[Differential diagnosis of pure ground glass nodules in lung with spectral CT imaging].

Objective: To evaluate the differential diagnostic performance of quantitative parameters derived from the spectral CT imagingin pure ground-glass nodules. Methods: A total of 44 patients with pure ground glass nodules underwent chest energy spectrum CT and with known subsequently pathological findings in the Imaging Department of the Second Affiliated Hospital of Soochow University from August 2017 to September 2019 were retrospectively analyzed. Among them, there are 18 males and 26 females, aged from 26 to 79 (51±12) years. They were divided into as the inflammatory group (n=12), pre-invasive adenocarcinoma group (n=17) and invasive adenocarcinoma group (n=15). The aforementioned three groups were further reclassified as non-invasive adenocarcinoma group (inflammatory lesion+pre-invasive lesion) and invasive adenocarcinoma group in order to evaluating the values of water concentration (WC) for the determination of adenocarcinoma infiltration status. The values of WC derived from the arterial and venous phase of the lesion, iodine concentration (IC), standardized iodine concentration (NIC) were measured respectively.The slope of the energy spectral curve (K40-70KeV) derived from the arterial and venous phase of the lesion was also calculated. One-way ANOVA analysis was performed to compare the differences of the three groups and the multiple comparison method was used for further comparing. Intraclass correlation efficient (ICC) was used to assess the consistency of the three times of measurements. The area under curve(AUC) of Receiver Operating Characteristic (ROC) was conducted to evaluate the diagnostic performance of water based values. Results: The values of WC in the arterial and venous phases were significantly different. As in the inflammatory group, the pre-invasive lesion group and the invasive adenocarcinoma group, the values of WC was (291.95±58.66) mg/cm3, (297.61±63.96) mg/cm3and (374.52±60.62) mg/cm3 of the arterial phase, and (277.07±33.78) mg/cm3, (291.74±50.49) mg/cm3 and (373.33±75.12) mg/cm3 of the venous phase, respectively(all P<0.05). Further comparison demonstrated that no significant difference was observed for the values of WC derived from the arterial phases and venous phases between the inflammatory lesion group and the pre-invasive lesion group (all P>0.05).There were an significant differences between the invasive adenocarcinoma group, the inflammatory lesion group and the pre-invasive lesion group (all P<0.05). The values of WC derived from the venous phase achieved the largest AUC (0.770) for differentiating invasive adenocarcinoma from non-invasive adenocarcinoma (inflammatory lesions+pre-invasive lesions) in the pure ground glass nodules. The sensitivity and specificity were 66.67% and 93.10%, respectively, when using 349.31 mg/cm³ as the optimal threshold. The slope of the spectral curve and iodine-related parameters (IC, NIC) derived from arterial or venous phases among the three groups were not significantly different (all P>0.05). Conclusion: The values of WC derived from the spectral CT can better distinguish inflammatory, pre-invasive lesions and invasive adenocarcinoma, which is helpful for the qualitative analysis for pure ground glass nodules.

[The value of 3.0T magnetic resonance -diffusion kurtosis imaging in the differential diagnosis of rectal mucinous adenocarcinoma and common adenocarcinoma].

Objective: To investigate the value of 3.0T MRI diffusion kurtosis imaging (DKI) quantitative histogram parameters in the differential diagnosis of rectal mucinous adenocarcinoma (MC) and common adenocarcinoma (AC). Methods: One hundred and ten patients from Department of Radiology, the Second Affiliated Hospital of Soochow University between September 2015 and September 2019 with complete magnetic resonance imaging (MRI) and DKI results confirmed by surgery and pathology were retrospectively analyzed, including 16 patients in MC group and 94 patients in AC group. Two physicians outlined the region of interest (ROI) on the DKI image with b=1 000 s/mm(2), and obtained quantitative DKI parameters, including the diffusion coefficient (D value) and kurtosis coefficient (K value) corrected for non-Gaussian distribution. The apparent diffusion coefficient (ADC) values of quantitative parameters of diffusion-weighted imaging (DWI) were obtained through image registration, and histogram analysis was performed to obtain the mean value, 25th percentile, 50th percentile, 75th percentile, skewness and kurtosis of the above parameters, respectively. The difference between the quantitative histogram parameter analysis results of the rectal MC group and the AC group was evaluated, and the main indicators and multivariate comprehensive analysis indicators was screened, and the effectiveness of quantitative histogram parameters related to histopathological classification in the differential diagnosis of rectal MC and AC was evaluated. Results: There was no significant differences in gender, age, lesion location, T stage or N stage between MC group and AC group (all P>0.05). The multivariate binary logistic stepwise regression screening showed that D50th percentile and K25th percentile are statistically significant indicators (B values were 2 966.166 and -4.550, respectively; Wals values were 9.000 and 15.720, respectively; and P values were 0.003 and <0.001, respectively). The combined area under the curve of the two indictors was 0.85, but there was no statistically significant difference in pairwise comparison using DeLong method (P>0.05). The results of histogram analysis of quantitative parameters measured by the two physicians were consistent, and the inter-group correlation coefficient ranged from 0.880 to 0.981. Conclusions: The quantitative parameter histogram analysis of the DKI double-index model is helpful for the differentiation of rectal MC and AC, in which the D50th percentile and K25th percentile have differential diagnosis significance, and are superior to the ADC value of the single-index model.

