apical Par complex by the adapter protein Inscuteable (29, 37). Reducing the level of Gα_i, Pins, or Mud prevents neuroblast mitotic spindle alignment. A second spindle orientation pathway involves Pins, the tumor suppressor Discs large (Dlg) and the microtubule plus-end-directed kinesin heavy chain 73 (Khc73). Khc73 binds Dlg and coimmunoprecipitates with Pins. Khc73 localized to astral microtubules can induce Pins-Dlg cortical polarity (27).In canonical G-protein signaling pathways, the binding of ligand to a seven-transmembrane receptor triggers a heterotrimeric G-protein α subunit (Gα) to exchange GTP for GDP, resulting in the dissociation of the Gα subunit from its associated Gβγ heterodimer (12, 20). This exposes interactive sites in the Gα and Gβγ subunits, allowing their binding to and activation of downstream effectors. Since Gα subunits possess an intrinsic GTPase activity, GTP hydrolysis leads to the reassembly of heterotrimeric G protein causing signaling to cease. In noncanonical G-protein signaling the seven-transmembrane receptor is replaced by an intracellular guanine nucleotide exchange factor, such as Ric-8 (37). In studies in Drosophila and Caenorhabditis elegans Ric-8 has been shown to positively regulate Gα_i activity and is essential for asymmetric cell divisions (1, 2, 5, 8, 11, 36). Although initially characterized as a guanine nucleotide exchange factor (GEF) for isolated G_αsubunits, more recent biochemical studies have shown that Ric-8A (the mammalian equivalent of Ric-8) also acts on a complex of GDP-Gα_i, the mammalian Pins homolog LGN, and NuMA (nuclear mitotic apparatus protein; the mammalian equivalent of Mud) catalytically releasing GTP-Gα_i and causing liberation of NuMA from LGN (30, 31). Ric-8A can also catalyze guanine nucleotide exchange on Gα_i1 bound to the GPR/GoLoco exchange inhibitor AGS3, a paralog of LGN (33). During mitosis the N-terminal portion of LGN binds NuMA and the C-terminal domain binds GDP-Gα_i and the trimolecular complex localizes to the cell cortex, where the dynamic release of NuMA from LGN may regulate aster microtubule pulling during cell division (3, 9, 10, 22).In the present study we examined the role of Ric-8A in mitotic spindle orientation in adherent cells and in polarized MDCK cells. In nonpolarized adherent cells cell such as HeLa, integrin mediated cell-substrate adhesion orients the mitotic spindle parallel to the substratum, and thereby both daughter cells remain attached. This requires the actin cytoskeleton, astral microtubules, the microtubule plus end tracking protein EB1, myosin X, cdc42, LIM kinase 1, and phosphatidylinositol(3,4,5)-triphosphate (PIP₃) (13, 18, 32, 34, 35). PIP₃ may direct dynein/dynactin-dependent pulling forces on the spindle midcortex to orient the mitotic spindle (34). In polarized cells such as Madin-Darby canine kidney (MDCK) cells, the mitotic spindle is constrained by the topology of the cell and cortical cues provided by adherens junctions (24). In contrast to HeLa cells these cues are insensitive to phosphatidylinositol 3-kinase (PI3K) inhibition, which blocks the generation of PIP₃ (34). We found that inhibiting either Ric-8A or Gα_i expression impairs the orientation of the metaphase mitotic spindle in HeLa cells and pertussis toxin, which blocks Ric-8A triggered nucleotide exchange, disrupts the normal mitotic spindle alignment of both HeLa and MDCK cells. Impairment of Ric-8A expression or function inhibits the localization of Gα_i1, LGN, NuMA, and dynein to the metaphase cortex opposite the spindle poles.     

Major urinary metabolites of 6-keto-prostaglandin F2α in mice

Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF_2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF_2α urinary metabolites included dinor-6-keto-PGF_2α (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF_1α (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI₃ by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF_2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases.     

Systemic administration of antisense oligonucleotides simultaneously targeting CK2?? and ???? subunits reduces orthotopic xenograft prostate tumors in mice

CK2 is a highly conserved, ubiquitous, signal responsive protein serine/threonine kinase. CK2 promotes cell proliferation and suppresses apoptosis, and increased CK2 expression is observed in all cancers examined. We previously reported that direct injection of antisense (AS) CK2α phosphorothioate oligonucleotides (PTO) into xenograft prostate tumors in mice significantly reduced tumor size. Downregulation of CK2α in tumor cells in vivo appeared to result in overexpression of CK2α' protein. This suggested that in cancer cells downregulation of CK2α might be compensated by CK2α' in vivo, prompting us to design a bispecific (bs) AS PTO (bs-AS-CK2) targeting both catalytic subunits. bs-AS-CK2 reduced CK2α and α' protein expression, decreased cell proliferation, and induced apoptosis in cultured cells. Biodistribution studies of administered bs-AS-CK2 oligonucleotide demonstrated its presence in orthotopic prostate xenograft tumors. High dose injections of bs-AS-CK2 resulted in no damage to normal liver or prostate, but induced extensive cell death in tumor tissue. Intraperitoneal treatment with bs-AS-CK2 PTO decreased orthotopic tumor size and downregulated both CK2 mRNA and protein expression. Tumor reduction was accomplished using remarkably low doses and was improved by dividing the dose using a multi-day schedule. Decreased expression of the key signaling pathway proteins NF-κB p65 and AKT was also observed. We propose that the molecular downregulation of CK2 through bispecific targeting of the two catalytic subunits may be uniquely useful for therapeutic elimination of tumors.     

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.     

A crucial role of IL-17 and IFN-γ during acute rejection of peripheral nerve xenotransplantation in mice

Nerve injuries causing segmental loss require nerve grafting. However, autografts and allografts have limitations for clinical use. Peripheral nerve xenotransplantation has become an area of great interest in clinical surgery research as an alternative graft strategy. However, xenotransplant rejection is severe with cellular immunity, and Th1 cells play an important role in the process. To better understand the process of rejection, we used peripheral nerve xenografts from rats to mice and found that mononuclear cells expressing IFN-γ and IL-17 infiltrated around the grafts, and IFN-γ and IL-17 producing CD4+ and CD8+ T cells increased during the process of acute rejection. The changes of IL-4 level had no significant difference between xenotransplanted group and sham control group. The rejection of xenograft was significantly prevented after the treatment of IL-17 and IFN-γ neutralizing antibodies. These data suggest that Th17 cells contribute to the acute rejection process of peripheral nerve xenotransplant in addition to Th1 cells.