Neuroblastoma cells provoke Schwann cell proliferation in vitro

BACKGROUND: A subset of human neuroblastomas (NBs) has the capacity to mature completely, imitating sympathetic ganglia. Previously, we showed that the neuronal population in spontaneously maturing NBs usually has a near-triploid DNA content without 1p deletions, and we concluded that the constantly diploid Schwann cells (SCs) do not belong to the neoplastic component of these tumours. We therefore hypothesised that NB cells are able to stimulate SC proliferation, and that SCs trigger NB differentiation. PROCEDURE: We performed in vitro experiments to test this model and to test whether SCs can also influence the growth of aggressive NBs. Human SCs were co-cultivated with NB tumours and cell lines, and were harvested after defined time intervals. Proliferative activity of the SCs and the NB cells was determined by visualisation of 5-bromo-2'-deoxyuridine (BrdU) incorporation or Ki-67 staining. Neurite outgrowth and neurofilament (NF) expression were analysed immunocytochemically and apoptotic rate was determined by a terminal deoxynucleotidyl transferase-mediated dUTP-X fluorescein nick end labelling (TUNEL) assay. RESULTS: Human NB tumours or cell lines unequivocally increased the proliferation of SCs in vitro. In cocultivated NB cells, the proliferative activity was not altered in the first days of cocultivation, although neurite outgrowth and NF expression were enhanced. However, after 10 days, the mitotic rate of neuroblastic cells decreased and the apoptotic rate showed a marked increase. CONCLUSIONS: The results of the cocultivation experiments provide an experimental hint that the in vivo growth of SCs in NBs is caused by the neoplastic neuroblasts, and they also indicate that cells from peripheral nerves can influence the growth of aggressive NB cells if cocultivated.     

Expression of neurotrophin receptor TrkA inhibits angiogenesis in neuroblastoma

BACKGROUND: Mechanisms regulating the expression of angiogenic factors in tumor cells are largely unknown. High expression of the neurotrophin receptor TrkA in neuroblastomas (NB) is associated with favorable prognosis, whereas TrkB is expressed on aggressive, MYCN-amplified NB. PROCEDURE: To investigate the biological effects of TrkA and TrkB expression on angiogenesis in NB, we examined the expression of angiogenic factors in the human NB cell line SY5Y and its TrkA and TrkB transfectants. RESULTS: In comparison to parental SY5Y cells, mRNA and protein levels of angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium (CM) of parental SY5Y and SY5Y-TrkB cells induced endothelial cell proliferation, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model, and was associated with reduced angiogenic factor expression and less vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay.     

脂肪间充质干细胞的体外诱导和成血管作用

Objective To study in vitro induction of adipose-derived mesenchymal stem cells (ADMSCs) into endothelial cells and blood vessel formation on three-dimensional (3D) media． Methods The 3rd passage ADMSCs were induced in vitro into endothelial cells on conditional medium and grew on Matrigel medium． The cell surface antigens CD31 and CD44 were detected with flow cytometric analysis before and after induction． HE staining, FⅧ-RAg immunohistochemical staining, inverted microscope and transmission electron microscope were used for morphological study.Results The expression of CD44 was positive and CD31 negative in ADMSCs in flow cytometric analysis.After induction,CD31 became positive while CD44 was negative． Paving-stone-like cell appearance was seen under inverted microscope 14 days after induction.The cells were FⅧ-RAg positively stained with immunohistological method,and Weibel-Palade body was observed under transmission electron microscope.On Matrigel media,the induced cells migrated in lumping with pseudopodia protrusion after 24 hours,and formed grid structure on the 7th day.Long vasculature was observed on the 13th day,and the vessels branched on the 20th to 30th day.The vessels stained positive for FⅧ-Rag． Conclusions ADMSCs have the potential to give rise to endothelial cells with in vitro induction and to form vessel structure in 3D media.ADMSCs have potential role in vascularized tissue engineering grafting．     

