Tbr1影响大脑皮质发育及细胞分化与迁移

目的 探究Tbr1基因在大脑新皮质与海马发育过程中的功能。 方法 分别取胚胎165d、185d,出生后0d、3d、7d、14d昆明小鼠的脑组织,每个年龄点取材8～10 (共52) 只,常规固定脱水后石蜡切片,用免疫荧光检测小鼠大脑皮质、海马及齿状回神经细胞迁移与片层化发育过程中Tbr1的表达与分布情况。结果 1.在大脑皮质,Tbr1最早在皮质板广泛表达,随日龄的增加其表达逐渐向皮质板下层移动且最终定位于皮质的第Ⅵ层;2.相似地,齿状回处Tbr1在颗粒细胞层表达,P7之后其表达定位于颗粒细胞下层;3.根据其表达位置及组织发生规律,推测Tbr1阳性细胞即是皮质板内迁移中的新生神经元。 结论 Tbr1是影响小鼠大脑皮质发育及神经细胞迁移与分化的关键分子。作为新生神经元的标记物,Tbr1参与细胞分化与迁移以及大脑皮质片层化的形成过程。     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

 We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND 10, decreased until PND 30, and thereafter remained at the same level as on PND 0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND 10 and PND 15, after which the immunoreactivity gradually decreased and reached a plateau at PND 30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED 20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND 0. Immunoreactivity in the neurons of the subiculum was found after PND 5 and that of the polymorphic cell layers was found after PND 15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions. Accepted: 13 June 1996     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND?10, decreased until PND 30, and thereafter remained at the same level as on PND?0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND?10 and PND?15, after which the immunoreactivity gradually decreased and reached a plateau at PND?30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED?20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND?0. Immunoreactivity in the neurons of the subiculum was found after PND?5 and that of the polymorphic cell layers was found after PND?15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions.     

Contrary roles of kainate receptors in transmitter release at corticothalamic synapses onto thalamic relay and reticular neurons

Corticothalamic fibres, which originate from layer VI pyramidal neurons in the cerebral cortex, provide excitatory synaptic inputs to both thalamic relay neurons and reticular neurons; reticular neurons in turn supply inhibitory inputs to thalamic relay neurons. Pyramidal cells in layer VI in the mouse somatosensory cortex highly express mRNA encoding kainate receptors, which facilitate or depress transmitter release at several synapses in the central nervous system. We report here that contrary modulation of transmitter release from corticothalamic fibres onto thalamic relay and reticular neurons is mediated by activation of kainate receptors in mouse thalamic ventrobasal complex and thalamic reticular nucleus. Exogenous kainate presynaptically depresses the synaptic transmission at corticothalamic synapses onto thalamic relay neurons, but facilitates it at corticothalamic synapses onto reticular neurons. Meanwhile, the lemniscal synaptic transmission, which sends primary somatosensory inputs to relay neurons, is not affected by kainate. In addition, GluR5-containing kainate receptors are involved in the depression of corticothalamic synaptic transmission onto relay neurons, but not onto reticular neurons. Furthermore, synaptically activated kainate receptors mimic these effects; high-frequency stimulation of corticothalamic fibres depresses synaptic transmission onto relay neurons, but facilitates it onto reticular neurons. Our results suggest that the opposite sensitivity of kainate receptors at the two corticothalamic synapses is governed by cortical activity and regulates the balance of excitatory and inhibitory inputs to thalamic relay neurons and therefore their excitability.     

Transient co-localization of calretinin, parvalbumin, and calbindin-D28k in developing visual cortex of monkey

