Particulate endocytosis mediates biological responses of human mesenchymal stem cells to titanium wear debris.

Continual loading and articulation cycles undergone by metallic (e.g., titanium) alloy arthroplasty prostheses lead to liberation of a large number of metallic debris particulates, which have long been implicated as a primary cause of periprosthetic osteolysis and postarthroplasty aseptic implant loosening. Long-term stability of total joint replacement prostheses relies on proper integration between implant biomaterial and osseous tissue, and factors that interfere with this integration are likely to cause osteolysis. Because multipotent mesenchymal stem cells (MSCs) located adjacent to the implant have an osteoprogenitor function and are critical contributors to osseous tissue integrity, when their functions or activities are compromised, osteolysis will most likely occur. To date, it is not certain or sufficiently confirmed whether MSCs endocytose titanium particles, and if so, whether particulate endocytosis has any effect on cellular responses to wear debris. This study seeks to clarify the phenomenon of titanium endocytosis by human MSCs (hMSCs), and investigates the influence of endocytosis on their activities. hMSCs incubated with commercially pure titanium particles exhibited internalized particles, as observed by scanning electron microscopy and confocal laser scanning microscopy, with time-dependent reduction in the number of extracellular particles. Particulate endocytosis was associated with reduced rates of cellular proliferation and cell-substrate adhesion, suppressed osteogenic differentiation, and increased rate of apoptosis. These cellular effects of exposure to titanium particles were reduced when endocytosis was inhibited by treatment with cytochalasin D, and no significant effect was seen when hMSCs were treated only with conditioned medium obtained from particulate-treated cells. These findings strongly suggest that the biological responses of hMSCs to wear debris are triggered primarily by the direct endocytosis of titanium particulates, and not mediated by secreted soluble factors. In this manner, therapeutical approaches that suppress particle endocytosis could reduce the bioreactivity of hMSCs to particulates, and enhance long-term orthopedic implant prognosis by minimizing wear-debris periprosthethic osteolysis.     

直肠癌切除前后腹腔冲洗液脱落细胞学对比研究

目的研究直肠癌术中腹腔多次冲洗，对于降低患者腹腔内脱落癌细胞阳性率的临床意义。方法对63例直肠癌患者术中腹腔冲洗分6次进行，肿瘤切除前3次，切除后3次；将6次腹腔冲洗液行常规病理学涂片细胞学检查结果进行对比。结果肿瘤切除前，全组患者腹腔冲洗液脱落癌细胞均呈阳性表达。肿瘤切除后第1次腹腔冲洗液脱落癌细胞阳性40例，第2次阳性33例，第3次阳性13例。肿瘤切除后第1次冲洗液的脱落癌细胞阳性结果与切除后第2次冲洗液阳性结果比较，P〉0.05，差异无统计学意义；但第3次冲洗液的脱落癌细胞阳性结果与第2次比较，P〈0.01，差异有统计学意义。结论对直肠癌患者进行术中多次腹腔冲洗可降低腹腔内脱落癌细胞的阳性率。     

巯基化合物对内毒素诱导大鼠枯否细胞NF—kB活性和肿瘤坏死因子释放的影响

目的 观察巯基化合物对内毒素诱导大鼠枯否细胞（KC）NF-kB活性和肿瘤坏死因子-a（TNF-a）释放的影响。方法采用胶原酶灌注和percoll密度梯度离心法分离得到高纯度大鼠KC，在过夜培养后冲洗两遍（不含血清），加入内毒素（LPS）10ng／ml刺激原代培养的KC，观察不同浓度谷胱甘肽乙基酯（GSHEE）及N-乙酰半胱氨酸（NAC）对大鼠KCTNF-a释放和KC内谷胱甘肽（GSH）及NF-kB活性的影响。并观察使用谷胱甘肽合成抑制剂BSO后，对NAC抑制炎症因子的影响，采用凝胶滞留电泳法测NF-kB活性，采用高效液相色谱分析法测GSH水平。结果 LPS诱导KC中GSH水平无变化；GSHEE和NAC可提高KC中GSH水平（P〈0．05），降低NF-kB活性和TNF-a释放（P〈0．05）。NAC与BSO合用时KC中GSH水平无变化，TNF-a释放降低（P〈0．05）。结论 NAC通过抑制KC中NF-kB的激活和TNF-a的释放调节KC功能，但这种抑制作用并非通过增加GSH介导。     

