[The crucial toxic components of ambient fine particles promoted the maturation and differentiation of megakaryocytes].

Objective: To reveal the crucial toxic components of ambient fine particles (PM2.5) that affect the maturation and differentiation of megakaryocytes. Methods: Human megakaryocytes were exposed to the organic fractions, metallic fractions and water-soluble fractions of PM2.5 at two exposure doses (i.e. actual air proportion concentration or the same concentration), respectively. The cell viability was performed to screen the non-cytotoxic levels of toxic components of PM2.5 using the CCK-8 assay. CellTiter-Blue assay, morphological observation, flow cytometry analysis and WGA staining assay were used to evaluate the cell morphological changes, occurrence of DNA ploidy, alteration in the expressions of biomarkers and platelet formation, which were key indicators of the maturation and differentiation of megakaryocytes. Results: Compared to the control group, both metallic and organic components of PM2.5 resulted in a lag in megakaryocytes with an increase in cell volume and the onset of DNA ploidy. Flow cytometry analysis showed that CD33 (the marker of myeloid-specific) decreased and CD41a (a megakaryocyte maturation-associated antigen) increased in metallic and organic components of PM2.5 treatment groups. Moreover, compared to the control group, budding protrusions increased in metallic and organic components of PM2.5 treatment groups. The water-soluble components had no effect on the maturation and differentiation of macrophages. Conclusion: Metallic and organic components of PM2.5 are the crucial toxic components that promote the maturation and differentiation of megakaryocytes.

[Clinicopathological characteristics of NTRK-rearranged mesenchymal tumors in childhood].

Objective: To investigate the clinical and pathological features of pediatric NTRK-rearranged tumors. Methods: Four NTRK-rearranged soft tissue tumors and one renal tumor at Shanghai Children's Medical Center, Shanghai Jiaotong University and Singapore KK Women's and Children's Hospital from January 2017 to September 2019 were identified. Pan-TRK immunohistochemistry, and the ALK and ETV6 gene break-apart fluorescence in situ hybridizations (FISH) were performed. NTRK gene rearrangement was detected using sequencing-based methods. Results: There were 3 males and 2 females in this study. The patients were between 3 months and 13 years of age. Histologically, the tumors were infiltrative spindle cell tumors with variable accompanying inflammatory cells. Immunohistochemistry showed positive reactivity for pan-TRK in all tumors, with nuclear staining for NTRK3 fusion, and cytoplasmic staining for NTRK1 fusion. The molecular testing revealed NTRK gene fusions (one each of TPM3-NTRK1, ETV6-NTRK3 and DCTN1-NTRK1, and two cases of LMNA-NTRK1). Two patients were receiving larotrectinib. The others were are well without disease, with follow-up durations of 9 to 29 months. Conclusions: NTRK-rearranged mesenchymal tumors from soft tissue sites and kidney are identified. A novel DCTN1-NTRK1 fusion is described. Pan-TRK immunohistochemistry is useful for diagnosis. NTRK-targeted therapy may be an option for unresectable, recurrent or metastatic cases.

[Application of the pathological classification of "CCCG-WT-2016" (2019 revision) for treatment of Wilms tumors].

Objective: To describe our experiences in application of the 2019 revision of "CCCG-WT-2016" for the diagnosis of Wilms tumors. Methods: Ninety-one cases of Wilms tumor diagnosed at Shanghai Children's Medical Center from January 2015 to December 2018 were collected. All cases were reviewed by two senior pathologists, including one from China and the other from Singapore, according to the 2019 revision of "CCCG-WT-2016." Results: The specimens were obtained by core biopsy (n=21), primary nephrectomy (n=41), post-chemotherapy nephrectomy/resection (n=18), or biopsy/resection of metastatic/relapse/post-chemotherapy metastatic lesion(s) (n=11). The specimens of core biopsy and primary nephrectomy (n=62) all had favorable histology.Twelve post-chemotherapy nephrectomy cases were subdivided into three risk groups: low risk (n=0), intermediate risk (n=10) and high risk (n=2). Six post-chemotherapy resection cases were subdivided into 3 risk groups:low risk (n=0), intermediate risk (n=5) and high risk (n=1). The remaining 11 cases were comprised of metastatic, relapse, and post-chemotherapy metastatic lesions. The concordance rate of the two senior pathologists was 100%(91/91). Conclusions: The 2019 revision of "CCCG-WT-2016" is clearly written and easy to use. It can serve as the basis of accurate classification for clinical treatment.

[A randomized controlled study of erlotinib versus pemetrexed combined with cisplatin in neoadjuvant therapy of stage â ¢A EGFR-mutant lung adenocarcinoma].

