The effect of PEG-coated gold nanoparticles on the anti-proliferative potential of Specific Nutrient Synergy.

The role of PEG-coated gold nanoparticles (Au NPs) on the anti-proliferative effect of Specific Nutrient Synergy (SNS) on HTLV-1 infected (C91-PL and HuT-102) and non-infected (CEM and Jurkat) malignant T-lymphocytes cells, was investigated. When PEG-coated Au NPs (of different molecular weights) were added alone, there was no effect on either viability or proliferation of the leukemic cell lines studied. Treatment of cells with SNS and PEG (5 or 10 kDa) coated Au NP reduced significantly the proliferation in all cell lines tested; this reached more than 50% reduction as compared to the control for cells treated for 96 h. Data showed that the best anti-proliferative effect was obtained using SNS and Au NP coated with PEG of molecular weights of 5 and 10 kDa with almost no effect of PEG of lower molecular weights (0.75 and 2 kDa) or higher ones (20 kDa). This was true as well for HTLV-1 infected as for non-infected malignant T-lymphocytes. Electron microscopy results showed uptake of the gold particles to Jurkat cells. All described effects are specific to leukemia cell lines, and no effects were observed with freshly activated human mononuclear lymphocytes as control.

Effects of dietary protein and calcium on thymus apoptosis induced by fluoride in female rats (Wistar rats).

Our previous studies showed that excessive fluoride (F) ingestion seriously damaged the nonspecific immune function in rabbits. However, the underlying mechanisms of the F-induced damage to the immune system are unclear. The purpose of this study was to investigate whether F induces thymus apoptosis in female rats and its underlying mechanisms by monitoring ultrastructural changes and DNA fragmentation. The results showed that excessive F induced ultrastructural changes and significantly increased the tail length and tailing ratio in thymus lymphocytes. Protein (Pr) supplementation markedly decreased the tailing ratio in thymus lymphocytes in the case of malnutrition. Furthermore, molecular analysis showed that excessive F ingestion significantly up-regulated the expression levels of caspase-3 and caspase-9 mRNA, whereas Pr and calcium (Ca) supplementation down-regulated the expression levels induced by excessive F in the case of malnutrition. In conclusion, these results indicate that excessive F up-regulates the expression levels of caspase-3 and caspase-9 mRNA and induces thymus apoptosis in female rats. Pr and Ca play key roles in process of F-induced thymus apoptosis in malnourished female rats.

Melatonin supplementation restores cellular proliferation and DNA synthesis in the splenic and thymic lymphocytes of old rats.

OBJECTIVES: In this study we investigated the effect of melatonin treatment on the proliferative activity, the rate of DNA synthesis and the histopathological changes of splenic and thymic lymphocytes in old rats. METHODS: Two subgroups of old rats (25-months-old) were used in this study. One subgroup was given melatonin in the drinking water (250-300 mg/day/rat) for 3 months while the second subgroup was given water containing diluent. A third group consisted of young rats (3-months-old) which served as an additional control. RESULTS: A (3)H-thymidine autoradiographic investigation showed a reduction in both the proliferative activity and the rate of DNA synthesis in splenic and thymic lymphocytes in old rats. In addition, light and electron microscopy showed severe histopathological changes in these cells from diluent-treated old rats. Melatonin administration increased the proliferative activity and the rate of DNA synthesis in the lymphocytes of both the spleen and thymus of the old animals. Also, histopathological changes were partially reversed by melatonin treatment with the tissues appearing similar to those in the young rats. CONCLUSION: The stimulation of the lymphocyte activity by melatonin is a beneficial response, especially in old rats, since aging results in an inhibition in lymphocytic functions.

Malondialdehyde production in Jurkat T cells subjected to oxidative stress.

OBJECTIVE: We investigated the relation between membrane lipid peroxidation, as evaluated by malondialdehyde (MDA), and oxidative stimuli in the Jurkat T-cell line and designed a cellular model to assess the antioxidant potential of compounds. METHODS: Jurkat T cells were subjected to different concentrations of Fe(2+) ions (from 25 to 150 micromol/L) or H(2)O(2) (from 0.1 to 5 mmol/L), and MDA was determined after separation in high-performance liquid chromatography of the adduct with thiobarbituric acid. MDA production also was investigated in cells supplemented with epigallocatechin gallate and genistein and subjected to Fe(2+) oxidative treatment. RESULTS: MDA production increased with the concentration of Fe(2+), whereas H(2)O(2) had no effect at any concentration. Oxidative stress for 15 min or 2 h produced similar MDA levels. The supplementation of epigallocatechin gallate partly prevented MDA production (about 40%, P < 0.05), whereas genistein exerted no preventive effect on lipid peroxidation. CONCLUSION: We propose this cellular model, consisting of Jurkat T cells subjected to 100 micromol/L of Fe(2+) for 15 min, to study the protective effect of antioxidant supplementation against membrane lipid peroxidation.

[Comparative study of modulating effect of kidney tonifying recipe and spleen invigorating recipe on T-lymphocyte apoptosis in corticosterone treated rats].

OBJECTIVE: To compare the modulating effect of Kidney tonifying recipe (KTR) and Spleen invigorating recipe (SIR) on T-lymphocyte apoptosis (TLA) in corticosterone treated rats (CTR). METHODS: Qualitative and quantitative analysis on activation-induced TLA was conducted by transmission electron microscope and TUNEL labelled flow cytometry detection. RESULTS: TLA percentage of CTR was 45.87 +/- 7.22%, while that of normal control rats was 34.25 +/- 6.47%, the difference between them was significant (P < 0.01). TLA percentage of Youguiyin treated rats was 35.90 +/- 7.39%, and that of Bushen Yishou capsule treated rats was 36.20 +/- 9.14%, compared with that of CTR, P < 0.01 and P < 0.05 respectively. TLA percentage of the SIR treated group was 36.92 +/- 11.82%, which was different insignificantly as compared with that of CTR. CONCLUSION: TLA susceptibility was significantly enhanced in CTR. Both KTRs (Youguiyin and Bushen Yishou capsule) had down-regulating effect on TLA, while the effect of SIR was insignificant, suggesting that down-regulating activation-induced TLA may be one of the important mechanisms of KTR in improving T-lymphocyte function in CTR.

Effect of electroacupuncture on the activities of tyrosine protein kinase in subcellular fractions of activated T lymphocytes from the traumatized rats.

The present study was to observe the dynamic changes of tyrosine protein kinase (TPK) activity in subcellular fractions in the early stage of activation of T lymphocytes from normal and traumatized rats, and the regulatory effect of electroacupuncture (EA) stimulation on it. The results showed that the activities of TPK in membranous and cytosolic fractions of activated T lymphocytes were increased on second 5, and the peak was on scond 45 after ConA stimulation. Then it was decreased gradually. Comparing with the control group, the activities of TPK in membranous and cytosolic fractions of activated T lymphocytes from the traumatized rats were inhibited in various degrees especially in membrane. EA of "Zusanli"(ST-36) and "Lanwei"(Extra 33) points could enhance the activity of TPK in subcellular fractions of activated T lymphocytes from the traumatized rats. The results indicated that EA stimulation could prevent the inhibition of activation of TPK induced by trauma stress, and contribute to transmembrane signal transduction of T lymphocytes.

Restoration of thymocyte development and function in zap-70-/- mice by the Syk protein tyrosine kinase.

The Syk family of protein tyrosine kinases, consisting of ZAP-70 and Syk, associate with the pre- and alphabeta T cell antigen receptors (TCRs) and undergo tyrosine phosphorylation and activation following receptor engagement. Thymocyte development in zap-70-/- mice is blocked at the CD4+CD8+ TCR(lo) stage. The presence of Syk in the thymus has raised the possibility that Syk may be able to mediate TCR function. To determine if Syk can play a role in thymocyte development, we generated zap-70-/- mice expressing a human syk cDNA. Syk expression restored both thymocyte development and function. In addition, Syk function required the CD45 transmembrane protein tyrosine phosphatase. Hence, ZAP-70 and Syk can play overlapping functions and exhibit similar regulatory mechanisms in mediating alphabeta T cell development.

Population-based biomonitoring in the Czech Republic--the system and selected results.

In the framework of the system of monitoring the environmental impact on population health, the concentration of lead, cadmium and selenium in blood and cadmium in urine was measured in adults (n = 670), children (n = 599) and umbilical blood (n = 549) using atomic absorption spectrophotometry. Furthermore, cytogenetic analysis of peripheral lymphocytes in all population groups under study was investigated. The median blood Pb level for the overall group of adults (47.8 micrograms/l, i.e. 0.23 mumol/1) was significantly higher in men (51.5 micrograms/l, i.e. 0.25 mumol/l). Smoking significantly influenced the blood Pb level in women. The 90th percentile in no group exceeded the value of 100 micrograms/l (0.48 mumol/l). The median blood Cd level in adults (0.9 microgram/l, i.e. 0.008 mumol/l) depends on smoking habit (1.25 micrograms/l, i.e. 0.01 mumol/l). The median urine Cd level was 0.585 microgram/g creatinine (0.59 mumol/mole creatinine) in adults and 0.37 microgram/g creatinine (0.37 mumol/mole creatinine) in children. The median blood Se level (53.5 micrograms/l, i.e. 0.68 mumol/l) was found to be higher in the group of non-smokers (57.5 micrograms/l, i.e 0.73 mumol/l). Lead and selenium level were significantly lower in the umbilical blood. Cytogenetic analysis results showed age-dependent average percentages of aberrant cells: 1.1% in umbilical blood, 1.27% in children and 1.71 in adults in line with the reference values for the Czech population.

