Microwaves and cellular immunity. II. Immunostimulating effects of microwaves and naturally occurring antioxidant nutrients

The effect of 8.15-18 GHz (1 Hz within) microwave radiation at a power density of 1 microW/cm2 on the tumor necrosis factor (TNF) production and immune response was tested. A single 5 h whole-body exposure induced a significant increase in TNF production in peritoneal macrophages and splenic T cells. The mitogenic response in T lymphocytes increased after microwave exposure. The activation of cellular immunity was observed within 3 days after exposure. The diet containing lipid-soluble nutrients (beta-carotene, alpha-tocopherol and ubiquinone Q9) increased the activity of macrophages and T cells from irradiated mice. These results demonstrate that irradiation with low-power density microwaves stimulates the immune potential of macrophages and T cells, and the antioxidant treatment enhances the effect of microwaves, in particular at later terms, when the effect of irradiation is reduced.     

Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.     

Effect of microwave irradiation on polyphenolic compounds from Satureja hortensis L.

Summer savory (Satureja hortensis L.) is most often used as a culinary herb, but it also has medicinal benefits. The extracts from control and irradiated savory were obtained by ultrasound extraction for 30 minutes in an ethanol — water (80:20, v/v) mixture. Polyphenolic compounds from savory were identified and characterized by high-performance liquid chromatography coupled with a photodiode array detector and mass spectrometer. The separation was performed using an Altima C18 column (100×3 mm, 3 μm) and as mobile phase two solvent mixture: A — acetonitrile and B — water-formic acid (99.9:0.1, v/v). Peaks were identified with authentic standards in accordance to retention time, UV spectra and molecular mass. It was identified as caffeic acid, rosmarinic acid, luteolin, naringenin and apigenin. A quantitative determination of polyphenolic compounds was performed applying the external standard method. Our study showed large quantitative differences between the control plant and the irradiated plant.      

Nuclear factor of activated T cells negatively regulates expression of the tumor necrosis factor receptor-related 2 gene in T cells

Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.     

Synergistic induction of polypeptides by tumor necrosis factor and interferon-gamma in cells sensitive or resistant to tumor necrosis factor: assessment by computer based analysis of two-dimensional gels using the PDQUEST system

Tumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon-gamma (IFN-gamma) in both TNF-sensitive and TNF-resistant variants of the cervical carcinoma line, ME-180. TNF alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two-dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN-gamma enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, TNF and IFN-gamma induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of TNF and IFN-gamma in ME-180 cells.     

Effect of bredinin on the primary culture of fetal mouse cells and adult mouse lung cells.

The effects of bredinin on the primary culture of fetal mouse cells (fetal cells) and adult mouse lung cells (lung cells) were compared. Bredinin inhibited the growth of both cells, and this inhibition was found to be caused, at least in part, by the inhibition of the S phase and/or the transition from the G1 to S phase of the cell cycle. Bredinin inhibited both DNA and RNA synthesis without affecting protein synthesis. However, the inhibitory effect of bredinin differed between the two cell lines; the fetal cells were more sensitive than the lung cells, and bredinin inhibited DNA synthesis 100 times more potently in the fetal cells. The inhibition of DNA synthesis by bredinin in the fetal cells was gradually lowered by in vitro aging of the fetal cells to a level similar to that in the lung cells. There was no difference in the rate of incorporation of bredinin into the cells between the fetal cells and the lung cells. When fetal tissue was used as an enzyme source, bredinin was converted to bredinin 5'-monophosphate (BMP), but when lung tissue was used, bredinin was not converted. This is in agreement with the finding that bredinin has selective toxicity on fetuses in vivo but is hardly toxic to adult cells, which suggests the involvement of BMP in the selective toxicity of bredinin on the fetus.     

Microwaves in organic synthesis: Synthesis of pyridazinones,phthalazinones and pyridopyridazinones from 2‐oxo‐arylhydrazones under microwave irradiation

The phenylhydrazones 1a‐d condensed with ethyl cyanoacetate to yield pyridazinones 2a‐d that reacted with sulphur in presence of piperidine to yield the aminothienopyridazineones 3a,b that reacted with electron poor olefins and acetylenes to yield phthalazines 10‐12. The condensed aminothiophenes 3a,b reacted with dimethylformamide dimethylacetal to yield amidines 13a,b. Compounds 2a,b condensed with dimethylformamide dimethylacetal to yield the trans enamines 16a,b that cyclized readily into the pyridopyridazinones 17a,b on treatment with ammonium acetate in presence of acetic acid. Compounds 2a‐d reacted also with benzylidenemalononitrile to yield the phthalazinones 21a‐d. The reactions were conducted both by microwave heating and conventional heating. Better yields in much shorter reaction times were achieved by microwave heating.     

Effect of microwave irradiation on the reactivity of chloroarenes in Suzuki—Miyaura reaction

Simple catalytic systems for the Suzuki—Miyaura reaction of aryl chlorides under microwave conditions were tested. Microwave irradiation facilitated the reaction course. The catalyst, base, and solvent effect were studied and two reaction systems offered reasonable to high yields within a short time.     

Effect of nitrite on cell growth and antibody production in mouse splenic B cells stimulated with lipopolysaccharide and B cell hybridomas.

Nitric oxide, nitrite and nitrate are released by activated macrophages in an immune response. We showed here that nitrite influenced cell growth and antibody production in mouse lipopolysaccharide (LPS)-stimulated splenic B cells and B cell hybridomas. The addition of 10(-7) and 10(-6) M nitrite enhanced deoxyribonucleic acid (DNA) synthesis of LPS-stimulated splenic B cells. However, DNA synthesis and antibody production in the case of total spleen cells stimulated with LPS were suppressed by nitrite in a dose dependent-manner. These phenomena were also observed in a similar experiment involving mouse B cell hybridomas. Antibody production of all B cell hybridomas was significantly suppressed by the addition of nitrite. This suppressing effect could not be explained by changes in viable cell yields. This data suggests that the antibody production and cell proliferation of B cells may be influenced by nitrite from activated macrophages in the immune response.     

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.     

Cytokine production after helium-neon laser irradiation in cultures of human peripheral blood mononuclear cells.

The effects of laser light on the immune system have not been extensively characterized. Low-power laser sources, such as the helium-neon (He-Ne) laser with a wavelength of 632.8 nm, have been found to produce photobiological effects with evidence of interference with immunological functions. We have investigated the effects of He-Ne laser irradiation on Ficoll-Hypaque-isolated human peripheral blood mononuclear cells (PBMC). Cultured cells were irradiated for various times at two selected intensities and then stimulated with different mitogens. The rate of incorporation of 3H-thymidine into the DNA of stimulated cells decreased with increasing energy density. The levels of interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in supernatants of the cultures were determined (irradiated either before or after stimulation). When stimulating cells after irradiation, significantly increased levels of all cytokines were detected after 30 min of irradiation (18.9 J cm-2), whereas after 60 min of irradiation (37.8 J cm-2) cytokine levels were found to be significantly decreased.     

Porphyrins in Diels-Alder reactions. Improvements on the synthesis of barrelene-fused chlorins using microwave irradiation

The microwave irradiation technique was applied to the Diels-Alder reaction of tetrakis(pentafluorophenyl)porphyrin with pentacene and naphthacene. Both reactions proceed within minutes to afford the corresponding monoadducts in 83% and 23% yield, respectively. When compared with the yields obtained under classical heating (22% and no reaction, respectively), this represents an impressive improvement of these reactions. Bisadducts (bacteriochlorins and isobacteriochlorins) are also obtained in the reaction with pentacene; these compounds are not formed under classical heating.     

Liquiritigenin inhibits tumor growth and vascularization in a mouse model of HeLa cells

Angiogenesis is one of the crucial steps in the transition of a tumor from a small, harmless cluster of mutated cells to a large, malignant growth, capable of spreading to other organs throughout the body. Vascular endothelial growth factor (VEGF) that stimulates vasculogenesis and angiogenesis is thought to be as an anti-angiogenic target for cancer therapy. Liquiritigenin (LQ), a flavanone existing in Radix glycyrrhiza, shows extensive biological activities, such as anti-inflammatory and anti-cancer properties. In our studies, liquiritigenin effectively inhibited the growth of tumors xenografted in nude mice from human cervical cancer cell line HeLa cells, and microvascular density (MVD) of the tumor exposed to liquiritigenin was reduced in a dose dependent manner, especially in the high dose group. Moreover, the expression and secretion of VEGF were down-regulated by the drug in vivo and in vitro. Therefore, liquiritigenin can be further studied on cancer and other diseases associated with VEGF up-regulation.     

Effect on topical 5-methoxypsoralen on tumorigenesis induced in albino and pigmented hairless mouse skin by UV irradiation

A new line of the Skh:HRII hairless pigmented mouse (black juvenile coat) is described which has been selectively bred for the capacity to respond consistently to simulated solar UV radiation with a continuous and strong tan. This mouse demonstrates a degree of protection from chronic UV-induced tumorigenesis when compared with the Skh:HRI hairless albino mouse, and has been used here to study the effect of induced melanogenesis on phototumorigenesis. Mice were irradiated for 10 weeks with incremental doses of simulated solar UV radiation (UVA + B) from a fluorescent tube source which induced tumours in 100% of albino mice and 93% of black mice by 200 days (minimally oedemal), or with 60% of this dose (sub-oedemal) which induced tumours in 85% of albino mice and 65% of black mice. Mice were also exposed to the UVA component of these radiation sources, obtained by window glass filtration. The effect of topical 5-methoxypsoralen (5-MOP) was examined, at either 0.003% with minimally oedemal UVA + B or its UVA component alone, or at 0.01% with sub-oedemal UVA + B or its UVA component alone, in both albino and black mice. The 5-MOP concentrations were selected as the maximum concentration which did not increase the erythema and oedema responses after a single exposure to minimally oedemal or sub-oedemal UVA + B. At 200 days, the tumorigenic response to sub-oedemal UVA + B was significantly increased by topical 5-MOP, to 100% in albinos and 93% in black mice. In contrast, tumorigenesis in response to minimally oedemal UVA + B was unaffected by topical 5-MOP. The UVA component alone of either irradiation regime was not tumorigenic under these conditions. When combined with topical 5-MOP, the UVA of minimally oedemal UVA + B became moderately tumorigenic, and resulted in a tumour incidence of 23% in albinos and 14.5% in black mice. However, the UVA component of sub-oedemal UVA + B, when combined with topical 5-MOP, was highly tumorigenic specifically in albino mice, inducing tumours in 93% of albino mice but in only 27% of black mice. Tan intensity resulting from minimally oedemal UVA + B was not enhanced by topical 5-MOP, and its UVA component combined with 5-MOP resulted in only a minimal tan. However, the tan intensity resulting from sub-oedemal UVA + B with topical 5-MOP was strongly increased, although its UVA component combined with 5-MOP did not produce a perceptible tan.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of helium/neon laser irradiation on nerve growth factor synthesis and secretion in skeletal muscle cultures

Low energy laser irradiation therapy in medicine is widespread but the mechanisms are not fully understood. The aim of the present study was to elucidate the mechanism by which the light might induce therapeutic effects. Skeletal muscle cultures were chosen as a target for light irradiation and nerve growth factor (NGF) was the biochemical marker for analysis. It was found that there is a transient elevation of intracellular calcium in the myotubes immediately after irradiation (P<0.001). Preincubation of the myotubes with either the photosensitizers 5-amino-levulinic acid (5-ALA), or with hematoporphyrin (Hp) enhanced the elevation of cytosolic calcium (P<0.001) after helium/neon irradiation (633 nm) with an energy of 3 J/cm(2). In addition, helium/neon irradiation augmented the level of NGF mRNA fivefold and increased NGF release to the medium of the myotubes. Thus, it is speculated that transient changes in calcium caused by light can modulate NGF release from the myotubes and also affect the nerves innervating the muscle. The NGF is probably responsible for the beneficial effects of low-level light.     

Ultraviolet-A irradiation to the eye modulates intestinal mucosal functions and properties of mast cells in the mouse

We previously reported that topical irradiation of the eye by ultraviolet-B (UVB) activated hypothalamo-pituitary-adrenal axis (HPA-A) of the mouse to increase 3, 4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the skin by an inducible nitric oxide synthase (iNOS)-dependent mechanism. This work demonstrates that irradiation of the eye by ultraviolet-A (UVA) specifically increased DOPA-positive cells in the mucosa of the jejunum and colon of C57BL/6J mice by some HPA- and iNOS-independent mechanism. UVA-induced increase in DOPA-positive cells in the intestine was inhibited by the administration of hexamethonium or prazosin plus propranolol, blockers for the sympathetic nervous system. UVA irradiation of the eye increased DOPA- and histidine decarboxylase (HDC)-positive cells in the intestinal mucosa of both C57BL/6J and WBB6F1/J mice but not in the mutant strain W/Wv of the latter that lack mast cells. UVA irradiation of the eye suppressed the intestinal peristalsis of control, hypophysectomized or iNOS(-/-) C57BL/6J mice by the mechanism that was inhibited by hexamethonium or prazosin plus propranolol. These observations suggest that UVA irradiation of the eye stimulated the sympathetic nervous system to increase the mucosal DOPA- and HDC-positive mast cells and suppressed the peristalsis of the small intestine of the mouse.