IL-7-mediated protection of pro and pre-B cells from the adverse effects of corticosterone

The studies herein demonstrate that Interleukin-7 (IL-7) promotes survival of murine pro- and pre-B cells against stress levels of corticosterone (Cs). In short-term, 16-h, bone marrow cultures IL-7 abrogated Cs-induced apoptosis and cell cycle arrest in pro-B cells by decreasing apoptosis 60% and completely restoring the cell cycle. IL-7 also reduced Cs-induced apoptosis by 36% in pre-B cells and 24% in IgM(+) B cells, but did not restore deficits in the cell cycle. Among pro- and pre- B cells, substantial protection against high, pharmacological, levels of Cs was also provided by IL-7. Interestingly, stem cell factor, while reducing spontaneous apoptosis in pro-B cells, did not protect against Cs-induced death, either alone or with IL-7. In conclusion, IL-7 has potential immunotherapeutic value since it provides substantial protection to pro- and pre-B cells against the adverse effects of Cs.     

急性高原肺水肿患者血清中VEGF,TNF-α,IL-6及NO的含量变化

目的：检测高原肺水肿患者发生及转归过程中内皮生长因子（VEGF）,肿瘤坏死因子-α（TNF-α）,白介素-6（IL-6）和一氧化氮（NO）的含量变化,探讨高原肺水肿发生发展可能的病理生理机制。方法：收集10例高原肺水肿患者治疗前和转归后的血清样本,采用酶联免疫法和硝酸还原测定VEGF,TNF-α,IL-6和NO的含量。结果：VEGF在高原肺水肿发病时血清的含量为（167.9±26.5）pg/ml,而转归之后其含量显著降低为（53.1±17.0）pg/ml,（P〈0.01）;TNF-α在血清中的含量从发病时的（86.2±24.1）pg/ml减少到转归后的（29.2±6.8）pg/ml,（P〈0.05）,有显著性差异。而IL-6的水平也从发病时的（32.3±16.5）pg/ml变化为转归后的（12.5±8.0）pg/ml,虽然有下降的趋势,但没有统计学差异。而高原肺水肿患者血清中的NO水平从发病时的（33.8±3.3）μmol/L明显的升高到转归后的（74.1±6.2）μmol/L,（P〈0.01）。结论：VEGF,TNF-α,IL-6和NO参与了高原肺水肿的发生和转归过程。     

腹腔镜与开腹手术对胃癌根治术患者TNF-alpha和IL-6 水平的影响研究

目的:探讨腹腔镜与开腹手术对胃癌根治术患者肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平的影响.方法:选取自2011年3月至2013年5月期间来我院就医并行胃癌根治术的72例胃癌患者作为研究对象.并将所有患者随机平均分成腹腔镜组和传统开腹组各36例.其中,腹腔镜组行腹腔镜辅助下胃癌根治术,传统开腹组行传统开腹胃癌根治术.比较两组手术时间、术中出血量、肛门排气时间及住院时间;比较两组患者术前及术后血清TNF-α和IL-6水平.结果:腹腔镜组术中出血量、肛门排气时间及住院时间均明显低于对照组(P＜0.05)腹腔镜组术后TNF-α和IL-6水平明显低于开腹组(P＜0.05),两组比较有显著性差异(P＜0.05).结论:腹腔镜辅助下胃癌根治术较传统开腹胃癌根治术术后血清TNF-α和IL-6水平低,对机体免疫功能影响较小,可减少患者术后感染机会,值得在临床中推广应用.     