Photofrin II induces cytokine secretion by mouse spleen cells and human peripheral mononuclear cells.

The aim of our study was to find out if Photofrin II, a cytotoxic drug used routinely in photodynamic therapy (PDT), can induce immune responses in vitro, and to compare its effects with those of the protoporphyrin 9, hemin, which also has antitumor properties. We tested the effect of these porphyrins on lymphocyte proliferation and secretion of interleukin-2, interleukin-3, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), by human or murine mononuclear cells (MNC) without an activating light. Both the Photofrin II- and hemin-treated cells showed a significant increase in cytokine secretion in the presence of suboptimal concentrations of mitogen. Moreover, Photofrin II and hemin significantly increased production of TNFalpha and IFNgamma even in the absence of mitogen. The cellular binding sites of Photofrin II and hemin to MNC were localized by electromicroscopy or fluorescence. Combined stimulation of cells by mitogens and porphyrins maintained optimal vital ionic balance of potassium, sodium and chlorine in the lymphocytes. In the cells thus treated there was a significant increase in intracellular calcium, a vital second messenger for lymphokine secretion. We demonstrate that the effect of Photofrin II on the immune system involves enhanced cytokine secretion which may account for the subsequent tumor eradication by PDT.

[Effect of HE-NE laser acupuncture on the spleen in rats].

The splenic sinuses, lymphocytes and antigen presenting cells in the spleen of rat stimulated by He-Ne laser acupuncture were observed by using TEM to investigate the ultrastructural changes of them. The width of endothelial interstice was increased, hence a lot of blood cells oozed out of the splenic sinuses. There were numerous activated T cells which contained abundant mitochondria and ribosome in the periarterial lymphatic sheaths. The B cells were gradually differentiated into large lymphocytes, immature and mature plasmatic cells with a lot of endoplasmic reticula. They were prominently increased in the splenic nodules. The macrophages had short processes with numerous folds and microvilli and tended to neighboring lymphocytes. The nucleus pores were increased. There were a lot of vacuoles, phagosome and mitochondria were closed to macrophages and lymphocytes. In conclusion the activities of the cellular immunity and humoral immunity were enhanced by laser.

Steroidogenic and morphogenic characteristics of human peritubular cells in culture.

We have explored the morphogenic and functional characteristics of human peritubular cells originating from seminiferous tubule (ST) fragments isolated from the testes of two prepubertal patients with the androgen insensitivity syndrome. These ST were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 supplemented with antibiotics, transferrin, hydrocortisone, vitamin E, and 3% fetal bovine serum. A centrifugal growth of elongated fibroblast-like cells peripheral to the ST explants was observed. Muscle-specific actin and 3 beta-hydroxysteroid dehydrogenase were evident in the peritubular area and in the elongated cells growing from the tubules. Histochemistry was negative in the tubules themselves, revealing the mixed nature of these cultures. The ST fragments were lost after subculturing, leaving a homogeneous monolayer of fibroblast-like cells. The steroidogenic potential of these cells was demonstrated by the secretion of testosterone (T) to the culture medium. T secretion was stimulated by hCG in a time-dependent fashion (patient 1: Day 11, 84% and Day 15, 200%; patient 2: Day 8, 73% and Day 11, 32% over basal). FSH also stimulated T secretion (patient 1: Day 5, 136% and Day 8, 89%; patient 2: Day 8, 117% and Day 11, 129% over basal). Furthermore, T secretion by these cultures was 100% higher than that observed in mesenchymal cells obtained from the testicular intertubular space in the same patient. Spontaneous T secretion and hormone responses declined progressively to cease by 25 days in culture. These results suggested the involvement of Sertoli cell (SC)-secreted factor(s) in the regulation of T secretion by peritubular cells. In order to further explore possible paracrine interactions between peritubular and Sertoli cells, we carried out heterologous cocultures with rat SC. After 72 h a striking redistribution of both cell types was observed with the formation of cord-like structures. Ultrastructural examination of these cords showed the formation of a basement membrane between epithelial (Sertoli) and mesenchymal cells of peritubular origin. No resumption of T secretion was observed, but an increase in androgen-binding protein (ABP) production by rat SC under basal (37%) and FSH-stimulated (52%) conditions was evident. Our results show that in the human peritubular compartment, cells exist that can alternatively express steroidogenic functions, associate with SC in a specific mesenchymal-epithelial interaction, and exert regulatory influences on ABP secretion by SC. In addition they indicate that communicating events in the testis are preserved throughout evolution.

[Immune function and abdominal surgery in blood stasis syndrome in patients with gastrointestinal diseases].

52 Patients with Blood Stasis Syndrome (BSS) in abdominal surgical diseases were divided into 3 types according to their symptoms, signs and natures of diseases. Peripheral blood T lymphocyte subsets of these BSS patients and 12 healthy persons were studied with Flow Cytometry and monoclonal antibodies. The immunoglobulins and complements of these cases were also studied. There were no difference in T cell subsets, immunoglobulins and complements between Qizhi-BSS group and normal control. The Shire-BSS group showed that CD8 cell, IgG, IgM, and C1, C3c were increased. These results showed that the immune response increased in this type of BSS patients. In the Qixu-BSS group, the CD3 was nearly normal, and the CD4+, CD4+/CD8+ ratio, and CD16+ were statistically decreased. However, the CD8+ cells markedly increased, the IgG, IgM and IgD were also lowered significantly in this type. The marked morphologic abnormal changes in ultrastructures of T lymphocyte were found in 6 patients with Qixu-BSS group. These results showed that the Qixu type of Blood Stasis Syndrome patients were in the immuno-suppressive status.

[Effect of He-Ne laser acupuncture on lymph-nodes in rats].

The lymphocytes and antigen presenting cells in lymph node of rats stimulated by He-Ne laser acupuncture were observed by using TEM and SEM to investigate the ultrastructural changes of them. There were numerous activated T-cells which showed deeply indented nucleus, abundant small void mitochondria and free ribosomes in the paracortex area. The B-cells were gradually differentiated into large lymphocytes, immature and mature plasmatic cells which with a lot of rough endoplasmic reticulum. They were prominently increased in the pulp area. The macrophages had short processes with numerous folds and microvilli and tended to neighboring lymphocytes. The nucleus pores were increased. There were a lot of pinocytosomes, phagosomes, lysosomes in various size of macrophages. The bundles (5-6 nm in diameter) of microfilaments of the macrophages were extended from the cytoplasm to the processes. The interdigitating cells which contained the characterized single layer of rER, numerous polysomes, mitochondria and well-developed Golgi-complex were closed to macrophages and lymphocytes. In conclusion the activities of the cellular immunity and humoral immunity were enhanced by laser.

Reentry of T cells to the adult thymus is restricted to activated T cells.

To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media.

CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 micrograms/ml each transferrin and insulin + 5 ng/ml sodium selenite +/- 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo. with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability greater than or equal to 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium.

Pharmacophysiology of atopic dermatitis.

Atopic dermatitis is clearly characterized by altered cutaneous physiologic responses. There is a tendency to acral vasoconstriction. Rubbing causes skin pallor and white dermographism. Vascular instability is demonstrated by responses to cholinergic agents, histamine, and nicotinates. Psychophysiologic studies demonstrate exaggerated vasodilator responses to emotional stress with consequent pruritus and scratching. The itch threshold is low, duration is prolonged, and nighttime scratching movements may be frequent or almost continuous. Regardless of the inciting trigger factors, the scratching causes the damage and the severe dermatitis. Thermal as well as emotional stimuli to sweating cause severe itching in AD, yet the concept of a miliaria-type, poral occlusion mechanism remains unproven. Some studies suggest actually increased sweating along with erythema and pruritus during acute flares of AD. The concept of sweat-borne allergens causing skin reactions during sweating is interesting but has never been proven. Studies of sweat responses to pharmacologic agents have produced conflicting data, and attempts to link these responses to Szentivanyi's beta-adrenergic blockade theory are not convincing. The numerous variables of climate, season, sex, age, and habitus affect sweating greatly. Future studies must carefully control for each of these factors before pharmacologically induced sweat responses can be interpreted clearly. A number of lines of evidence suggest involvement of histamine and other mediators in the evolution of erythema, pruritus, and scratching in AD. Flares of the condition have been reproducibly evoked by only two incitants: experimental emotional stress interviews and specific food challenge in selected sensitive individuals. In the latter, increased plasma histamine has been demonstrated, presumably generated by antigen/IgE stimulated degranulation of mast cells in the gut and/or skin. The demonstrated increased histamine releasability of basophils from atopic individuals may be the result of defective cellular regulatory mechanisms. Recent studies have demonstrated increased cyclic AMP-phosphodiesterase activity in leukocytes from atopic individuals. The resultant decreased intracellular cyclic AMP removes an inhibitory factor, which in turn causes net cellular hyperresponsiveness. This effect has been shown to account, at least in part, for increased histamine release from leukocytes of patients with AD. These and other studies focused upon cell functional regulation are providing better understanding of basic biochemical abnormalities and may lead to improved diagnostic and therapeutic approaches in managing atopic disease.