人骨髓间充质干细胞分离培养及血管内皮生长因子对其产生的诱导分化作用

目的：目前有关骨髓间充质干细胞向内皮细胞诱导分化的研究较少。本实验分离和培养人骨髓间充质干细胞，用带有VEGF165的质粒转染人骨髓间充质干细胞，探讨血管内皮生长因子对其体外诱导分化的作用。 方法：实验于2005—04／2006—04在吉林大学人兽共患病教育部重点实验室完成。取成人的已排除血液系统肿瘤疾病的新鲜骨髓（自愿提供），采用Percoll梯度分离培养骨髓间充质干细胞，于倒置显微镜下观察细胞形态变化和生长情况。原代细胞培养至增殖接近融合状态时，单克隆培养法分离传代培养，扩增骨髓间充质干细胞。采用流式细胞术检测细胞免疫学表型。在原核细胞大肠杆菌DH5α中复制扩增和提取，纯化、克隆pcDNA3．0-VEGF165质粒。用脂质体转染法转染骨髓间充质干细胞：应用流式细胞术检测诱导后骨髓间充质干细胞免疫学表型变化j并采用免疫荧光染色鉴定转染情况，并设质粒空载和未转染的骨髓间充质干细胞为对照。 结果：人骨髓间充质干细胞原代培养1周后，造血细胞消失，贴壁细胞体积增大，呈现梭形外观，有粗大的细胞突起伸出。2周后细胞融合成单层，梭形突起变长，排列有明显的方向性，细胞排列成旋涡状、网状、辐射状。流式细胞术显示，人骨髓间充质干细胞免疫学表型CD44、CD29阳性，CD34、CD31、CD45阴性。VEGF165诱导骨髓间充质千细胞后CD44表达明显降低，CD31明显升高。免疫荧光染色显示，用FITC标记后的VEGF抗体使细胞显现绿色荧光，用cy3标记的CD31抗体使细胞显现了红色荧光。 结论：转染后的骨髓间充质干细胞细胞表型发生明显转变，CD31表达率明显增高，呈现典型的内皮细胞的表型特征，这说明骨髓间充质干细胞具有向内皮细胞分化的潜能。     

Microlaryngeal surgery on a day case basis

The aim of this study was to assess the clinical safety of performing microlaryngeal surgery (MLS) under general anaesthesia in selected patients in the ambulatory setting. Twenty-two adult patients were scheduled to have tissue specimens of the larynx taken by biopsy (54%), for vocal cord polypectomy (41%) or for vocal cord cyst excision (5%). Twenty-one ASA I and II patients (95%) were discharged home the same day of the procedure. Two of them presented with laryngospasm after extubation of the trachea. One ASA III patient (5%) had to be admitted overnight because of severe laryngospasm and bronchospasm, but was discharged the day after the operation. None of the patients had significant complications after leaving the recovery room (mean stay 85 min). There were no re-admissions to the hospital. Our data suggests that microlaryngeal surgery in selected patients can be safely performed on a day case basis.     

Polycythemia is associated with bone loss and reduced osteoblast activity in mice

Summary

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO).

Introduction

PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO.

Methods

Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2^V617F knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO.

Results

Compared to wt, both JAK2^V617F and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P?<?0.01) suggesting a more severe bone phenotype than JAK2^V617F. Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice.

Conclusions

This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

     

Intra-rater Reliability of a Qualitative Landing Scale for the Single-Hop Test: A Pilot Study

BackgroundThe test battery classically used for return-to-sport (RTS) decision-making after anterior cruciate ligament (ACL) reconstruction (ACLR) may not be sufficient, as it does not include a qualitative analysis of movement. Therefore, the Landing Error Scoring System (LESS) scale was adapted to a primary functional test in the typical RTS test battery: the single leg hop for distance (SHD).Hypothesis/ PurposeThe aim of this study was to determine the intra-rater reliability of the LESS scale adapted to the SHD (SHD-LESS scale) in healthy young athletes.Study DesignReliability analysisMethodsNineteen healthy individuals (14 men, 5 women; mean age: 22.4 years) participated in the study. Participants performed the SHD tasks on both limbs (dominant and non-dominant) using a standardized protocol in two sessions that were one week apart (single reviewer; 2-dimensional video). Intra-class correlation coefficients (ICC2,1) were used to measure the reproducibility of the scale in the dominant (dom) and non-dominant (nondom) limbs. Additionally, limb data (dom and nondom) were pooled and evaluated collectively with intra-class correlation coefficients. The Kappa coefficient was used to assess the reproducibility of each individual item of SHD-LESS scale.ResultsThe intra-rater reliability was good (ICCdom = 0.77; ICCnondom = 0.87; ICCpooled = 0.87) for the overall SHD-LESS scale scores. Agreement of SHD-LESS individual items ranged from 62% to 100%. Dorsiflexion at initial contact (97% agreement; kappa value=0.79) and knee valgus after landing (88% agreement; kappa value=0.65) had excellent agreement and kappa values.ConclusionThe newly-adapted SHD-LESS scale showed good intra-rater reliability overall. Further studies should evaluate the impact of using the SHD-LESS scale within the RTS test battery on outcomes in patients after ACLR.Level of Evidence3     