[The quantitative evaluation of early radiation-induced changes in the salivary glands using MRI].

Objective: To quantitatively evaluate the early radiation injury of salivary glands in patients with nasopharyngeal carcinoma (NPC) after intensity-modulated radiotherapy (IMRT). Methods: Twenty patients with NPC between 2014 and 2015 from the Second Affiliated Hospital of Soochow University were retrospectively analyzed.All patients underwent an MRI scan before and after IMRT.The volumes, T(1)WI, T(2)WI signal intensity(SIs) and apparent diffusion coefficient (ADCs) of the parotid and submandibular glands were measured.The relative signal intensity (RSIs) of each salivary gland was calculated with cerebrospinal fluid as control.The quantitative parameters of salivary glands were compared before and after radiotherapy. Results: The volumes (cm(3)) and T(1)WI RSIs of the parotid and submandibular glands (14.88±6.00, 5.21±1.76, 2.98±1.05, 1.88±0.42, respectively) were significantly lower than those before radiotherapy (22.26±8.26, 7.76±2.45, 3.58±1.02, 2.27±0.50, respectively) (t=9.921, 4.013, 10.126, 4.202, respectively, P=0.000 for all). The T(2)WI RSIs and ADCs (×10(-3) mm(2)/s) of the parotid and submandibular glands (0.50 ± 0.08, 0.41±0.04, 1.31±0.19, 1.50±0.13, respectively) were significantly higher than those before radiotherapy (0.45±0.07, 0.33±0.05, 1.02±0.21, 1.23±0.13, respectively) (t=-4.846, -9.276, -9.957, -10.679, respectively, P=0.000 for all). The volumes of parotid and submandibular glands were correlated with ADCs (r=-0.512, P=0.000; r=-0.358, P=0.001; respectively). The volumes and ADCs of submandibular glands were correlated with T(1)WI RSIs and T(2)WI RSIs(P<0.05). Conclusion: MRI can quantitatively evaluate the early changes of salivary glands after radiotherapy of nasopharyngeal carcinoma as a noninvasive method, and has high clinical application potential.

Protein phosphatase 2A interacts with and directly dephosphorylates RelA.

Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors are key regulators of a variety of genes involved in inflammatory responses, growth, differentiation, apoptosis, and development. There are increasing lines of evidence that NF-kappa B/Rel activity is controlled to a great extent by its phosphorylation state. In this study, we demonstrated that RelA physically associated with protein phosphatase 2A (PP2A) subunit A (PR65). Both the N- and C-terminal regions of RelA were responsible for the PP2A binding. RelA co-immunoprecipitated with PP2A in melanocytes in the absence of stimulation, indicating that RelA forms a signaling complex with PP2A in the cells. RelA was dephosphorylated by a purified PP2A core enzyme, a heterodimer formed by the catalytic subunit of PP2A (PP2Ac) and PR65, in a concentration-dependent manner. Okadaic acid, an inhibitor of PP2A at lower concentration, increased the basal phosphorylation of RelA in melanocytes and blocked the dephosphorylation of RelA after interleukin-1 stimulation. Interestingly, PP2A immunoprecipitated from melanocytes was able to dephosphorylate RelA, whereas PP2A immunoprecipitated from melanoma cell lines exhibited decreased capacity to dephosphorylate RelA in vitro. Moreover, in melanoma cells in which I kappa B kinase activity was inhibited by sulindac to a similar level as in melanocytes, the phosphorylation state of RelA and the relative NF-kappa B activity were still higher than those in normal melanocytes. These data suggest that the constitutive activation of RelA in melanoma cells (Yang, J., and Richmond, A. (2001) Cancer Res. 61, 4901-4909) could be due, at least in part, to the deficiency of PP2A, which exhibits decreased dephosphorylation of NF-kappa B/RelA.

Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization.

Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with beta-arrestin 1 but continues to exhibit association with adaptin 2 alpha and beta subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca(2+) mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to beta-arrestin 1 but exhibit decreased binding to adaptin 2alpha and beta. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.

Suppression of phospholipase C blocks Gi-mediated inhibition of adenylyl cyclase activity.

The potential effect of inhibition of phospholipase C on the response of Gi-coupled receptors was investigated in neuroblastoma x glioma hybrid (NG108-15) cells. The phospholipase C specific inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (U73122), which did not affect basal and forskolin-stimulated adenylyl cyclase activities, time- and dose-dependently blocked delta-opioid receptor-mediated inhibition of adenylyl cyclase activity, the EC50 (0.5 microM) of which was consistent with that for inhibition of bradykinin-dependent phospholipase C activation (EC50 = 1 microM). U73122 treatment also blocked functional responses of m4 muscarinic receptor and alpha2-adrenoceptor in NG108-15 cells and three opioid receptors (mu, delta and opioid receptor-like receptor (ORL1)) in human neuroblastoma SK-N-SH cells. 1-[6-((17Beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidinedione (U73343), an inactive analog of U73122, did not show any effect, which suggests that the blockade by U73122 of Gi-coupled receptor-mediated signaling is probably mediated through inhibition of phospholipase C, although a possible direct modification of G proteins can not be excluded. Furthermore, treatment with U73122 but not U73343 blocked the GTP-induced inhibition of adenylyl cyclase, indicating blockade at the level of Gi proteins.

N-Methyl-D-aspartate attenuates opioid receptor-mediated G protein activation and this process involves protein kinase C.

The effects of N-methyl-D-aspartate (NMDA) on opioid receptor-mediated G protein activation were explored in neuroblastoma X glioma hybrid (NG108-15) cells. Treatment of the cells with NMDA resulted in a remarkable attenuation of [35S]guanosine-5'-O-(3-thio)triphosphate binding stimulated by [D-Pen2,D-Pen5]-enkephalin (DPDPE), a delta-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased approximately 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by approximately 60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of delta-, mu-, and kappa-opioid receptors in primarily cultured neurons also were attenuated significantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadministration of the protein kinase C (PKC) inhibitors or by elimination of the extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated Gialpha2 phosphorylation. Transient transfection into NG108-15 cells of the wild-type Gialpha2 and a mutated Gialpha2 (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DPDPE responses were greatly inhibited by NMDA treatment in the wild-type Gialpha2-transfected cells but much less affected in the mutant Gialpha2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.

Inhibition of phospholipase C blocks opioid receptor-mediated activation of Gi proteins.

Activation of phospholipase C (PLC) in response to stimulation of delta-opioid, m4 muscarinic and alpha2 adrenergic receptors was observed in NG108-15 cells. Treatment with PLC specific inhibitors, U73122 and ET-18-OCH3, blocked delta-opioid receptor-mediated activation of G proteins with no effect on opioid binding to the receptors. U73122 treatment also suppressed functional responses of m4 muscarinic and alpha2 adrenergic receptors in NG108-15 cells. Furthermore, the G protein activation mediated by mu- and delta-opioid receptors and opioid receptor-like receptor (ORL1) were abolished by U73122 in SK-N-SH neuroblastoma cells. Inhibition of adenylyl cyclase induced by high concentrations of GTP was blocked by U73122, suggesting that blockade is at the level of G proteins. Our results thus indicated that inhibition of PLC leads to blockade of Gi protein activation mediated by opioid receptors or other Gi-coupled receptors.

Endogenous opioid receptor-like receptor in human neuroblastoma SK-N-SH cells: activation of inhibitory G protein and homologous desensitization.

Endogenous expression of opioid receptor-like receptor (ORL1), in human neuroblastoma SK-N-SH cells was demonstrated by binding with nociceptin/ orphanin FQ (N/OFQ). Scatchard analysis of [3H]N/ OFQ saturation binding data gave Kd = 1.3 +/- 0.1 nM and Bmax = 1.58 +/- 2.5 fmol/mg protein. N/OFQ stimulation increased [35S]GTP gamma S binding to cell membranes and attenuated forskolin-induced cAMP accumulation in a concentration-dependent manner. The effects of N/OFQ were eliminated by the pretreatment of pertussis toxin (PTX) but not by the antagonists of opioid receptors, revealing mediation of N/OFQ signal transduction by ORL1 receptor and PTX sensitive G protein(s). The ability of N/OFQ to inhibit cAMP production was greatly reduced after prechallenging with N/OFQ, indicating that ORL1 undergoes homologous desensitization in neuronal cells.

