Following allergen exposure, sensitized Brown-Norway rats develop airway hyperresponsiveness (AHR) and eosinophilic inflammation together with an increase in activated T cells (CD25+) in the airways. We tested the hypothesis that CD4+ T cells are involved directly in the acquisition of AHR. Spleen T cells from animals that were injected intraperitoneally on three consecutive days with ovalbumin/Al(OH)3, showed a dose-dependent proliferative response in vitro to ovalbumin, but not to bovine serum albumin, as measured by [3H]thymidine uptake. For total T-cell transfer, spleen cells obtained from donor rats 4 days after sensitization were depleted of adherent cells by a nylon wool column separation. CD4+ and CD8+ T cells were purified by immunomagnetic beads cell separation. Recipient naive rats were injected intravenously with 50 x 10(6) total T cells, 20 x 10(6) and 5 x 10(6) CD4+ cells, and 5 x 10(6) CD8+ cells, and were exposed to ovalbumin aerosol 24 hr afterwards. After a further 24 hr, airway responsiveness to acetylcholine (ACh) was measured and provocative concentration (PC) values PC100, PC200 and PC300) (the ACh concentration needed to achieve 100, 200 and 300% increase in lung resistance above baseline) were calculated. Airway responsiveness was significantly increased in recipients of sensitized total T cells compared with recipients of cells from saline-injected donor rats (P
In this study, the antioxidant, cytotoxic, and antitumorigenic activities of a fractionated, ethanol extract derived from Rhus verniciflua Stokes (RVS), a plant indigenous to Korea, China, and Japan, were determined. Physicochemical analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the active component of a Sephadex G-150-fractionated RVS extract (PII fraction) was a copper-containing glycoprotein, possibly a plant laccase. Antioxidant activity of the fractionated RVS extract, observed in both aqueous and lipid in vitro oxidation reactions using 1,1-diphenyl 2-picrylhydrazyl (DPPH) radical, site-specific Fenton-reaction deoxyribose, and a model lipid emulsion test system, indicated an affinity for protection against hydroxyl and peroxyl radicals. Cultured mouse brain neurons were protected against glucose oxidase-induced hydroxyl radical in the presence of the fractionated RVS extract (e.g., 58% protection at 4.9 microM and 95% protection with 22.7 microM RVS). RVS was further shown to protect against in vitro Fenton-reaction-induced single- and double-strand scission in supercoiled plasmid DNA. Further testing for bioactivity of the fractionated RVS extract was based on the affinity to inhibit cell proliferation in cultured HeLa and CT-26 tumor cells. The presence of RVS resulted in 70% cell death after 24 h of incubation in both cell lines at a minimum concentration of 2.48 microM RVS. Data demonstrate multiple bioactive chemopreventative properties of a Sephadex G-150-fractionated extract derived from RVS.
The possibility of transplanting adult stem cells into damaged organs has opened new prospects for the treatment of several human pathologies. The purpose of this study was to develop a culture system for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free media formulation and ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy. Somatic stem cells were isolated from the human periodontium using a minimally invasive periodontal access flap surgery in healthy donors. Expanded hPDLSCs in a xeno-free culture showed the morphological features of stem cells, expressed the markers associated with pluripotency, and a normal karyotype. Under appropriate culture conditions, hPDLSCs presented adipogenic and osteogenic potential; indeed, a very high accumulation of lipid droplets was evident in the cytoplasm of adipogenic-induced cells, and indisputable evidence of osteogenic differentiation, investigated by transmission electron microscopy, and analyzed for gene expression analysis has been shown. Based on these data, the novel xeno-free culture method might provide the basis for Good Manufacturing Procedure culture of autologous stem cells, readily accessible from human periodontium, and can be a resource to facilitate their use in human clinical studies for potential therapeutic regeneration.
The comparative study of extracellular ATP (ATP0) effect on free cytosolic calcium concentration ([Ca2+]i) in normal (isolated rat thymocytes) and transformed (leukosis MT-4 line) T-cells was carried out. Addition of 1 mM ATP to Ca-free incubation medium of both types of cells, loaded with indo-1, had no effect on [Ca2+]i level. Upon subsequent addition of 1 mM CaCl2 to the incubation medium the rapid and significant increase of [Ca2+]i in MT-4 cells was registered. This effect was maintained within 10 min and was not inhibited by phospholipase C inhibitor 0.2 mM neomycin, that was induced by cation entry into the cells from the extracellular medium. Both types of cells were shown to demonstrate ecto-ATPase activity in the presence of 1 mM MgCl2 or CaC12 in the incubation medium. Estimation of kinetic parameters has indicated that the maximum rate of extracellular ATP hydrolysis by MT-4 cells is higher and Mg2+ and Ca2+ activation constants are lower as compared to respective parameters of ATP hydrolysis by thymocytes. The possible functional significance of the increased level of ecto-ATPase activity in malignantly transformed cells is discussed.