Summary This paper reports a double-labelling immunocytochemical study of the three calcium-binding proteins calretinin, parvalbumin, and calbindin-D28k in developing and adultMacaca primary visual cortex. In adult visual cortex, each protein marks a subset of GABAergic neurons with a characteristic laminar distribution and virtually no co-localization was found between these three proteins, suggesting that each calcium-binding protein may serve as a marker for one or more cortical subcircuits. The immature visual cortex, immunostained using identical techniques was then analysed to determine if each calcium-binding protein could serve as a developmental marker for these circuits. The Cajal-Retzius cells of layer 1 contained all three proteins during development. Calbindin-D28k and calretinin were co-localized starting at Fd (foetal day) 45 and after Fdl25, parvalbumin also was present in the same Cajal-Retzius cells. All three proteins continued to be expressed until the Cajal-Retzius disappeared postnatally. In layers 2–6 calbindin-D28k and calretinin were never co-localized. In contrast, parvalbumin and calretinin were found in neurons of deep layer 3 from Fd 155 to postnatal (P6) weeks with a few persisting even later. Before birth almost all PV⁺ neurons in layers 4–6 were CaB⁺, but by P3 weeks only a few PV⁺/CaB⁺ neurons remained in layer 4C and these completely disappeared by P6 weeks. Co-localization in layer 4 neurons overlaps the period of ocular dominance segregation, suggesting that the onset of cortical maturity coincides with segregation of calcium-binding proteins within the GABA interneurons.     

Highly prevalent colorectal cancer-infiltrating LAP+ Foxp3− T cells exhibit more potent immunosuppressive activity than Foxp3+ regulatory T cells

Although elevated CD4⁺Foxp3⁺ regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4⁺Foxp3⁺ T cells (Tregs) were Helios⁺ and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ∼30% of intratumoral CD4⁺Foxp3⁻ T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and was ∼50-fold more suppressive than Foxp3⁺ Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies.     

Localization of IL-17+Foxp3+ T Cells in Esophageal Cancer

The etiology of cancer is unclear. Recent studies indicate that some cytokines, such as interleukin (IL)-17, and regulatory T cells are involved in the development of cancer. This study aims to detect a subset of T cell, IL17+Foxp3+ T cell, in the pathogenesis of esophageal cancer (Eca). Twelve patients with squamous Eca were recruited in this study. The surgically removed Eca tissue was collected. Cells isolated from Eca tissue were analyzed by flow cytometry. The results showed that 2–10% Eca tissue-derived CD4⁺ T cells expressed Foxp3; only 0.2–0.8% non-ca tissue-derived CD4⁺ T cells expressed Foxp3. Further analysis showed that 3–15% Eca-isolated CD4⁺ T cells were also IL-17 positive whereas only 0.4–1.5% non-ca tissue-isolated CD4⁺ T cells were IL-17 positive. We also found that about 4.8–11.2% Foxp3⁺ IL-17⁺ T cells in isolated CD4⁺ T cells from Eca tissue that were significantly less than in non-ca tissue derived CD4⁺ T cells. Less than 1% Foxp3⁺ IL-17⁺ T cells in isolated CD4⁺ T cells in both Eca patients and healthy controls. Treatment with hypoxia markedly increased the expression of IL-6 in peripheral CD68+ cells. Coculturing CD68+ cells and Foxp3+ T cells under hypoxic environment resulted in abundant expression of IL-17 in Foxp3+ T cells that could be blocked by pretreatment with either anti-IL-17 or anti-transforming growth factor beta antibodies. We conclude that IL-17+Foxp3+ T cells may contribute to the development of Eca.     

Foxp3+ regulatory T cells promote lung epithelial proliferation

Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality each year. There is a paucity of information regarding the mechanisms necessary for ARDS resolution. Foxp3⁺ regulatory T cells (Foxp3⁺ T_reg cells) have been shown to be an important determinant of resolution in an experimental model of lung injury. We demonstrate that intratracheal delivery of endotoxin (lipopolysaccharide) elicits alveolar epithelial damage from which the epithelium undergoes proliferation and repair. Epithelial proliferation coincided with an increase in Foxp3⁺ T_reg cells in the lung during the course of resolution. To dissect the role that Foxp3⁺ T_reg cells exert on epithelial proliferation, we depleted Foxp3⁺ T_reg cells, which led to decreased alveolar epithelial proliferation and delayed lung injury recovery. Furthermore, antibody-mediated blockade of CD103, an integrin, which binds to epithelial expressed E-cadherin decreased Foxp3⁺ T_reg numbers and decreased rates of epithelial proliferation after injury. In a non-inflammatory model of regenerative alveologenesis, left lung pneumonectomy, we found that Foxp3⁺ T_reg cells enhanced epithelial proliferation. Moreover, Foxp3⁺ T_reg cells co-cultured with primary type II alveolar cells (AT2) directly increased AT2 cell proliferation in a CD103-dependent manner. These studies provide evidence of a new and integral role for Foxp3⁺ T_reg cells in repair of the lung epithelium.     