IL-6, LIF, and TNF-alpha regulation of GM-CSF inhibition of osteoclastogenesis in vitro

During pathological bone loss, factors that are both stimulatory and inhibitory for osteoclast differentiation are over-expressed. Despite the presence of inhibitory factors, osteoclast differentiation is significantly enhanced to bring about bone loss. To examine the hypothesis that stimulatory growth factors overcome the effects of inhibitory factors, we have examined the ability of IGF-I, IGF-II, IL-6, LIF, and TNF-alpha to overcome osteoclast differentiation inhibition by GM-CSF in vitro. Osteoclast numbers were significantly elevated by treatment with IGF-I, IGF-II, IL-6, LIF, or TNF-alpha alone whereas GM-CSF treatment of stromal cell and osteoclast co-cultures inhibited osteoclast formation. IL-6, LIF, or TNF-alpha, individually overcame GM-CSF inhibition whereas neither IGF-I nor IGF-II treatment overcame GM-CSF inhibition. Interestingly, GM-CSF addition with either IL-6 or TNF-alpha increased osteoclast numbers beyond that seen with either IL-6 or TNF-alpha alone. Combined treatment with TNF-alpha and IL-6 showed a significant increase in osteoclast numbers with GM-CSF addition. Examination of the impacts of these growth factors individually or in combinations on stromal cell M-CSF, RANKL, and OPG expression revealed a complex pattern involving alterations in the ratio of RANKL to OPG and/or M-CSF expression as candidate mechanisms of action.     

Cytokine-mediated induction of metallothionein in Hepa-1c1c7 cells by oleanolic acid

Oleanolic acid (OA), a pentacyclic triterpene acid, has been reported to possess inducing activity of hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of OA on the regulation of MT expression in an in vitro model. OA that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the OA-treated conditioned media from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with OA and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in a decrease in the effects of OA on the MT induction. In the OA-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. However, the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the OA-treated conditioned media from the RAW 264.7 cells. These results suggest that the up-regulation of MT expression by OA was mediated by the TNF-alpha and IL-6 released from UA-activated macrophages.     

Expression of glutamyl aminopeptidase by osteogenic induction in rat bone marrow stromal cells

Glutamyl aminopeptidase (GluAP, EC 3.4.11.7, ENPEP) is a 130-kDa homodimeric zinc metallopeptidase which specifically cleaves the N-terminal glutamate or aspartate residue of peptidic substrates such as cholecystokinin-8 or angiotensin (Ang) II, in vitro. We used a DNA microarray hybridization (Genechip Rat Expression Array 230A, Affymetrix Inc., Santa Clara, CA, USA) to demonstrate that GluAP was upregulated in osteogenic induced rat bone marrow stromal cells (BMSCs). To compare the expression of GluAP in the osteogenic differentiation and non-osteogenic differentiation of rat BMSCs in vitro, the cells were osteogenic induced in vitro. We also performed an MTT assay, alkaline phosphatase assay, alizarin red staining, and an immunohistochemical analysis to determine the osteogenic differentiation of BMSCs. The expression of GluAP was examined by real-time polymerase chain reaction (PCR). The real-time PCR results showed that GluAP was upregulated in osteogenic differentiated BMSCs in vitro, suggesting that GluAP may be correlated with the osteogenic differentiation of BMSCs.     

The role of calcitonin and alpha-calcitonin gene-related peptide in bone formation

The Calca gene encodes two polypeptides, calcitonin (CT) and α-calcitonin gene-related peptide (α-CGRP), generated through alternative splicing. While CT, a hormone mainly produced by thyroidal C cells, has been described as a major regulator of bone resorption, α-CGRP, a neuropeptide expressed in the cells of the central and peripheral nervous system, is mostly known as a regulator of vascular tone. Surprisingly, the generation and skeletal analyses of two mouse deficiency models has recently uncovered a physiological function for both peptides in the regulation of bone formation. In the first model, where the replacement of exons 2-5 of the Calca gene resulted in the combined deficiency of CT and α-CGRP, an increased bone formation rate (BFR) was observed, whereas decreased BFR was found in the second model, where the introduction of a translational termination codon into exon 5 of the Calca gene resulted in the specific absence of α-CGRP.     