Development of the long-term culture models of haematopoietic stem cells (HSCs) is one of the important tasks in modern biotechnology. It has been suggested that stromal presence is important for haematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive and definitive haematopoietic cells, or direct cell-to-cell contacts of the cultured material with underlying stromal base would be required. During present studies, influence of various feeder layers and feeder layer conditioned media on proliferative, differentiative and clonogenic activity of human AC133+ derived from human umbilical cord blood was investigated. Cell extracts for feeder layers were prepared from 4-6 weeks old human embryos and co-cultured feeder cells. Effects of the conditioned media were also determined. Culture and feeder layer media were additionally supplemented with commonly implemented factors such as GM-CSF, IL-3 and LIF. Estimation of morpho-functional properties of AC133+ cultivated suspension cultures was performed in subculture experiments using semisolid agar culture conditions. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analyzed. Our data suggest that haematopoiesis can be sustained for prolonged cultivation periods in the presence of feeder layer cells or conditioned media supported culture models. Prolonged support of primitive haematopoietic cells and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and suggests that direct cell-to-cell contacts may not be exclusively required for successful long-term in vitro haematopoiesis.
SETTING: Department of Pulmonary Medicine P, Bispebjerg Hospital, Copenhagen, Denmark. OBJECTIVE: To study the ability of two proteins secreted from Mycobacterium tuberculosis, MPT-64 and MPT-59 to induce delayed type hypersensitivity (DTH) reactions following intradermal administration. DESIGN: In a small scale clinical investigation, skin reactions to these antigens were compared to reactions to tuberculin PPD RT23 in 1) patients with active tuberculosis, 2) BCG vaccinated healthy subjects with close contact with tuberculous patients, and 3) BCG vaccinated healthy subjects without contact with tuberculous patients. Tests for in vitro reactivity to these antigens were carried out in similar groups. RESULTS: All subjects gave positive reaction to tuberculin PPD RT23, whereas approximately half of the subjects in each of the three groups reacted to MPT-59. Two subjects (one patient with tuberculosis and one healthy bacille Calmette-Guérin vaccinated subject without patient contact) reacted to MPT-64. The studies of cell proliferation and induction of interferon-gamma (IFN-gamma) following stimulation with tuberculin PPD and MPT-64 supported this profile of reactivity. CONCLUSION: None of the experimental skin test antigens had properties superior to tuberculin PPD RT23 in humans. The failure of MPT-64 to induce delayed type hypersensitivity reactions in the majority of tuberculosis patients is discussed, in view of the potent reactivity to MPT-64 in tuberculous guinea pigs.
WHO Collaborating Centre for Gonorrhoea and other STIs, Swedish Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine, Microbiology, Orebro University Hospital, Orebro, Sweden.
We describe a Neisseria gonorrhoeae strain, found in Sweden in 2011, that harbours a N. meningitidis porA gene causing false-negative results in PCRs targeting the gonococcal porA pseudogene. Furthermore, the strain had no prolyliminopeptidase (PIP) activity that many commercial biochemical kits for species verification in culture rely on. Enhanced awareness of the spread of such strains and screening for them can be crucial.
BACKGROUND AND AIMS: Insulinoma is a very rare type of islet cell tumour, but nevertheless the most common endocrine tumour of the pancreas. We aimed at reviewing our clinical experience with this tumour type and to assess whether organ culture could be obtained from surgically resected insulinoma material. MATERIAL AND METHODS: All patients with insulinomas (6 men and 10 women) referred to Haukeland University Hospital between 1986 and 2006 were included in the study. Median age of onset was 53 years (range 21-74). Biochemical diagnosis was established during a 72 h fast test. Imaging and localization of the tumours were performed with intra-operative ultrasonography, endoscopic ultrasonography, CT-scan and/or transcutaneous ultrasonography. For six patients, organ cultures were set up from tumour tissue fragments. RESULTS: The annual incidence of insulinoma was 0.8 per million. The patients generally presented with non-specific, episodic symptoms, which often were mistaken for cardiovascular, neurological or diabetic disease and in some cases delayed the diagnosis with several years. Two patients had diabetes prior to the diagnosis of insulinoma. Patient weight gain was probably due to increased food intake, compensating for the hypoglycemia. Intra-operative ultrasonography detected all tumours correctly, whereas 73% were detected by endoscopic ultrasonography and 38% by CT scan. Five insulinomas were located in the head, eight in the body and three in the tail of the pancreas. All were removed by open-access surgery, eleven cases by resection and five by enucleation. One tumour was malignant with liver metastases and two patients had tumours defined as borderline. Insulinoma tissue fragments developed into spheroids during the first week of culturing and insulin secretion into the media was demonstrated. CONCLUSIONS: Insulinomas are rare and diagnostically challenging tumours. Intra-operative ultrasonography was superior to other imaging modalities to locate the lesion. In organ culture, insulinomas readily form spheroids which may be used to yield insight into beta-cell biology.
To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community.
A retrospective observational study of the routine testing for N gonorrhoeae by analysis of test results compiled from the laboratory information system in two departments of clinical microbiology.
Altogether, 158 male and female subjects with NAAT-positive results for N gonorrhoeae were included in the study. Samples for culture of N gonorrhoeae were collected from 102/158 (64.6%) subjects recalled after a NAAT assay was found positive. Growth of N gonorrhoeae was seen in the samples from 54/102 (52.9%) of the re-examined NAAT-positive subjects. Among subjects with samples collected within the first week after the positive NAAT test, 34/44 (77.3%) were confirmed positive by culture.
This study shows that it is possible for the general practitioner to recall a substantial number of NAAT-positive subjects to collect swabs for culture of N gonorrhoeae to confirm gonococcal infection in the community. Most recall samples are culture positive if collected within a week of the NAAT-positive test, and may provide a sufficient monitoring of the drug susceptibility of N gonorrhoeae strains in the community.