Effect of low doses and high homeopathic potencies in normal and cancerous human lymphocytes: an in vitro isopathic study

OBJECTIVES: Biologic effects of high homeopathic potencies can be studied in cell cultures using cell lines or primary cells. We hypothesized that primary cells would be more apt to respond to high potencies than cell lines, especially cancer cell lines. We set out to investigate the effects of low doses and high homeopathic potencies of cadmium chloride, respectively, in an intoxication model with human primary lymphocytes compared to a human leukemia cell line (Jurkat). DESIGN: Cells were pretreated with either low concentrations (nM-microM) or high potencies (pool 15-20c) of cadmium for 120 hours, following which they were exposed to a toxic treatment with a range of cadmium concentrations (8-80 microM) during 24 hours. Cell viability was eventually assessed by use of the MTS/PES assay. Controls included a vehicle (NaCl 0.9%) for the low concentrations of cadmium or water 15-20c for cadmium 15-20c. A total of 34 experiments were conducted, 23 with low concentrations and 11 with high potencies of cadmium. Data were analyzed by analysis of variance. RESULTS: Pretreatment with low concentrations or high potencies of cadmium significantly increased cell viability in primary lymphocytes after toxic challenge, compared to control cells (mean effect +/- standard error = 19% +/- 0.9% for low concentrations respectively 8% +/- 0.6% for high potencies of cadmium; p < 0.001 in both cases). The pretreatment effect of low doses was significant also in cancerous lymphocytes (4% +/- 0.5%; p < 0.001), albeit weaker than in normal lymphocytes. However, high homeopathic potencies had no effect on cancerous lymphocytes (1% +/- 1.9%; p = 0.45). CONCLUSIONS: High homeopathic potencies exhibit a biologic effect on cell cultures of normal primary lymphocytes. Cancerous lymphocytes (Jurkat), having lost the ability to respond to regulatory signals, seem to be fairly unresponsive to high homeopathic potencies.

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

PURPOSE: To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, (177)Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. METHODS: In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand (177)Lu-AMBA was used and compared with established bombesin radioligands such as (125)I-Tyr(4)-bombesin and (125)I-[D: -Tyr(6),beta-Ala(11),Phe(13),Nle(14)]-bombesin(6-14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. RESULTS: (177)Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. (177)Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with (177)Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with (177)Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with (177)Lu-AMBA than with the conventional bombesin radioligands. CONCLUSION: The present in vitro study suggests that (177)Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels.

Cbfa-1 (Runx-2) and osteocalcin expression by human osteoblasts in heparin osteoporosis in vitro

Heparin may cause adverse effects on bone formation following long-term application. The exact pathomechanism is unclear, but in vitro data suggest an impaired osteoblast function. The transcription axis of Cbfa-1 (Runx-2) and osteocalcin is crucial in maintaining an equilibrium of bone formation and resorption in vivo. We used a human osteoblast cell culture model to further investigate the effect of heparin (low-molecular-weight heparin, dalteparin) on the expression of these two regulators of osteoblast differentiation. At high doses, dalteparin caused a significant inhibition of both osteocalcin and Cbfa-1 expression in vitro. Our data support the hypothesis of a direct inhibition of osteoblast function underlying heparin osteoporosis.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

Impact of culture conditions on the proliferative lifespan of human T cells in vitro

BACKGROUND: In human T cells, telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control the lifespan of T cells. To improve T-cell expansion it is of critical importance to understand the effects of culture parameters on telomerase activity and lifespan. METHODS: We investigated the influence of culture condition (FCS, human AB serum and autologous serum) and stimulation (PHA/feeder cells, anti-CD3/CD28 beads) on the lifespan, clonogenicity (number of positive wells), cell cycle, telomerase activity and telomere length of T cells in vitro. RESULTS: The proliferative lifespan of T cells expanded with PHA/feeder cells and autologous serum from different donors was doubled compared with stimulation with PHA/feeder cells and AB serum. No or only a small difference was found for T cells expanded with anti-CD3/CD28 beads and autologous or AB serum. The use of autologous serum also increased the clonogenicity to about three-fold compared with the use of AB serum or FCS, without any signs of differences in the fractions of cycling cells. Interestingly, T cells cultured with autologous serum exhibited a significantly higher telomerase activity at day 6 after stimulation and a reduced decline of telomerase activity compared with cultures with AB serum. DISCUSSION: The use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity and increases the telomerase activity of human T cells in vitro. This might be useful for applications where large numbers of specific T cells are required.