脂肪间充质干细胞的体外诱导和成血管作用

目的 研究在体外诱导脂肪间充质干细胞(ADMSCs)向内皮细胞分化、在立体培养基上的血管形成情况.方法 将传至第三代的ADMSCs用内皮细胞诱导液、Matrigel三维培养基进行诱导培养,对ADMSCs和诱导细胞选用CD31、CD44细胞表面抗原在流式细胞仪检测表达情况,用HE染色、FⅧ-RAg免疫组织化学染色及倒置显微镜、透射电镜等进行观察鉴定.结果流式细胞仪上检测ADMSCs的表达为CD44阳性、CD31阴性,诱导的细胞CD31阳性、CD44阴性.诱导细胞14d倒置显微镜下呈铺路石样形态,FⅧ-RAg染色细胞呈阳性,透射电镜下在细胞浆内见到内皮细胞特有标志物Weibel-Palade小体。ADMSCs在Matrigel三维培养基上诱导,24h细胞迁徙成团、有伪足伸出,诱导7d伸出的细胞形成交叉网格状,13d形成较长血管,20d较长血管粗而多、出现小分叉,30d长血管、分叉血管变粗厚;FⅧ-RAg染色后诱导血管亦呈阳性.结论 脂肪间充质干细胞在体外经诱导能向内皮细胞分化、能形成血管,可作为促进组织工程移植物血管化的良好的种子细胞.

Abstract：

Objective To study in vitro induction of adipose-derived mesenchymal stem cells (ADMSCs) into endothelial cells and blood vessel formation on three-dimensional (3D) media． Methods The 3rd passage ADMSCs were induced in vitro into endothelial cells on conditional medium and grew on Matrigel medium． The cell surface antigens CD31 and CD44 were detected with flow cytometric analysis before and after induction． HE staining, FⅧ-RAg immunohistochemical staining, inverted microscope and transmission electron microscope were used for morphological study.Results The expression of CD44 was positive and CD31 negative in ADMSCs in flow cytometric analysis.After induction,CD31 became positive while CD44 was negative． Paving-stone-like cell appearance was seen under inverted microscope 14 days after induction.The cells were FⅧ-RAg positively stained with immunohistological method,and Weibel-Palade body was observed under transmission electron microscope.On Matrigel media,the induced cells migrated in lumping with pseudopodia protrusion after 24 hours,and formed grid structure on the 7th day.Long vasculature was observed on the 13th day,and the vessels branched on the 20th to 30th day.The vessels stained positive for FⅧ-Rag． Conclusions ADMSCs have the potential to give rise to endothelial cells with in vitro induction and to form vessel structure in 3D media.ADMSCs have potential role in vascularized tissue engineering grafting．     

Retroviral vector-producer cell mediated angiogenesis inhibition restricts neuroblastoma growth in vivo

BACKGROUND: The purpose of this study was to determine whether gene therapy-mediated delivery of an angiogenesis inhibitor, a truncated, soluble vascular endothelial growth factor receptor (Flk-1/KDR, VEGFR-2), could suppress tumor growth in a murine model of neuroblastoma. METHODS: Murine fibroblasts producing a replication-defective retrovirus encoding this mutant form of flk-1 were made. These producer cells were mixed with neuroblastoma cells and injected subcutaneously into SCID mice. Subsequent tumor growth was then measured. RESULTS: Murine neuroblastoma growth was decreased by 95% after 25 days. Similar tumor growth inhibitory effects were observed when the flk-1 producer cells were co-injected with cells from two different human neuroblastoma cell lines. CONCLUSIONS: Neuroblastoma growth can be significantly restricted in vivo with a single injection of cells that produce a retroviral vector encoding the gene for an angiogenesis inhibitor. This suggests that gene therapy-mediated delivery can be an effective alternative to chronic administration of these cytostatic anticancer agents.     

The somatostatin analogue octreotide inhibits neuroblastoma growth in vivo.

Neuroblastoma, a neural crest-derived childhood tumor of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. However, the majority of neuroblastomas are diagnosed as metastatic tumors with a poor prognosis despite intensive multimodal therapy. The neuropeptide somatostatin (SOM) has been shown to inhibit neuroblastoma growth and induce apoptosis in vitro. Therapeutic effects of SOM analogues are dependent on tumor expression of high-affinity receptors. In the present study, human neuroblastoma SH-SY5Y cells were grown as xenografts in nude rats. In vivo SOM receptor expression in the xenografts was identified using scintigraphy with 111In-pentetreotide. Rats were randomized to treatment with the long-acting SOM analogue octreotide (10 microg s.c. every 12 h), 13-cis-retinoic acid (4 mg orally every 24 h), or vasoactive intestinal peptide (40 microg s.c. every 24 h) and compared with controls. Tumor volume was assessed every second day and tumor weight after 10-12 d. Octreotide treatment inhibited neuroblastoma growth significantly with reduced tumor volumes at 10 and 12 d compared with untreated controls (mean 3.56 and 4.24 versus 6.48 and 8.01 mL, respectively; p < 0.01). Also, tumor weights after 10-12 d were reduced in octreotide-treated animals (n = 8, median weight 2.90 g, range 1.67-5.57 g) compared with untreated rats (n = 14, 7.54 g, 1.65-10.82 g, p = 0.005). Serum IGF-I decreased significantly over time both in rats treated with octreotide and in untreated controls. It is concluded that treatment with the SOM analogue octreotide may significantly decrease neuroblastoma tumor growth in vivo. Further studies are warranted to establish the role of SOM analogues in the treatment of children with unfavorable neuroblastoma.     

Allergy testing: in vivo versus in vitro

Table 4 briefly summarizes the relative advantages and disadvantages of skin tests versus in vitro tests for detecting allergen-specific IgE. The skin test remains unexcelled as a sensitive and cost efficient test for specific IgE. The high degree of skin test sensitivity is very important when a patient must be evaluated for potentially life-threatening allergies such as to penicillin or stinging insects. The results of both skin tests and in vitro assays depend very much on the quality of the allergen extracts used for the tests. Although the quality of extracts is improving, there is still little standardization. Both skin tests and in vitro assays are difficult to quality control. Practicing allergists rely on experience, and the correlation between patient histories and skin tests results for quality control of the results. Although this system suffices for common allergens, the results for uncommon allergens may be misleading. Quality control is also difficult for in vitro tests. Participation in quality control programs, such as that being offered by the College of American Pathologists, will increase and lead to better quality and standardization of in vitro test results. At the present time, properly performed skin tests are the best available method for detecting the presence of allergen specific IgE. They are rapid, sensitive, and inexpensive on a per test basis. In vitro tests are acceptable substitutes for skin tests in some circumstances. If the patient does not have normal skin, cannot discontinue interfering medications, or is so sensitive by history that anaphylaxis seems possible, in vitro tests are preferred. In vitro tests are better when it is necessary to test a difficult patient such as a combative, mentally retarded adult. In vitro tests also have been invaluable in many allergy research studies. Physicians must remember that positive tests for allergen-specific IgE do not diagnose allergy. They only indicate the presence of IgE molecules with a particular immunologic specificity. A decision whether the specific IgE molecules are responsible for clinically apparent disease must be made by a well-trained physician. The ultimate standard for the diagnosis of allergic disease remains the combination of: (1) positive double-blind challenge, (2) the presence of specific IgE, and (3) demonstration that the symptoms are the result of IgE-mediated inflammation.     

Inflammation inhibits muscarinic signaling in in vivo canine colonic circular smooth muscle cells

We investigated the effects of experimental colitis on the muscarinic signaling properties and contractile behavior of canine colonic circular smooth muscle. The hypotheses that inflammation 1) inhibits in vivo muscarinic receptor mediated contractions, and 2) alters receptor density or receptor-binding affinities were tested. Muscarine was infused close-intra-arterially in seven conscious dogs during normal and experimental colitis states. Colonic circular muscle contractions were recorded via surgically attached strain gauge transducers. Muscarine stimulated phasic contractions in a dose-dependent manner, whereas colitis was inhibited. The inhibitory concentration 50% dose of M(3) receptor inhibitor was several times lower than that of M(1), M(2), and M(4) inhibitors during normal and colitis. However, inflammation induced a significant leftward shift in the circular muscle inhibitory dose-response curve of M(2) inhibitor. Muscarinic receptor density and binding analyses in isolated circular muscle cells was done in normal and colitis states. Inflammation significantly decreased maximum binding from 4082 fmol/mg to 2708 fmol/mg, whereas affinity constant remained unaffected. The conclusions were that 1) spontaneous and muscarine-activated in vivo phasic contractile activity of colonic circular muscle cells is primarily mediated by M(3) receptors; 2) inflammation was associated with a shift in M(2) receptor potency, due chiefly to a decrease in receptor density; and 3) this inhibitory effect was seen in normal and inflamed states, suggesting the importance of M(2) receptor. These findings suggest that changes in muscarinic response during colitis may contribute to the abnormal motility seen with inflammatory bowel disease.     

In vitro and in vivo effects of erythrocyte phototherapy on newborns

The photodynamic action of the bilirubin is associated with severe consequences observed during 'in vitro' irradiation of the erythrocytes. This paper is designed to evaluate the bilirubin photodynamic effects which occur 'in vitro' and 'in vivo' on erythrocytes in healthy and jaundiced infants. The in vitro bilirubin sensitized photoreaction damages the erythrocytes mainly at the membrane level. In particular, a dramatic decrease of ATPase activity and an increased susceptibility to lipid peroxidation, expressed as malondialdehyde production, were observed. For in vivo studies, specific fluorescent probes have been used to verify probable changes on the functional architecture of the erythrocyte membrane in the phototherapy-treated infants. Our results showed that specific areas of the membrane are differently affected, mainly at lipid/protein interface. Although the role of the erythrocyte membrane is an important factor of the hemorheological behavior, the measurement of blood viscosity and erythrocyte aggregation and filtration did not show significant alterations during the overall time of phototherapy.     

Hypoxia inhibits proliferation of fetal pulmonary arterial smooth muscle cells in vitro

Normal pulmonary arterial development in the relatively hypoxic intrauterine environment and pulmonary arterial remodeling in hypoxic infants include extension of the smooth muscle layer into normally nonmuscular arteries and thickening of the arterial media in the muscular arteries. These changes require proliferation of immature smooth muscle cells or differentiation of smooth muscle cell precursors. Because the mechanisms that regulate these processes have not been clearly defined, we asked whether decreased oxygen tensions could promote either hyperplasia or hypertrophy of smooth muscle cell precursors in vitro. We have studied cells that proliferate and migrate out of explants from the media of the pulmonary arteries of near-term bovine fetuses, because these cells are representative of those that are involved in normal arterial development and possibly also in arterial remodeling. Decreases in oxygen tension within and below the physiologic range do not cause hyperplasia or hypertrophy of these cells. Instead, cell proliferation decreased at oxygen tensions below 60 mm Hg. The effects of hypoxia on proliferation of aortic and pulmonary arterial smooth muscle cells were identical, but effects on proliferation of dermal fibroblasts and endothelial cells were smaller in magnitude and evident only at lower oxygen tensions. These findings suggest that hypoxia does not act directly on smooth muscle cells to produce increased quantities of these cells in the pulmonary arteries during normal prenatal development or during remodeling of the pulmonary arteries of the hypoxic neonate, implying that other factors mediate these phenomena.     

Double-blind, placebo-controlled Alternaria alternata immunotherapy: in vivo and in vitro parameters.

Few studies have been published on the efficacy and safety of immunotherapy with fungal extracts, possibly because of difficulties arising from antigenic variability among different strains of fungus. The aim of the study was to analyze changes in the in vivo and in vitro parameters in response to immunotherapy with an Alternaria alternata extract. We studied 28 patients with rhinitis, bronchial asthma, or both caused by Alternaria. The patients were randomized to the active immunotherapy or placebo group, and a conventional schedule of immunotherapy was used. We recorded changes for a year in skin reactivity (skin prick test), conjunctival reactivity (conjunctival provocation test), and in vitro parameters (serum-specific IgE, IgG, IgG1 and IgG4 for A. alternata complete extract and for natural and recombinant Alt a 1). Twenty-three patients completed the study and all attained the maintenance dose. There were no changes in skin reactivity in the active treatment group, and reactivity increased at the end of the study period in the placebo group. Conjunctival sensitivity decreased only in the active treatment group when the maintenance dose was reached. Allergen-specific IgE decreased, and IgG, IgG1 and IgG4 increased in all periods of study in the active treatment group, with no changes in the placebo group. Allergen-specific immunotherapy with the A. alternata extract tested here led to a decrease in conjunctival reactivity and induced a significant immunologic response.     

In vivo and in vitro studies on the sonographical detection of Ascaris lumbricoides

Intestinal ultrasound, a frequently applied diagnostic tool in industrialized nations, has recently also been introduced in tropical regions. This study attempts to describe the anatomical and sonographical features of Ascaris lumbricoides in the human intestine. In the course of a schistosomiasis morbidity study in Madagascar, 581 inhabitants of a rice-farming village on the high plateau of the island had their stools examined by means of a modified Kato-Katz thick smear technique (four slides per sample); 53 % had eggs of Ascaris lumbricoides in their stools. Twenty-two individuals underwent intestinal ultrasound examination and, in six cases, Ascaris lumbricoides was visualized. All six patients showed eggs upon stool examination. At ultrasound, the parasite was seen as a large, curved echogenic strip (4–6 mm in diameter) with an inner, anechoic, longitudinal canal. The image resembled a winding highway, the central structure representing the pseudocoel of the parasite. Patients were treated with mebendazole. The excreted worms of one patient were scanned under water, showing the same characteristics as in vivo. We conclude that Ascaris lumbricoides has a characteristic sonographical appearance and should not be a confounding factor in studies using intestinal ultrasound. Received: 4 July 1996 Accepted: 19 August 1996     

In vitro and in vivo antimicrobial activity of topical ofloxacin and other ototopical agents

BACKGROUND: Ototopical agents are extensively used for otitis externa (OE), acute otitis media identified by otorrhea in patients who have tympanostomy tubes (AOM-TT) and chronic suppurative otitis media (CSOM). The quinolones have particular value as ototopical agents because of the broad spectrum of antibacterial activity of importance in otic diseases and the high concentrations of antibacterial activity at the site of infection. METHODS: A survey of literature on in vitro activity and microbiologic efficacy in clinical trials of quinolone otic products for OE, AOM-TT and CSOM. RESULTS: OE: Floxin otic and Cortisporin TC otic suspension were equally effective in eradicating the three major pathogens Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus aureus. CSOM: Ofloxacin otic was effective in an open label trial in uniform eradication of S. aureus, P. aeruginosa, Proteus mirabilis and Enterobacter spp. AOM-TT: Ofloxacin otic and amoxicillin/clavulanate (by mouth) were equivalent clinically; rates of eradication of initial pathogens were similar for Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis, but ofloxacin otic was superior in eradication of S. aureus and P. aeruginosa CONCLUSIONS: In each of the studies of OE, CSOM and AOM-TT, ofloxacin otic solution was effective in eradicating the bacterial pathogen from the site of infection: equivalent to Cortisporin for children with OE; superior to amoxicillin/clavulanate for patients with AOM-TT who had acute drainage; and effective in eradicating bacterial pathogens from the external canal of patients with CSOM.     

Experimental study of thymidine kinase gene therapy of neuroblastoma in vitro and in vivo

Neuroblastoma arises as a direct result of genetic disorder; therefore, it should be well treated and conquered by gene therapy in future. In this study, neuroblastoma cell line SH-SY5Y experiments, in vitro and in nude mice in vivo, were subjected to research thymidine kinase suicide gene to treat neuroblastoma. The plasmid LXpsp-hytk and a plasmid LXSH were transduced separately by lipofectin into human neuroblastoma cell line SH-SY5Y. SH-SY5Y-hy and SH-SY5Y-hytk were selected by hygromycin B. Different ganciclovir (GCV) concentration was given to SH-SY5Y-hytk to determine optimal GCV concentration. The cytotoxic effect of GCV on SH-SY5Y-hytk, SH-SY5Y-hy, and SH-SY5Y cells was determined. Scapular subcutaneous tumors were established in nude mice by inoculating 2.5×10⁶ SH-SY5Y-hytk on their left sides and 2.5×10⁶ SH-SY5Y-hy cells on their right sides for every mouse of treatment group and control group, respectively. After 1 week, mass grew in both sides of all the mice, and from the eighth day on, every mouse in treatment group received daily intraperitoneal injection of GCV 50 mg/kg body weight for 14 days; every mouse in control group received daily intraperitoneal injection of 1 ml saline for 14 days. On day 22 tumors were excised and weighed on the left and right sides, respectively, and apoptosis was detected by TUNEL method. Apoptotic index was calculated on the left and on the right sides, respectively, for every mouse in treatment group and control group. The lowest concentration of hygromycin B was 60 g/ml. The cytotoxic effect of GCV on SH-SY5Y-hytk cells was obvious (IC₅₀=0.03 M), whereas GCV showed almost no cytotoxic effect on SH-SY5Y and SH-SY5Y-hy cells (IC₅₀>400 M). SH-SY5Y-hytk was killed by concentrations of 30 M GCV effectively and it obviously showed the bystander effect, when SH-SY5Y-hytk remained at least 18% in the mixture culture cells. The tumor on the left side was much smaller than that of the right side in control group (p<0.05), and apoptotic index of the left was higher than that of the right in control group (p<0.01). SH-SY5Y-hytk has the bystander effect over 18% SH-SY5Y-hytk of the mixture culture cells at the concentration of 30 M GCV. The HSV-tk/GCV system was effective in treating SH-SY5Y neuroblastoma cell line in vivo as well. Our findings suggest that thymidine kinase gene therapy could be a potential method for treating neuroblastoma in the future.     

In vivo and in vitro sensitivity of Falciparum malaria to quinine in Thai children

The study was carried out to assess the efficacy of quinine in children with Falciparum malaria in relation to in vitro sensitivity (measured in terms of minimum inhibitory concentration: MIC) and to trough serum levels of quinine during the course of treatment. Fifty children aged ten months to 12 years with Falciparum malaria were randomly divided into two groups. Group I: 24 children were treated with quinine 10 mg base per body weight every eight hours for 14 days. Group II: 26 children were treated with quinine at the dosage adjusted to the body surface area based on an adult dose of 500 mg base eight hourly for 14 days. There were three treatment failures, one RI and one RII in group I, and one RI in group II. The serum concentrations of quinine reached a peak level on day two and levelled off by the end of the first week. Concentrations in group II were higher than in group I. The mean minimal inhibitory concentration (MIC) of quinine in the two groups was 14.89 nmol per ml ranging from 8-26 nmol per ml. In cases with treatment failure, the trough serum quinine levels became lower than the corresponding MIC after day six (RI) and after day two (RII). The rise of MIC suggests that sensitivity of Falciparum malaria parasites to quinine may be decreasing in Thailand. Failures of treatment in standard dosage may occur in cases infected by parasites with high MIC, in which trough serum quinine levels cannot be maintained above the MIC longer than six days during the course of treatment. However in one cured case, the trough serum quinine levels were below the MIC throughout treatment. More research is needed on the real relationship between serum quinine concentrations, the MIC, and clinical and parasitological response to quinine.     

Cysteamine in the treatment of cystinosis in children. In vitro and in vivo studies

The effect of cysteamine was studied in 6 children with nephropathic cystinosis. In 3 of them an in vitro study on fibroblasts was performed. The cystine content of fibroblasts was immediately diminished (about 90% of total cystine content) as soon as the concentration of cysteamine in the medium was greater than or equal to 0,1 mmole/l. In vivo, 50 to 89 mg/kg/day of cysteamine was administered for 9 to 37 months (mean 21,3). There was no adverse reaction. In all cases a dramatic decline in leukocyte cystine level was observed (in 5 cases the level was within the range seen in clinically unaffected heterozygotes). Growth was not improved. The renal function was stabilised in 3 cases. Photophobia which was present in 4 children decreased in 2 cases or disappeared in 2 cases.