Objective: To evaluate the feasibility, efficacy and safety of epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs) for neoadjuvant therapy. Methods: Eighty-six patients with stage ⅢA EGFR-mutant lung adenocarcinoma were assigned to 2 groups (n=43 in each group) according to the random number table method: neoadjuvant targeted therapy group (single oral dose of erlotinib 150 mg per day, for 9 weeks) and neoadjuvant chemotherapy group (2 cycles of pemetrexed combined with cisplatin chemotherapy followed by 3- week discontinuation). Surgical treatment was underwent after imaging efficacy evaluation. Results: In neoadjuvant targeted therapy group, 4 achieved complete response (CR), 25 achieved partial response (PR), giving an objective response rate (ORR) of 67.4%. In pathological response, 8 patients had grade Ⅰ, 20 patients had grade Ⅱ, giving a pathological response rate of 65.1%. The most frequent adverse events (AEs) were rash and diarrhea. In neoadjuvant chemotherapy group, 2 had CR and 17 had PR, giving an ORR of 44.2%. In pathological response, 3 patients had grade Ⅰ, 15 patients had grade Ⅱ, giving a pathological response rate of 41.9%. The main AEs were hematologic toxic effects. The ORR, histological efficacy and hematologic toxicity showed statistical significance between the two groups (P<0.05). The neoadjuvant targeted therapy group had 90.7% resection rate, (299.8±23.4) ml of hemorrhage volume during operation, (5.2±0.4) days of extubation time and 9.3% postoperative complication rate. Corresponding results were 83.7%, (308.9±22.7) ml, (5.4±0.6) days and 11.6% in neoadjuvant chemotherapy group, which showed no statistical significance (P>0.05). Conclusions: Neoadjuvant targeted treatment for stage ⅢA lung adenocarcinoma harboring EGFR mutations. The regimen could be considered as a choice of neoadjuvant treatment for patients with stage ⅢA EGFR-mutant lung adenocarcinoma.

Activation of presynaptic kainate receptors suppresses GABAergic synaptic transmission in the rat globus pallidus.

The globus pallidus (GP) plays a central integrative role in the basal ganglia circuitry. It receives strong GABAergic inputs from the striatum (Str) and significant glutamatergic afferents from the subthalamic nucleus (STN). The change in firing rate and pattern of GP neurons is a cardinal feature of Parkinson's disease pathophysiology. Kainate receptor (KAR) GluR6/7 subunit immunoreactivity is expressed presynaptically in GABAergic striatopallidal terminals which provides a substrate for regulation of GABAergic transmission in GP. To test this hypothesis, we recorded GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) in the GP following electrical stimulation of the Str. Following blockade of AMPA and N-methyl-d-aspartate receptors with selective antagonists, bath application of kainate (KA) (0.3-3 microM) reduced significantly the amplitude of evoked IPSCs. This inhibition was associated with a significant increase in paired-pulse facilitation ratio and a reduction of the frequency, but not amplitude, of miniature inhibitory postsynaptic currents (mIPSCs), suggesting a presynaptic site of KA action. The KA effects on striatopallidal GABAergic transmission were blocked by the G-protein inhibitor, N-ethylmaleimide (NEM), or protein kinase C (PKC) inhibitor calphostin C. Our results demonstrate that KAR activation inhibits GABAergic transmission through a presynaptic G protein-coupled, PKC-dependent metabotropic mechanism in the rat GP. These findings open up the possibility for the development of KA-mediated pharmacotherapies aimed at decreasing the excessive and abnormally regulated inhibition of GP neurons in Parkinson's disease.

Activation of Group III mGluRs increases the activity of neurons in area 17 of the cat.

Activation of Group III metabotropic glutamate receptors (mGluRs) by L(+)-2-amino-4-phosphonobutyric acid (L-AP4) has different effects on in vitro slice preparations of visual cortex (Jin & Daw, 1998) as compared with in vivo recordings from somatosensory cortex (Wan & Cahusac, 1995). To investigate the role of Group III mGluRs in the cat visual cortex, in vivo recordings were made of neurons in area 17 of the visual cortex of kittens and adult cats at different ages and the effect of iontophoretic application of L-AP4 (100 mM) was examined. Application of L-AP4 resulted in an increase of the spontaneous activity and visual response of neurons to visual stimulation, the former more than the latter. The effect of L-AP4 was greatest at 3-5 weeks of age with the effect on the visual response declining more rapidly than the effect on spontaneous activity. Consistent with work in rat cortex (Jin & Daw, 1998), the effect of L-AP4 was significantly greater in upper and lower layers than in middle layers. Whole-cell in vitro recordings from slices of rat visual cortex indicated that L-AP4 (50 mM) did not increase the number of spikes elicited by increasing levels of current injections. These results confirm that L-AP4 increases activity in vivo and reasons for the discrepancy with the in vitro results are discussed.

Effect of the group I metabotropic glutamate agonist DHPG on the visual cortex.

Metabotropic glutamate receptors have a variety of effects in visual cortex that depend on the age of the animal, the layer of the cortex, and the group of the receptor. Here we describe these effects for group I receptors, using both in vivo and in vitro preparations. The metabotropic group I glutamate receptor agonist 3,5 dihydroxyphenylglycine (DHPG) potentiates the responses to N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in slices of rat visual cortex. It also increases, initially, the visual response in the cat visual cortex. Both these effects are largest at 3-4 wk of age and decline to insignificance by 10 wk of age. Both are also largest in lower layers of cortex, which explains why the facilitatory effects found with the general metabotropic glutamate agonist 1S,3R aminocyclopentane-1,3-dicarboxylic acid (ACPD) are observed only in lower layers. Prolonged application of DHPG in the cat visual cortex, after the initial excitatory effect, produces depression. We also found that DHPG facilitates the NMDA response in fast-spiking cells, which are inhibitory, providing a partial explanation for this. Thus there are multiple effects of group I metabotropic glutamate receptors, which vary with layer and age in visual cortex.

2R,4R-4-Aminopyrrolidine-2,4-dicarboxylate (APDC) attenuates cortical EPSPs.

We studied the effect of the Type II metabotropic glutamate receptor (mGluR 2,3) agonist APDC on the response of neurons in slices of rat visual cortex. In all cortical layers, APDC attenuated the EPSP produced by stimulation of the predominant excitatory input. This EPSP attenuation was seen in both younger and older rat slices and was present with G-protein blockade in the cell recorded, demonstrating that it was a presynaptic effect. Further, this EPSP attenuation was blocked by the mGluR 2,3 antagonist EGLU. A postsynaptic depressive effect of APDC on the NMDA response was seen in layers 2 and 3, but not in layers 5 and 6. Thus, the predominant action of Type II mGluRs in the visual cortex is a presynaptic reduction of glutamate release which persists through development. This regulation may be important in the setting of excitatory tone in visual cortex and in the extraction/processing of visual information.

Serotonin receptors modulate rod signals: a neuropharmacological comparison of light- and dark-adapted retinas.

Previous physiological studies have shown that serotonin (5-HT) reciprocally modulates ON and OFF channels in the mammalian retina. This study was undertaken to determine if the serotoninergic system is exclusively associated with the rod pathway. We tested drugs specific to 5-HT3 receptor, a serotonin-gated ion channel, in both dark- and light-adapted retina. Consistent with previous studies, we demonstrated that 5-HT3 receptors modulate the light-evoked responses of ganglion cells in the dark-adapted state. Moreover, we have extended these prior studies and shown that activation of the 5-HT3 receptor is capable of completely blocking the light-evoked response of OFF-center cells whereas inactivation of the 5-HT3 receptor is capable of completely blocking the light-evoked responses of ON-center cells. In contrast, in light-adapted retinae, serotonin agents failed to have any effect on retinal processing. These data suggest that the serotoninergic system in retina is (1) specifically associated with rod-related pathways; and (2) exerts a powerful modulatory force over information transfer in the retina. Together these observations suggests that serotonin plays an important physiological role in modulating retinal processing.

A differential effect of APB on ON- and OFF-center ganglion cells in the dark adapted rabbit retina.

The glutamate analog, 2-amino-4-phosphonobutyric acid (APB) is a proven tool in exploring the retinal circuit; it has been shown to interfere specifically with the transmission from photoreceptor to depolarizing bipolar cell. Consequently, in photopic retinae, the application of APB disrupts the ON-channel leaving the OFF-channel undisturbed; on the other hand, in the scotopic state, APB application blocks all ganglion cell responses. In this paper, we will show that the ON- and OFF-channels have a differential sensitivity to application of APB. That is to say, APB blocks center responses in ON-ganglion cells at mean concentration of 22 +/- 5.1 microM (mean +/- standard error of the mean; n = 15) and in OFF-ganglion cells at mean concentration of 91 +/- 15.5 microM (n = 16). Since considerable data rule out direct effects of APB on ganglion cells, we hypothesize that this effect is due to a difference in the synaptic gain of ON and OFF pathways in the inner retina.

A role for 5HT3 receptors in visual processing in the mammalian retina.

We investigated the role of 5HT3 receptors in the mammalian retina using electrophysiological techniques to monitor ganglion cell activity. Activation of 5HT3 receptors with the selective agonist 1-phenylbiguanide (PBG) increased the ON responses of ON-center ganglion cells, while decreasing the OFF responses of OFF-center cells. The application of a selective 5HT3 antagonist had a reciprocal effect, namely it reduced the center response in ON-center cells and concomitantly increased the center responses in OFF-center cells. Since putative serotoninergic amacrine cells in the retina are connected specifically to the rod bipolar cell, these agents most likely affect the rod bipolar terminal. These data, together with previous studies, suggest that both 5HT2 and 5HT3 receptors mediate an excitatory influence which serves to facilitate the output from rod bipolar cells, the former via a phosphatidyl inositol second-messenger system, and the latter via a direct ion channel.