Activation of N-methyl-D-aspartate receptor attenuates acute responsiveness of delta-opioid receptors.

Coadministration of antagonists of N-methyl-D-aspartate (NMDA) receptor and opioids has been shown to prevent development of opiate tolerance in animal and clinical studies, but its cellular and molecular mechanisms are not understood. In this study, the effect of NMDA on delta-opioid receptor (DOR)-mediated signal transduction was investigated in neuroblastoma x glioma NG108-15 cells that functionally express both DOR and NMDA receptors. Acute incubation of NG108-15 cells with NMDA, a specific agonist of NMDA receptor, significantly attenuated the ability of DOR agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) to inhibit forskolin-stimulated cAMP production. The attenuation caused by NMDA was dose-dependent, and the EC50 of DPDPE increased 100-fold (from 4.6 nM to 500 nM) after NMDA treatment. The NMDA effect on responsiveness of delta-opioid receptors to DPDPE could be blocked by ketamine, a NMDA receptor-specific antagonist. This NMDA attenuation effect on DOR activity was also observed in neuronal primary cell cultures from fetal mouse brain but not in the Chinese hamster ovary cell line stably transfected with DOR alone. Interestingly, NMDA pretreatment reduced the cellular response to epinephrine but not to that of prostaglandin E1 in NG108-15 cells, which suggests differential modulation of NMDA on different G protein-coupled receptors. Pretreatment of NG108-15 cells with ketamine along with DPDPE greatly attenuated DPDPE-induced acute desensitization of DOR. Furthermore, the specific inhibitors of protein kinase C, either chelerythrine chloride or Go 6979, effectively blocked the NMDA effect, which indicates the involvement of protein kinase C in the process. In conclusion, the activation of NMDA receptors can attenuate acute responsiveness of DOR in neuronal cells, whereas its blockage leads to reduction of DOR desensitization. These results have thus provided an insight into cross-talk between NMDA and opioid signal transduction.

Functional expression, activation and desensitization of opioid receptor-like receptor ORL1 in neuroblastoma x glioma NG108-15 hybrid cells.

Neuroblastoma x glioma NG108-15 hybrid cells have been examined for the expression of opioid receptor-like receptor (ORL1). [3H]Nociceptin/orphanin FQ (OFQ) bound to the cell membrane specifically (Kd = 3.6 +/- 0.6 nM) and inhibited forskolin-stimulated cAMP accumulation (EC50 = 0.72 +/- 0.3 nM). The responsiveness of NG108-15 cells to nociceptin/OFQ was blocked by pertussis toxin but not by naltrindole. The inhibitory activity of nociceptin/OFQ was significantly reduced after a prechallenge with the same peptide but was not influenced by DPDPE pretreatment, indicating acute and homologous desensitization of ORL1 receptors. Naltrindole caused the overshoot of cAMP in DPDPE-pretreated cells but not in nociceptin/OFQ-pretreated cells. The results indicate that ORL1 is functionally expressed and does not cross-interact with specific ligands of the delta opioid receptor in NG108-15 cells.

Modulation by calcium/calmodulin-dependent protein kinase II of functional response of delta opioid receptor in neuroblastoma x glioma hybrid (NG108-15) cells.

The potential modulation of opioid receptor signaling by calcium/calmodulin-dependent protein kinase II (CaMKII) has been investigated in NG108-15 cells. Both CaMKII specific inhibitors used, KN62 and KN93, time- and dose-dependently blocked inhibition of cAMP accumulation by [D-Pen2, D-Pen5]enkephalin (DPDPE), with an IC50 of about 1.2 microM and 0.8 microM, respectively. In the presence of 1 microM KN62 or KN93, the DPDPE dose-response curve shifted to the right (IC50 from 0.7 to 20 nM for KN62 and from 0.65 to 10 nM for KN93, respectively), and the maximal response was also significantly reduced. KN92, an inactive analogue of KN93, showed no significant impact, while ionomycin, an activator of CaMKII, greatly potentiated the opioid receptor response, suggesting that the effects of KN62, KN93 and ionomycin were likely mediated through CaMKII. In addition, KN62 did not affect ligand binding, receptor/Gi coupling, or basal and forskolin-stimulated adenylyl cyclase activity, suggesting its possible interference in the Gi/adenylyl cyclase interaction. Furthermore, a CaMKII inhibitor potently blocked the functional responses of other Gi-coupled receptors (m4 muscarinic and alpha2 adrenergic receptors) tested, but not that of Gs-coupled receptors (prostaglandin E1 and adenosine receptors). Our results clearly demonstrate that CaMKII modulates the signaling of opioid receptor and other Gi-coupled receptors.