DC expressing transgene Foxp3 are regulatory APC

Tolerogenic DC and suppressive Foxp3⁺ Treg play important roles in preventing autoimmunity and allograft rejection. We report that (adenovirus mediated) ectopic expression of Foxp3 in human DC (i.e. DC.Foxp3) yields an APC that severely limits T‐cell proliferation and type‐1 immune responses from the naïve, but not memory, pool of responder T cells in vitro. In marked contrast, the frequencies of type‐2 and Treg responses were dramatically increased after stimulation of naïve T cells with DC.Foxp3 versus control DC. DC.Foxp3‐induced CD4⁺CD25⁺ Treg cells potently suppressed the proliferation of, and IFN‐γ production from, CD4⁺ and CD8⁺ responder T cells. Notably, the immunosuppressive biology of DC.Foxp3 was effectively normalized by addition of 1‐methyl‐tryptophan or neutralizing anti‐TGF‐β1 Ab during the period of T‐cell priming. These data suggest the potential utility of regulatory DC.Foxp3 and/or DC.Foxp3‐induced CD4⁺CD25⁺ Treg as translational agents for the amelioration or prevention of pathology in the setting of allograft transplantation and/or autoimmunity.     

A light- and electron-microscopic study of GluR4-positive cells in cerebral cortex,subcortical white matter and corpus callosum of neonatal,immature and adult rats

The distribution of the [³H]alpha-amino-3-hydroxy-5-methylisoxzalepropionic acid (AMPA) receptor subunit GluR4 was studied in frontal, parietal and temporal cerebral cortex, subcortical white matter and corpus callosum of neonatal, immature and mature rats. In 1-to 2-day-old rats, a few oligodendrocyte progenitors and amoeboid microglia in the supraventricular part of the corpus callosum were immunolabelled for GluR4. At 7 to 10 days, the number of amoeboid microglia and oligodendrocyte progenitors in white matter increased; many neurons in cortex, including pyramidal neurons, were also moderately labelled for GluR4. The pattern of GluR4 immunostaining in 14-day-old rats was different from that in 7-to 10-day-old rats, but similar to the adult, in that there was no immunoreactivity in microglia and oligodendrocyte progenitors in subcortical white matter. A proportion of non-pyramidal neurons in cortex were moderately labelled, while some pyramidal neurons were lightly labelled. A population of small glial cells with features of oligodendrocyte progenitors were densely labelled in cortex.     

Distribution of glutamate receptor subunit GluR1 and GABA in human cerebral neocortex: a double immunolabelling and electron microscopic study

Specimens of human cerebral neocortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the glutamate receptor subunit GluR1 and gamma-aminobutyric acid (GABA). Many GluR1-positive pyramidal neurons and fewer GluR1-positive nonpyramidal neurons were present in the cortex. Non-pyramidal neurons were more heavily labelled for GluR1 than pyramidal neurons. Most GABAergic neurons were labelled for GluR1. The white matter was unstained, except for occasional labelled neurons. This pattern of GluR1 immunostaining is similar to that in rat cerebral cortex, but is different from that in the hippocampus and amygdala, where large numbers of pyramidal or projection neurons, but few non-pyramidal or GABAergic neurons, were labelled for GluR1.     

Localization of prolyl oligopeptidase in the thalamic and cortical projection neurons: A retrograde neurotracing study in the rat brain

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyses proline-containing peptides shorter than 30-mer. POP is believed to be associated with cognitive functions via neuropeptide cleavage. POP has been also connected to the inositol 1,4,5-triphosphate (IP₃) signalling but the effects of POP-inhibition to the IP₃ accumulation in vivo are still unclear. However, little is known about the physiological role of POP in the brain. We have previously found that in the rat brain POP was specifically expressed in the pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and corresponding projection areas in thalamus. Using a retrograde neurotracer we have now visualized the localization of POP in thalamocortical and corticothalamic projection neurons in ventrobasal complex and medial geniculate nucleus of thalamus and somatosensory/motor and auditory cortices. We observed that both in thalamus and cortex over 50% of projection neurons contained POP. These results support the hypothesis that POP is involved in thalamocortical and corticothalamic signal processing. We also propose, based on our neuroanatomical findings and literature, that POP may take part in the thalamocortical oscillations by interacting with IP₃ signalling in cells.     

Short‐term cytokine stimulation reveals regulatory T cells with down‐regulated Foxp3 expression in human peripheral blood

The identification of regulatory T cells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4⁺CD25⁺Foxp3⁺ are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine‐mediated STAT5 signals. Short‐term incubation of PBMCs or isolated CD4⁺ T cells, but not of lymph node cells, with IL‐2, ‐7, or ‐15 more than doubles the frequency of Foxp3⁺CD25⁺ among CD4⁺ T cells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up‐regulate CD25 and down‐regulate CD127, making them accessible to viable cell sorting. “Latent” Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non‐cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4⁺CD25⁺Foxp3⁺ cells reported in IL‐2 treated patients.     

Distinct neuronal populations specified to form corticocortical and corticothalamic projections from layer VI of developing cerebral cortex

Layer VI of the cerebral cortex contains heterogeneous populations of pyramidal neurons whose axons project either cortically or subcortically. It has been shown that a subset of layer VI neurons expressing latexin projects ipsilaterally to other cortical areas but does not contribute to the corticothalamic projections. Taking advantage of the connectional specificity of latexin-expressing neurons, we here determine whether corticocortical and corticothalamic neurons are generated at different times, and at which stage the connectional distinction develops in corticogenesis. Our experimental findings indicate that: (1) thalamic-projecting neurons in layer VI of the rat secondary somatosensory cortex (SII) are born at embryonic day 14 or before while latexin-expressing neurons in the same layer are generated at embryonic day 15 or later; (2) axonal invasion by SII neurons into ipsilateral cortical areas and into the posterior dorsal thalamus mainly takes place early in the postnatal period; (3) latexin-expressing neurons never project toward the dorsal thalamus in normal development; (4) presumptive latexin-expressing neurons in the neonatal SII are able to grow into a cortical slice in vitro, but do not invade a thalamic slice even transiently; (5) thalamic-projecting neurons, on the other hand, fail to simultaneously establish connections with a cortical slice. Taken together, our findings suggest that the time frame in which presumptive corticocortical and corticothalamic neurons are generated differs, and that the two populations are restricted in connectional fate potential by the perinatal period prior to target innervation.     

FTY720-induced conversion of conventional Foxp3- CD4+ T cells to Foxp3+ regulatory T cells in NOD mice

Citation Sun Y, Wang W, Shan B, Di J, Chen L, Ren L, Li W, Li D‐J, Lin Y. FTY720‐induced conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in NOD mice. Am J Reprod Immunol 2011; 66: 349–362 Problem FTY720 is known as an agonist of sphingosine‐1‐phosphate (S1P) receptor, but little is known about the possibility that FTY720 induces the conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in non‐obese diabetic (NOD) mice. Method of study FTY720 treatment was performed using Foxp3^?CD4⁺ T cells purified from NOD mice. Results FTY720 caused an increase in Foxp3⁺ Treg cells in lymphoid organs in NOD mice. FTY720 effectively induced Foxp3 expression in Foxp3^?CD4⁺ T cells both in vitro and in vivo, an effect that was inhibited by a TGF‐β‐neutralizing antibody or the proinflammatory cytokine IL‐6. T‐cell‐mediated embryo rejection in NOD mice was prevented upon FTY720 treatment. Conclusions The use of FTY720 along with Ag administration may represent a useful therapeutic strategy to selectively expand Ag‐specific Foxp3⁺ Tregs to intervene autoimmune and infectious diseases.