Social norms and obesity prevalence: From cohort to system dynamics models

Group‐level obesity can be seen as an emergent property of a complex system, consisting of feedback loops between individual body weight perception, individual weight‐related behaviour and group‐level social norms (a product of group‐level ‘normal' body mass index (BMI) and sociocultural ‘ideal' BMI). As overweight becomes normal, the norm might be counteracting health awareness in shaping individual weight‐related behaviour. System dynamics modelling facilitates understanding and simulating this system's emergent behaviour. We constructed six system dynamics models (SDMs) based on an expert‐informed causal loop diagram and data from six sociocultural groups (Dutch, Moroccan and South‐Asian Surinamese men and women). The SDMs served to explore the effect of three scenarios on group‐level BMI: ‘what if' weight‐related behaviour were driven by (1) health awareness, (2) norms or (3) a combination of the two. Median BMI decreased approximately 50% and 30% less in scenarios 2 and 3, respectively, than in 1. In men, the drop in BMI was approximately two times larger in scenario 1 versus 3, whereas in women, the drop was approximately equal in these scenarios. This study indicates that the overweight norm in men holds group‐level BMI close to overweight despite health awareness. Since norms are counteracting health awareness less strongly in women, other drivers of obesity must be more relevant.     

Brain biopsy in the diagnosis of cerebral mycosis fungoides

A case of cerebral mycosis fungoides co-existing with progressive multifocal leucoencephalopathy presented with dementia. Brain biopsy established the diagnosis of mycosis fungoides after cerebrospinal fluid examinations and computerised tomographic scanning of the brain produced non-specific abnormalities.     

High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.     

Tuberculosis in Patients Infected with Human Immunodeficiency Virus 1. A Retrospective Multicentre Study of 123 Cases in France

SUMMARY In order to study the epidemiological, clinical, and progressivecharacteristics of TB in HIV-infected individuals, a retrospectivestudy was conducted in nine infectious disease centres of universityhospitals located in the southern half of France. Among the5730 HIV-seropositive in- and out-patients, 123 (2.1 per cent)had TB (121 infections caused by M. tuberculosis, 2 by M. bovis).Tuberculosis was pulmonary in 53 patients (43.1 per cent), extrapulmonaryin 36 patients (29.3 per cent), and combined in 34 patients(27.6 per cent). There was no statistically significant differenceamong these three locations as to the mean CD4 count/mm³ (160±17),the type of antituberculosis therapy, the length of treatment(10.8±0.6 months) and the outcome. Fifty-two (45.2 percent) patients received an initial antituberculosis therapeuticregimen of four drugs: isoniazid, rifampicin, ethambutol, pyrazinamide;54 (46.9 per cent) were started on three drugs: isoniazid, rifampicin,ethambutol; and nine (7.8 per cent) received a two-drug combination:isoniazid, rifampicin. Fourteen of 75 patients subsequentlyreceived secondary preventive therapy. The mean follow-up timewas 252±290 days. Clinical healing was obtained in 57.7per cent of patients. Forty-six patients died, 33 during treatment:23 from AIDS and eight from TB (in the first 3 weeks of treatment).Five patients suffered from relapses due to poor treatment compliance.Patients had a good prognosis if tuberculosis was diagnosedearly.     

Pre-B cells and other possible precursor lymphoid cell lines derived from patients with X-linked agammaglobulinemia

A group of unique Epstein-Barr virus-containing cell lines was derived from the bone marrow of three patients with X-linked agammaglobulinemia. Efforts to obtain cell lines from the peripheral blood of these patients were uniformly unsuccessful. Immunofluorescence analyses as well as biosynthetic studies with [(35)S]methionine indicated unusual patterns of Ig synthesis in many of these bone marrow derived lines. Seven of the lines were of particular interest in that two produced no Ig of any type; two others showed no Ig by fluorescence but small amounts by [(35)S]methionine labeling; one expressed only cytoplasmic μ chains without any evidence of light chain synthesis, and two produced primarily μ chains with only slight amounts of light chains. One of the lines without membrane or cytoplasmic Ig studied in detail grew like a typical lymphoid line and was carried in intermittent culture over a period of 2 yr without Ig expression. One line grew quite differently and resembled the round cell type described previously, which has been obtained from a variety of sources. The cell line with cytoplasmic μ chains and no light-chain expression had the characteristic properties of pre-B cells. Three normal type Ig-producing cell lines also were obtained from the patients. The accumulated evidence obtained in the present study indicates that these unusual cell lines represent normal precursor cells of the B-cell lineage; these grew out in these cases because of the virtual absence of mature B cells that ordinarily overgrow the culture system. However, the possibility that in certain instances they reflect abnormal Ig synthesis characteristic of the disease has not been ruled out.