Endometrial cancer patients have a significant risk of harboring isolated tumor cells in histologically negative lymph nodes

In this study, we examine the prevalence of finding isolated tumor cells (ITCs) in negative lymph nodes of endometrial cancer patients using immunohistochemistry. Seventy-six endometrial cancer patients with lymph nodes histologically negative for metastatic disease were examined. Nodal tissue sections were stained with anticytokeratin antibodies AE-1 and CAM 5.2. Nodes with single or groups of cells (two to four cells) < or =0.2 mm and showing cytokeratin reactivity were positive for ITCs. Findings were compared to features of the primary tumor and patient outcome. ITCs were present in 31 of 1712 lymph nodes. Fifteen (19.7%) patients had ITC-positive nodes. ITCs involved only pelvic nodes in nine cases, only para-aortic nodes in five cases, and pelvic and para-aortic in one case. Tumor in adnexa was the only pathologic feature associated with nodal ITCs (P= 0.0485). All 15 patients with nodal ITCs were alive at follow-up. One (6.7%) patient suffered recurrent disease but was alive at last encounter. Disease recurred in 5 (8.8%) of 57 patients without nodal ITCs. Two are alive without disease, two alive with disease, and one died from her cancer. In summary, a significant proportion of endometrial cancer patients have ITCs detected by immunohistochemistry in histologically negative regional lymph nodes.     

扩张型心肌病家兔左室壁复极异质性的改变及意义

目的：观察离体心脏左心室三层心肌的单相动作电位的改变，以探讨扩张型心肌病易发心室颤动与三层心肌跨室壁复极不均一性的关系。方法：用阿霉素制作扩张型心肌病家兔模型，测定其室颤阈值（VFT）以及心外膜、中层心肌和心内膜心肌细胞的单相动作电位复极90％时程（APD90）、跨室壁复极离散度（TDR）。结果：扩张型心肌病VFT明显降低（P＜0．001），三层心肌细胞APD90均明显延长（P＜0．001），中层心肌细胞较心外膜、心内膜下心肌细胞延长更为显著（P＜0．05）；扩张型心肌病跨室壁复极离散度增加（P＜0．01）。结论：中层心肌细胞APD90明显延长、跨室壁复极离散度增加、三层心肌复极不均一性增加可能是扩张型心肌病容易发生心室颤动的重要原因。     

Effect of UVB 311 nm irradiation on normal human skin

Ultraviolet radiation B (UVB) on the skin induces erythema, inflammation and modifications of the immune system. These changes have been reported after excessive short-term or long-term exposure to broad spectrum UVB. In this study, we examined the effects of local repetitive UVB irradiation of 311 nm wavelength on the skin of seven young volunteers. Skin biopsies were taken before and after UVB irradiation, and we immunohistochemically analyzed the expression of CD1a and HLA-DR antigens of Langerhans cells (LC), the possible infiltration of dermis/epidermis by CD11b macrophages, the modifications or the induction of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) involved in the binding of leukocytes to the endothelial surface and the development of perivascular infiltrates of LFA-1⁺ mononuclear cells. We also determined the expression of substance P receptors (SPR) using biotinylated substance P (SPB). Exposure of UVB 311 nm induced a drastic reduction of CD1a⁺ cells and a moderate increase of HLA-DR⁺ dendritic cells in the epidermis without infiltration by CD11b macrophages. An increase of the binding of SPB to upper layer epidermal cells was noted in five of seven biopsies. In the dermis, vessel-associated ICAM-1 expression increased and an induction of E-selectin occurred on nearly 20 to 40% of endothelial cells, but VCAM-1 expression remained undetectable. The percentage of LFA-1⁺ cells did not change significantly after irradiation. These observations may be compatible with a selective role of UVB 311 nm on the skin immune response.     

Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells

Summary Cytochemical staining of normal human bone cells in monolayer cultures for alkaline phosphatase (ALP) indicated that the cultures contained mixed-cell populations. Time course evaluations of the cytochemical staining revealed, in addition to the ALP-negative cell population, at least two subpopulations of ALP-positive human bone cells with different levels of ALP. A cytochemical method has been developed which separates the ALP-positive cells into high and intermediate ALP subpopulations. In this method, human bone cells were stained for ALP using an azo-dye method and incubating at 4°C for 10 and 30 minutes, respectively. We defined the cell population that stained positively for ALP at 10 minutes as strong ALP-positive cells, and both strong and intermediate cells were stained at 30 minutes. The intermediate cells were determined from the difference between the values at the two time points. The intra- and interassay variations of the assay, with the same investigator in blinded investigations, were both less than 10% and the interobserver variation was approximately 25%. Analysis of the distribution of ALP levels in cells with a laser densitometer confirmed the presence of at least three cell subpopulations. 1,25(OH)₂D₃ treatment increased the proportions of both ALP-positive cell populations, whereas TGF-beta treatment increased only the intermediate ALP-positive cell population. On the contrary, fluoride increased the proportion of the strong ALP cells, and IGF-1 had no effect on the proportions of either ALP-positive subpopulation. When the ALP-specific activity was compared with the percentage of each ALP-positive subpopulations for the cells treated with effectors, the ALP-specific activity correlated with the total ALP-positive and with the strong ALP-positive populations but not with the intermediate ALP-positive subpopulation. In summary, this study represents the first evidence that normal human bone cells in monolayer cultures contained at least two subpopulations of ALP-positive cells, and that bone cell effectors could have differential effects on each cell population.     

Serotonin-induced increase in cAMP in ganglia isolated from the myenteric plexus of the guinea pig small intestine: Mediation by a novel 5-HT receptor

Serotonin (5-HT) is a mediator (through 5-HT_1P receptors) of slow EPSPs in myenteric ganglia of the small intestine. The effect of 5-HT can be mimicked by elevating cAMP; therefore, we tested the hypothesis that the slow EPSP-like response to 5-HT is cAMP-mediated. Guinea pig gut was enzymatically dissociated; myenteric ganglia remained intact and were collected by filtration. Neurons in the isolated ganglia retained their ability to manifest the slow EPSP-like response to 5-HT. Exposure to 5-HT raised the ganglionic level of cAMP (ED₅₀ 0.3 μM). This effect was not antagonized by the 5-HT_1P antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (100.0 μM), or mimicked by the 5-HT_1P agonist, 5-hydroxyindalpine (10.0 μM). Increases in cAMP were also evoked by the 5-HT₁ agonist, 5-carboxyamidotryptamine (10.0 μM), the 5-HT₂ agonist, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 1.0–10.0 μM), and by the 5-HT₄ agonists, renzapride (1.0–10.0 μM) and 5-methoxytryptamine (1.0–10.0 μM); however, neither the 5-HT₁/5-HT₂ antagonists, spiperone, methysergide, and methiothepin, nor the 5-HT₄ antagonist, tropisetron (ICS 205–930; 10.0 μM), were able to inhibit the rise in cAMP evoked by these compounds or by 5-HT (0.1–10.0 μM). The 5-HT-evoked elevation of cAMP was antagonized by ketanserin (10.0 μM), which also blocked the effects of 5-methoxytryptamine and DOI, but not those of renzapride. The effective concentration of DOI, however, was higher than that needed for activation of 5-HT₂ receptors, and Northern analysis using a cDNA probe encoding the rat 5-HT₂ receptor failed to reveal the presence of 5-HT₂ mRNA in myenteric ganglia, although it hybridizes with mRNA of the right size in the guinea pig brain. Compounds that failed to change levels of cAMP or to antagonize the action of 5-HT included 8-hydroxy-di-n-propylamino tetralin, R58639, R88226, and sumatriptan. It is concluded that the receptor responsible for the 5-HT-induced rise in cAMP in ganglia isolated from the guinea pig myenteric plexus is not a known subtype of 5-HT receptor. Since the pharmacology of this novel receptor is different from that of the slow EPSP-like response to 5-HT, the receptor probably does not mediate the slow EPSP. © 1993 Wiley-Liss, Inc.     

A HIGH FREQUENCY OF INHERITED DEFICIENCY OF COMPLEMENT COMPONENT C4 IN DARWIN ABORIGINES

Abstract A high frequency of serum complement component C4A deficiency may explain the higher prevalence and greater severity of systemic lupus erythematosus reported in Australian Aborigines. Inherited deficiencies of serum complement components C4A, C4B, and C2 were examined in two Australian Aboriginal populations from Darwin and Alice Springs and compared with the prevalence of complement deficiencies in white Australian blood donors. The frequency of C4A deficiency alleles was 29% in Darwin Aborigines compared with 12% in Alice Springs and 17% in Canberra blood donors. Partial C4B deficiency was also higher in Darwin Aborigines than in the other populations. Inherited deficiency of serum complement component C2 was not observed.