CO2 laser-irradiation through topically applied fluoride increases acid resistance of demineralised human enamel in vitro

PURPOSE: To evaluate the effect of CO2 laser treatment through topically applied amine fluoride solution on demineralised enamel. MATERIALS AND METHODS: Sixty extracted human molar crowns were selected and cut longitudinally into half. One half was subjected to a 10-day pH-cycling procedure to create caries-like lesions, whereas the other was left non-demineralised. The following treatments were randomly assigned (one treatment per tooth, on respective non-demineralised and demineralised matched specimens): exposure to a 1% amine fluoride solution for 15 s without irradiation (group I), irradiation for 15 s with a continuous-wave CO2 laser (group II), or laser-treatment for 15 s through the amine fluoride solution applied immediately beforehand (group III). Fluoride uptake (n = 30) and acid resistance (n = 30) were determined after treatment. Enamel surface alterations after laser irradiation were monitored using scanning electron microscopy. RESULTS: In groups I and III, an increased fluoride uptake was detected (p < or = 0.05). Laser irradiation through topical fluoride resulted in an increased acid resistance of sound and demineralised enamel specimens in deeper layers (p < or = 0.05). In addition, less surface alterations were observed in SEM examination of specimens irradiated through the amine fluoride solution compared with counterparts treated with laser only. CONCLUSIONS: CO2 laser light application through an amine fluoride solution may be instrumental in enhancing acid resistance of sound and demineralised enamel.

Intraindividual comparison of human radial and internal thoracic arteries in vitro and the effect of preoperative calcium blocker therapy

The patency rate of radial artery (RA) conduits is considerably lower than that of internal thoracic artery (ITA) grafts and the evidence suggests that this is due to a clinically suspected higher incidence of vasospasm. The aim of this study was, therefore, to compare intraindividually the pharmacological reactivity of RA with that of ITA. Both RA and ITA were taken from the same patients and investigated in parallel. Changes in tone were monitored isometrically on ring preparations from both arteries in organ baths with modified Krebs-Henseleit solution containing 1.25 mm calcium chloride at 1 g passive preload. In intraindividual comparisons maximal receptor-mediated contractile responses to noradrenaline and endothelin-1 and endothelium-dependent acetylcholine-induced relaxant responses revealed no differences between both arteries. By contrast, depolarization-induced contractions to potassium chloride (KCl) appeared to be significantly higher in RA than in ITA. Further analysis, however, revealed that this difference was due to preoperative calcium entry blocker (Ca(2+)eB) therapy. Compared with control tissues, maximal responses to KCl were significantly attenuated in the ITA but unchanged in RA when arteries were obtained from patients with preoperative Ca(2+)eB therapy. The present results suggested that functional responses to pharmacological stimuli of both RA and ITA were quite similar. Preoperative Ca(2+)eB therapy, however, attenuated markedly responses to KCl of the ITA leaving those of RA unchanged. These results, demonstrating a lower sensitivity to Ca(2+)eB of RA, therefore suggested that in addition to Ca(2+)eB other classes of drug may be more effective at reducing the propensity of RA conduits to vasospasm.

A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles

The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway. Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha. Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter

BACKGROUND: Several epidemiological studies show that inhalation of particulate matter may cause increased pulmonary morbidity and mortality. Of particular interest are the ultrafine particles that are particularly toxic. In addition more and more nanoparticles are released into the environment; however, the potential health effects of these nanoparticles are yet unknown. OBJECTIVES: To avoid particle toxicity studies with animals many cell culture models have been developed during the past years. METHODS: This review focuses on the most commonly used in vitro epithelial airway and alveolar models to study particle-cell interactions and particle toxicity and highlights advantages and disadvantages of the different models. RESULTS/CONCLUSION: There are many lung cell culture models but none of these models seems to be perfect. However, they might be a great tool to perform basic research or toxicity tests. The focus here is on 3D and co-culture models, which seem to be more realistic than monocultures.

Serum hyperglycosylated human chorionic gonadotropin to predict the gestational outcome in in vitro fertilization/intracytoplasmic sperm injection pregnancies

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted by the placenta in early pregnancy. Decreased H-hCG levels have been associated with abortion in spontaneous pregnancy. We retrospectively measured H-hCG and dimeric hCG in the sera of 87 in vitro fertilization patients obtained in the 3 weeks following embryo transfer and set the results in relation to pregnancy outcome. H-hCG and dimeric hCG were correlated (r(2) = 0.89), and were significantly decreased in biochemical pregnancy (2 microg/l and 18 IU/l, respectively) compared to early pregnancy loss (22 microg/l and 331 IU/l) and ongoing pregnancy (32 microg/l and 353 IU/l). Only H-hCG tended to discriminate between these last two groups.

Influence of the magnesium aspartate hydrochloride administration to the maternal circuit on the aspartate concentration of the fetal circuit under in vitro perfusion of human placenta

OBJECTIVES: Magnesium aspartate hydrochloride (Magnesiocard, Mg-Asp-HCl) is proposed as a substitute of magnesium sulfate for the treatment of preeclampsia and premature labor. After an i.v. administration of a dose equivalent to that used in the treatment of preeclampsia to nonpregnant volunteers, a 10-fold increase of aspartic acid (Asp) over the physiological level was observed. Animal experiments have demonstrated that highly increased fetal levels of acidic amino acids such as Asp could be associated with neurotoxic damage in the fetal brain. The influence of such an elevation of Asp concentration in the maternal circuit on the fetal level, using the in vitro perfusion model of human placenta, was investigated. STUDY DESIGN: After a control phase (2h), a therapeutic dose of Mg combined with Asp (Magnesiocard, Mg-Asp-HCl) was applied to the maternal circuit approaching 10 times the physiological level of Asp. The administration was performed in two different phases simulating either a peak of maximum concentration (bolus application, 2h) or a steady state level (initially added, 4h). RESULTS: In four experiments, during experimental phases (6h) a slow increase in concentration in the fetal circuit was seen for Mg, AIB (alpha-aminoisobutyric acid, artificial amino acid) and creatinine confirming previous observations. In contrast, no net transfer of Asp across the placenta was seen. A continuous decrease in the concentration of Asp on both maternal and fetal side suggests active uptake and metabolization by the placenta. Viability control parameters remained stable indicating the absence of an effect on placental metabolism, permeability and morphology. CONCLUSION: Elevation of Asp concentration up to 10 times the physiological level by the administration of Mg-Asp-HCl to the maternal circuit under in vitro perfusion conditions of human placenta has no influence on the fetal level of Asp suggesting no transfer of Asp from the maternal to fetal compartment. Therefore, the administration of Mg-Asp-HCl to preeclamptic patients would be beneficial for the patients without any impact on placental or fetal physiology.

Effects of a non-selective COX inhibitor and selective COX-2 inhibitors on contractility of human and porcine ureters in vitro and in vivo

BACKGROUND AND PURPOSE: Anti-inflammatory drugs are used in the treatment of acute renal colic. The aim of this study was to investigate the effects of selective COX-2 inhibitors and the non-selective COX inhibitor diclofenac on contractility of human and porcine ureters in vitro and in vivo, respectively. COX-1 and COX-2 receptors were identified in human ureter and kidney. EXPERIMENTAL APPROACH: Human ureter samples were used alongside an in vivo pig model with or without partial ureteral obstruction. COX-1 and COX-2 receptors were located in human ureters by immunohistochemistry. KEY RESULTS: Diclofenac and valdecoxib significantly decreased the amplitude of electrically-stimulated contractions in human ureters in vitro, the maximal effect (Vmax) being 120 and 14%, respectively. Valdecoxib was more potent in proximal specimens of human ureter (EC50=7.3 x 10(-11) M) than in distal specimens (EC50=7.4 x 10(-10) M), and the Vmax was more marked in distal specimens (22.5%) than in proximal specimens (8.0%) in vitro. In the in vivo pig model, parecoxib, when compared to the effect of its solvent, significantly decreased the maximal amplitude of contractions (Amax) in non-obstructed ureters but not in obstructed ureters. Diclofenac had no effect on spontaneous contractions of porcine ureter in vivo. COX-1 and COX-2 receptors were found to be expressed in proximal and distal human ureter and in tubulus epithelia of the kidney. CONCLUSIONS AND IMPLICATIONS: Selective COX-2 inhibitors decrease the contractility of non-obstructed, but not obstructed, ureters of the pig in vivo, but have a minimal effect on electrically-induced contractions of human ureters in vitro.