Differential effects of Zincum metallicum on cell models.

INTRODUCTION: Zinc is an essential trace element necessary for life. Traditional and complementary medicines use zinc-based formulations to treat different classes of diseases. Basic research on homeopathic preparations of zinc are rare and there are a few published clinical cases describing its effects on patients. The use of cell-based models in drug screening is a reliable source of evidence. METHODS: We sought to investigate experimental end-points using cell-based models to determine the effects of dilutions of Zincum metallicum prepared according to the Brazilian Homeopathic Pharmacopoeia. Murine RAW 264.7 macrophages and melanoma B16-F10 cell lines were cultured according to standard procedures. Cells were treated with either 5c, 6c or 30c Zincum metallicum and control cells with its respective vehicle (5c, 6c, or 30c Lactose). Macrophage activation by CD54 immunolabeling and intracellular reactive oxygen species (ROS) using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) were detected by flow cytometry. Phagocytic capacity (endocytic index) was quantified by light microscopy. Features of melanoma cells were analyzed by colorimetric assays to determine melanin content and cell proliferation rate. All obtained data were submitted to normality test followed by statistical analysis. RESULTS: Zincum metallicum 6c shifted high ROS-producing macrophages to a low ROS-producing phenotype. Macrophage CD54 expression was increased by Zincum metallicum 5c. No changes in endocytic index were observed. Melanoma cells were not affected by any treatment we tested. CONCLUSIONS: Differing responses and non-linearity were found on macrophages challenged with Zincum metallicum at high dilutions. No changes in melanoma cells were observed. Customised assays using target cells can be useful to investigate high-dilution effects. Other cell types and conditions should be explored.

[Effect of taurochenodeoxycholic acid on immune function in mice].

OBJECTIVE: To observe the influence of Taurochenodeoxycholic Acid (TCDCA) on immun, function in mice. METHODS: T-Cell subgroups were determined by using Flow cytometry; The content of anti-body in serum was assayed by using Spectrophotometry; The phagocytosis of mononuclear phagocyte system was determined by using Carbon particle clearance test and anti-sheep blood cell hemolysin was determined by using Turbidimetric method. RESULTS: TCDCA signifeantly enhanced the percentage of CD and CD19+ lymphocytes and CD4+/CD8+ value in peripheral blood and the content of serum hemolysin and lysozymem in mice. Moreover, TCDCA markedly improved the phagocytosis functions of mononuclear phagocyte system and observably inhibited delayed type hypersensitivity. CONCLUSION: TCDCA can significantly enhance the immune function in mice.

Sensitive flow cytometric method to test basophil activation influenced by homeopathic histamine dilutions.

OBJECTIVE: In an experimental setting, human basophil degranulation was triggered by anti-IgE to measure the effects from homeopathic solutions in an in-vitro cell system. A 3-color flow cytometric method with enhanced accuracy was established. As an example we looked at the influence of histamine on anti-IgE activation of basophils. METHODS: Basophils were identified in the flow cytometer by their physical properties in the forward and side scatter light depiction and by gating on CD2(-), CD14(-), CD16(-), CD19(-), HLA-DR(-) negative and CD123-positive cells. CD63 expression on the cell surface of the anti-IgE-activated basophils served as an activation marker. RESULTS: With this method we were able to study basophil function of the 0.6-3.9% basophils out of the mononuclear blood cell fraction and to document their activation status upon anti-IgE activation. Optimal activation occurs at 0.6 microg/ml final anti-IgE concentration; not less than 10,000 basophils have to be counted per batch to reduce the variation of the measurement. The fixation method was able to stabilize activation for two days. After investigation and reduction of the source of measurement variability, an unequivocally inhibited basophil activation was documented in a partly optimized system with homeopathic dilutions of histamine (10(-22)M, 10(-23)M, 10(-24)M, and 10(-25)M histamine). Dilutions greater than 10(-20)M histamine (Avogadro's number 6.02 x 10(23)) account for less than 1.36 molecules of histamine in the test sample, indicating a true homeopathic effect. CONCLUSIONS: This test system is adequate for studying the effects of highly diluted mediators on basophil activation by anti-IgE. The systematic application of this experimental arrangement is recommended to study the effects of homeopathic dilutions on basophils.

Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow.

Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody-selected FACS and has the advantage of a method directly transferable to human BMC.

Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy.

BACKGROUND: Anaphylactic reactions during anesthesia are mainly the result of muscle-relaxant (MR) drugs. Skin tests, serologic detection of specific IgE, and in vitro leukocyte histamine release are used to investigate MR allergy. OBJECTIVE: We describe a new assay that is based on the detection by flow cytometry of the altered expression of plasma membrane molecules of MR-activated basophils. METHODS: For this assay, which we have named the BASIC assay, basophils are incubated in vitro with MR, after which they are fixed and then triple labeled with fluorescein-conjugated anti-CD63, tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45, and R-phycoerythrin conjugated anti-IgE. The resulting B asophils' A ltered S urface I mmunofluorescence is detected by flow C ytometry (BASIC). RESULTS: Forty-one patients who had an allergic reaction during general anesthesia and 23 control subjects without such a history were studied. All included subjects' basophils were tested in the BASIC assay with at least 4 MR: suxamethonium, gallamine, vecuronium, and pancuronium. After reaction of the basophils of the MR-allergic patients with MRs, increased surface expression of CD63 and CD45 and decreased expression of IgE were detected. Increased expression of CD63 was observed most frequently and it was stronger than the alteration of the 2 other markers. Cross-reactivity between MRs commonly occurred. MRs diluted 10(-1) activate the basophils of the control subjects, suggesting that at relatively high concentrations MRs are also nonspecific basophil activators. CONCLUSION: In the diagnosis of MR allergy, the BASIC assay has a good specificity but a low sensitivity, and it correlates strongly with skin test results. It is currently appraised for the diagnosis of anaphylactic reaction induced by other classes of drugs.