IL-12重组质粒联合骨髓间充质干细胞对MCF-7人乳腺癌细胞侵袭及裸鼠移植瘤生长的影响分析

目的探讨IL-12重组质粒联合骨髓间充质干细胞（BMSC）对MCF-7人乳腺癌细胞侵袭及裸鼠移植瘤生长的影响。 方法分离纯化培养BMSC原代细胞,制备BMSC条件培养基,分析BMSC条件培养基对MCF-7细胞株体外增殖和凋亡的影响;建立裸鼠MCF-7转移瘤模型,分为对照组、BMSC组、IL-12组以及联合组,各组每3 d进行一次瘤内注射,共注射3次,15 d后处死裸鼠,比较各组裸鼠肿瘤、脾脏重量。采用χ²检验、t检验以及方差分析进行统计学分析。 结果BMSC条件培养基能够抑制MCF-7细胞增殖,且随着作用时间的延长其抑制作用升高[24 h（13.42±1.93）%,48 h（16.83±1.77）%,72 h（20.21±2.01）,F = 6.271,P = 0.000]。BMSC条件培养基处理MCF-7细胞,可有效促进MCF-7细胞凋亡,且随着作用时间的延长细胞凋亡率升高[24 h（10.82±2.18）%,48 h（18.73±2.95）%,72 h（28.15±3.52）%,F = 9.215,P = 0.000]。与对照组裸鼠肿瘤体积[1 d（124.12±9.28）mm³,3 d（582.41±17.28） mm³,7 d（983.42±42.10） mm³,15 d（793.46±109.38） mm³]比较,BMSC组[1 d（132.61±12.41） mm³,3 d（378.46±23.08） mm³,7 d（542.61±58.49）mm³,15 d（329.48±47.51） mm³]、IL-12组[1 d（119.85±13.10） mm³,3 d（322.75±26.49） mm³,7 d（518.37±67.54）mm³,15 d（302.17±68.53） mm³]以及联合组[1 d（123.41±8.94）mm³,3 d（217.85±24.03）mm³,7 d（318.29±47.32） mm³,15 d（189.27±37.58）mm³]裸鼠肿瘤体积生长速度均减慢,差异具有统计学意义（F_1d=0.827,F_3d=37.583、F_7d=31.472、F_15d=15.372,P < 0.001）;处死各组裸鼠后裸鼠肿瘤质量比较,BMSC组[（529.42±98.74） mg]、IL-12组[（544.01±117.85）mg]以及联合组[（327.55±78.56） mg]均低于对照组[（877.42±120.11）mg],且联合组裸鼠肿瘤质量最低,差异具有统计学意义（F = 10.821,P < 0.001）;而裸鼠脾指数BMSC组（7.58±1.21）、IL-12组（7.63±1.09）以及联合组（10.03±1.52）均高于对照组（5.37±0.89）,联合组裸鼠脾质量最高,差异具有统计学意义（F = 13.411,P < 0.001）。 结论BMSC在体内外均具有抑制MCF-7肿瘤增殖作用,联合使用pcDNA6/IL-12对裸鼠MCF-7转移瘤抑制具有着明显的协同作用。     

IL-1beta and TNF-alpha induce increased expression of CCL28 by airway epithelial cells via an NFkappaB-dependent pathway

CCL28 is a mucosal chemokine that attracts eosinophils and T cells via the receptors CCR3 and CCR10. Consequently, it is a candidate mediator of the pathology associated with asthma. This study examined constitutive and induced expression of CCL28 by A549 human airway epithelial-like cells. Real-time RT-PCR and ELISA of cultured cells and supernatants revealed constitutive levels of CCL28 expression to be low, whereas IL-1beta and TNF-alpha, induced significantly increased expression. Observations from induced sputum and human airway biopsies supported this. Signal transduction studies revealed that IL-1beta and TNF-alpha stimulation induced NFkappaB phosphorylation in A549 cells, but antagonist inhibition of NFkappaB p50-p65 phosphorylation correlated with marked reduction of IL-1beta or TNF-alpha induced CCL28 expression. Together these studies imply a role for CCL28 in the orchestration of airway inflammation, and suggest that CCL28 is one link between microbial insult and the exacerbation of pathologies such as asthma, through an NFkappaB-dependent mechanism.     

Calcitonin gene-related peptide immunoreactivity in airway epithelial cells of the human fetus and infant

Summary Calcitonin gene-related peptide-immunoreactive cells were identified within the epithelium of distal conducting airways in the human fetus and infant. Several peptides and amines, including calcitonin, have been identified previously within a specific population of airway epithelial cells. These cells, referred to as pulmonary neuroendocrine cells, are postulated to be airway chemoreceptors responsible for changes in ventilation and perfusion in response to changes in airway gas composition. Calcitonin gene-related peptide immunoreactive cells could be identified throughout the period of development studies (20 weeks gestation to 3 months of age), but were present in only limited numbers in less than 50% of individuals (n=23). In contrast, large numbers of calcitonin gene-related peptide immunoreactive cells were identified in 100% of infants (1–3 months, n=5) with bronchopulmonary dysplasia. The differential processing of mRNA transcribed from the calcitonin gene in neural and non-neural tissue suggests that calcitonin, rather than calcitonin gene-related peptide, is the primary product of translation in pulmonary neuroendocrine cells. However, considering the potent vasodilatory and bronchoconstrictive effects of calcitonin gene-related peptide, its presence in pulmonary neuroendocrine cells, even in small amounts, may be important in controlling pulmonary vaso- and/or bronchomotor tone. The presence of large numbers of calcitonin gene-related peptide immunoreactive cells in infants with bronchopulmonary dysplasia suggests that calcitonin gene-related peptide may be one further agent contributing to the pulmonary pathophysiology seen in this disease.     

Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction

Bone marrow comprises heterogeneous cell populations, of which certain progenitors have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. This study demonstrates the bone marrow stromal cells (BMSCs) with intrinsic plasticity to differentiate into hepatocyte-like phenotypes under in vitro induction of hepatocyte growth factor (HGF). BMSCs isolated from rat femurs and tibias were cultured and passaged 3-4 times in the presence of HGF. Cells were harvested on days 0, 10, and 20 and subjected to examination of any hepatocyte characteristics by flow cytometry, RT-PCR, Western blot, and immunocytochemistry. Expression of albumin and alpha-fetoprotein at both mRNA and protein levels was detectable on day 10. By contrast, c-Met mRNA was significantly decreased in BMSC in the course of HGF induction. Here BMSC was shown to differentiate into hepatocyte-like phenotypes given the HGF induction, as an alternative source for adult stem cell transplantation in liver repair.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

Noncanonical Wnt signaling in stromal cells regulates B-lymphogenesis through interleukin-7 expression

The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed Wnt signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by Wnt signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) 12 are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of Wnt signaling in regulating IL-7 and CXCL12 expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL12 mRNA was augmented by noncanonical Wnt5a. When mouse BM-derived cells were cultured on Wnt5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that Wnt5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical Wnt regulates B-lymphogenesis via IL-7 expression in stromal cells.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

Activation of bone marrow cells treated with Canova in vitro

Canova is a Brazilian complex homeopathic medication produced from Aconitum, Thuya, Bryonia, Lachesis and Arsenicum. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix, and progenitor cells that differentiate into mature blood cells. As it is the major site of blood cell formation, we studied in vitro Canova effects on bone marrow cells of mice. Swiss mouse femurs were dissected, cleaned, and the marrow was flushed. The cells were plated, treated or not, incubated for different times and processed for light, scanning electron, and confocal microscopy, and also flow cytometry. The treatment did not modify the expression of the analyzed surface markers or cytokine production. All microscopy techniques showed that a monocytic lineage (CD11b(+)) and stromal cells (adherent cells) were activated by treatment. Canova also increased cell clusters over adherent cells, suggesting proliferation areas.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship