Inhibitoty Effects of Local Anesthetics on Migration, Extracellular Release of Lysosomal Enzyme, and Superoxide Anion Production in Human Polymorphonuclear Leukocytes

This study examined the effects of four typical local anesthetics, lidocaine, prilocaine, procaine and tetracaine, on the functioning of human polymorphonuclear leukocytes (PMN). PMN were stimulated by fMet-Leu-Phe (FMLP) or phorbol myristate acetate (PMA) to elicit chemotaxis, extracellular release of beta-glucuronidase (BGL) and superoxide anion (SOA) production. the four agents inhibited chemotaxis efficiently and in a concentration-dependent manner but had only weak effects on the release of BGL. the effect of tetracaine was strongest, followed by lidocaine, then prilocaine, whereas the effect of procaine was blunt. the 50% inhibitory concentrations (IC₅₀ in molarity) of the four local aesthetics for chemetaxis were as follows: tetracaine=4.1×10^-4, lidocaine=3.2×10^-3, prilocaine=3.6×10, procaine=4.9×10^-3, those for SOA production induced by FMLP were : tetraaine=3.1×10^-4, lidocine=5.9×10^-3, prilocaine=1.9×10^-2, procaine=1.2×10^-2, those for SOA production indced by PMA were : tetracaine=1.1×10^-3, lidocine=1.2×10^-2, prilocaine=1.5×10-2, procaine=2.5×10^-2, and those for rlease of BGL were : tetracaine=1.6×10^-, lidocaine=5.3×10^-3, prilocaine=2.8×10^-2, procaine=1.2×10^-1. the IC₅₀ seemed to relate to the anesthetic's chemical structures and their inhibitory properties on PMN functions, as lidocaine and prilocaine, which are aminoamide type anesthetics, preferentially inhibited chemotaxis, whereas tetracaine and procaine, aminoester type anesthetics, inhibited SOA production induced by FMLP. the results suggest that the inhibitory effects of local anesthetics on human PMN functions are also correlated with local anesthetic potency and vary according to differences in their chemical structures.     

The Modulatory Effect of Exogenously Added Prostaglandin E1 Or E2 On the Delayed-Type Hypersensitivity Reaction of Normal or Tumored Mice

The effect of exogenously added prostaglandin (PG) E₁ or E₂ over concentration ranges of from 1 × 10^-4 to 1 × 10^-9 M were studied in order to determine their effect on the delayed-type hypersensitivity (DtH) reaction of normal or tumored mice. PGE or PGE₂. generally caused a stimulation over the control values of normal mice as detected by the footpad swelling assay. However, PGE¹ or PGE₂ at all concentrations tested were found to significantly inhibit the DtH reaction of CD₂F₁ tumored mice.     

Influence of Salbutamol and Isoproterenol on the Production of TNF and Reactive Oxygen Species by Bovine Alveolar Macrophs and Calcitriol Differentiated HL-60 Cells

The influence of the β-adrenergic agonists Salbutamol and Isoproterenol on the release of reactive oxyen species and Tumor Necrosis Factor (TNF) was tested with bovine alveolar macrophages and HL-60 cells differentiated to macrophages by calcitriol. The production of reactive oxygen species was analyzed by a microplate assay using dichlorofluorescein-diacetate. It could be shown that this method almost exclusively measures superoxide anions. TNF was determined by a bioassay with WEHI cells. The superoxide anion production was stimulated by Zymosan, the TNF release by LPS. By incubation with 5×10^-6 and 5×10^-7 M Salbutamol or 5×10^-7 and 5×10^-8 m lsoproterenol prior to the stimulation, the production of superoxide anions as well as of TNF was inhibited to a significant deree. The inhibitory effects of the adrenergic agonists were completely or at least partially inhibited by the respective antagonists, ICI 118.551 and Propanolol, respectively.     

Pretreatment of Human Peripheral Blood Lymphocytes with Interleukin-2 or Dexamethasone Does Not Alter Their Response to Met-Enkephn in A NK-Cmotoxic Assay

The effect of Met-Enkephalin (MENK; 10^-12 - 10^-8 M) on NK-activity of peripheral blood lymphocytes (PBL) after in vitro treatment (18 h, 37 °C) was examined in 30 young, healthy male donors. In the group as a whole (n = 30), no significant effect of MENK was detected. At the individual level, 18 of 30 donors (60%) responded to MENK either by mild enhancement (up to 8%, 8 responders), or by mild attenuation (up to 12%, 10 responders) of the basal NK-activity. The effect of MENK was donorrelated regarding the dose-response, E/T ratio, and direction of MENK action. The influence of pretreatment of PBL (1 h) with either graded doses of interleukin-2 (IL-2; 3, 25, 50 U/ml) or dexamethasone (Dex; 2.5 × 10^-9, 2.5 × 10^-8, 2.5 × 10^-7 M), on the effect of MENK was also tested. The idea was that pretreatment of PBL would result in predictable, and/or stronger response to MENK. In the group as a whole again no significant effect of MENK was detected on the NK-activity of PBL prestimulated by IL-2 (n = 16), or inhibited by Dex (n = 12). Further, pretreatment of PBL with IL-2/Dex did not significantly alter the intensity of modulation by MENK, which was generally mild. The data obtained have shown that pretreatment of PBL with IL-2 or Dex, regardless of their concentrations, did not significantly alter the frequency of responders to MENK being 50%, 62.5% and 64.3% with 3, 25 or 50 U/ml IL-2, respectively, and 50%, with all concentration of Dex used, as compared to that observed with resting PBL (60%). However, at the individual level physiological concentrations of MENK (10^-12 - 10^-9 M) induced enhancement or/and attenuation of the NK-activity pretreated with IL-2/Dex, respectively. The effect of MENK at the individual level was donor-related regarding the dose-response, E/T ratio, and direction of MENK action. Thus, pretreatment of PBL with graded concentrations of IL-2/Dex did not alter the effect of MENK on NK-activity, regarding the frequency and intensity, as well as the direction of modulation: it remained bidirectional, of low intensity, and independent of the grade of PBL preactivation/inhibition, therefore unpredictable.     

Expression of heat shock proteins 65, 72 and 90 in mercuric chloride-treated human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) were challenged with different concentrations of mercuric chloride and the expression of heat shock protein (hsp) 65, 72 and 90, were investigated by an indirect peroxidase method as well as the proliferation was measured by uptake of tritiated thymidine into DNA. Hsp 65 immunoreactivity was peripherally located in the cells at a mercuric chloride concentration of 5.5-2.8×10^-5 mol/L, which also gave a decreased DNA synthesis. Hsp 72 positive cells, with a nuclear and cytoplasmic immunoreactivity, were found at a concentration of 5.5-2.2×10^-5 mol/L. Hsp 90 positive cells also with an immunoreactivity at a nuclear and cytoplasmic level, were found at 1.1×10^-5-5.5×10^-6 mol/L. A stimulated DNA synthesis was obtained already at 2.2×10^-5 mol/L and was found down to 5.5×10^-6 mol/L. High concentrations of mercuric chloride might induce expression of hsp 65 and 72 as a protective mechanism. Hsp 90 as well as hsp 72 might be involved in the proliferation of human PBMC to mercuric chloride. Thus, this expression of hsp 72 may have a dual role.     

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte pha gocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10^-5 and 10^-6 M concentrations/ 4 × 10⁶ phagocytes. 10^-7 M con centration was not effective in all the instances.

These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.     

Effect of Thyroxine on the Immune Response of Mice in Vivo and in Vitro

The effect of thyroxine on the immune response of BALB/c mice to sheep erythrocytes was investigated. In mice rendered hypertiyroid by subcutaneous injections of T4 the primary immune response to an injection of SRBC in vivo did not show a consistent increase in splenic anti-SRBC plaque-forming cells. However, the total number of splenic cells in T4-treated mice was generally decreased, and thus, the number of PFC per 10⁶ splenic cells in T4-treated mice was higher than those of saline and buffer controls. In in-vitro primary response to SRBC PFC per culture (3×10⁷ splenic cells) increased significantly in T₄-injected animals as compared with controls. The calculated PFC per spleen also increased significantly. The addition of T₄ to normal splenic cell cultures enhanced the primary immune response to SRBC in vitro. The optimum concentration of T₄ was found to be 10^-8 M. These results indicate a direct enhancing effect of T₄ on the immune response of lymphoid cells. This enhancing effect, however, may be attenuated in vivo by the alteration of the number and/or composition of lymphoid cells brought about directly or indirectly by injections of exogenous thyroxine.     

Acetylcholine receptors in dissociated nucleus basalis of Meynert neurons of the rat

Electrical and pharmacological properties of acetylcholine (ACh)-induced currents in neurons dissociated from the nucleus basalis of Meynert (nBM) of immature (2-week-old) rats were investigated with the whole-cell mode of the patch-clamp technique. At a holding potential (V_H) of −50 mV, ACh (10⁻⁴M) evoked a transient inward current mimicked by nicotine (I_nACh), followed by a sustained outward current mimicked by carbamylcholine (I_mACh). The K_D values were 1.2 × 10⁻⁴ M for I_nACh) and 8.7 × 10⁻⁷ M for I_mACh. The reversal potenial of I_mACh was close to E_K. The I_mACh was determined to be elicited via the M₂ muscarinic receptor, based on the differences in sensitivity to muscarinic antagonists such as pirenzepine and AF-DX-116.     

The Effect of Delta-9-Tetrahydrocannabinol and 11-Hydroxy-Delta-9-Tetrahydrocannabinol on T-Lymphocyte and B-Lymphocyte Mitogen Responses

Previous studies have shown that delta-9-tetrahydrocanna-binol (THC) suppresses T-lymphocyte proliferation when added to human cell cultures. We report that THC when added to mouse splenocyte cultures suppressed T-lymphocyte (Con A, PHA) and B-lymphocyte (LPS) mitogen-induced proliferation. Although the ED₅₀ concentrations (5ug/ml; 1.6×10^-5 M) of THC were similar for suppressing all three mitogen responses, higher threshold concentrations of drug were required to effect suppression of the T-lymphocyte mitogen responses. Complete suppression of T- and B-lymphocyte responses was achieved with THC concentrations (8ug/ml or 2.6×10^-5 M) which were not directly toxic as judged by vital dye exclusion. The hydroxylated metabolite of THC, 11-hydroxy-THC, was observed to be much less potent in the inhibition of lymphocyte proliferation. However, as with the parent compound, B-lymphocyte responses appeared to be the most affected by the drug. Additional studies demonstrated that both T- and B-lymphocyte proliferation is rapidly suppressed following THC treatment, not affected by a 24 hr. pretreatment with THC, and not as readily suppressed by THC in cultures containing 20% serum. Thus, THC appears to inhibit both T- and B-lymphocyte proliferation with B-lymphocyte responses displaying greater inhibition at lower drug concentration. The 11-hydroxy metabolite is much less suppressive in this system than the parent compound.     

Control of the Production of Oxygen Intermediates of Human Polymorphonuclear Leukocytes and Monocytes by B-Adrenergic Receptors

The control by R-adrenergic receptors of the production of oxygen radicals by zymosan-stimulated human polymorphonuclear leukocytes (PMN) and monocytes (Mψ) was studied in vitro by means of chemiluminescence. In addition we asked whether PMN Mpsi; exhibit differential sensitivity to β-adrenergic stimulation. Eor β-adrenergic stimulation we applied fenoterol ranging from 10^-9 to 10 M × 2.7. We found a dose-dependent suppression of the production of oxygen radicals, the ID50 being approximately 10^-6 M both for PMN and Mpsi;. By assessment of lactic dehydrogenase release a cytotoxic effect of the drug could be ruled out. When incubated together with the β-adrenergic antagonist propranolol at 10^-6 and 10^-7 M the suppressive effect of fenoterol could be reversed in dose-dependency. Preincubation with fenoterol revealed that the inhibitory action on Mpsi; persisted, in contrast, no such suppression could be verified with PMN. Our findings indicate the control of the production of oxygen intermediates of human PMN and Mpsi; by β-adrenergic stimulation. Furthermore, selective functional modulation of resting PMN and Mpsi; by β-adrenoceptors is suggested. These effects may be of importance in vivo, in particular since fenoterol was applied in pharmacological doses.     

The Effect of Prostaglandin E1 or E2 OH the in Vitro Blastogenic Response of Lymphocytes from Normal and Tumor-Bearing Mice

The in vitro effect of exogenously added prostaglandin (PG) El or E₂ over concentrations ranges of from 1 × 10^--⁴ to 1 × 10-9 M were studied in order to determine their effect on the in vitro lymphocyte proliferation of thymic and splenic T and B cells from normal and tumor-bearing CD₂ F ₁ mice. It was found that PGE₁ generally caused greater inhibition of blastogenesis than did PGE₂ when reacted with splenic lymphocytes from normal mice. Indeed, PGE₂ was found to be stimulatory for both Con A^- and LPS-sensitive normal splenic lymphocytes. Both PGE_l and PGE₂ caused potent inhibition of Con A^- and PHA-sensitive splenic lymphocytes from the tumor-bearing mice. Additionally, PGE₂ was found to stimulate the LPS-reactive lymphocytes from the tumored mice. PGE₁ and PGE₂ both inhibited the Con A- and PHA-reactive thymic lymphocytes from normal mice at the lower concentrations studied, i.e., 10^--⁴ to 10^--⁶ M. Thereafter, at concentration ranoes of from 10^--⁷ to 10^--⁹ M both PGE₁ and PGE₂ were both found to be stimulatory. Finally, both PGE₁ and PGE₂ at all concentrations studied, strongly inhibited the thymic lymphocytes from tumored mice.     

数字化载银羟基磷灰石人工骨抗菌性及生物相容性

背景：载银珊瑚羟基磷灰石作为一种新型抗菌植骨材料,受到越来越多的关注,作为植入物需与人体具有良好的生物相容性。 目的：观察数字化载银珊瑚羟基磷灰石人工骨材料的抗菌性及生物相容性。 方法：将珊瑚羟基磷灰石粉末浸泡于不同浓度的硝酸银溶液,制备出不同含银量的载银羟基磷灰石,再将其与聚乳酸混合,并通过选择性激光烧结快速成形制备出具有特殊形状的数字化载银抗菌人工骨材料。 结果与结论：连续光源原子吸收光谱仪测定珊瑚羟基磷灰石浸泡于10^-2,10^-3,10^-4,10^-5 mol/L AgNO₃中制备的载银人工骨中Ag+的含量分别为2.31×10^-1%,3.18×10^-2%,6.75×10^-3%,6.05×10^-4%。体外抑菌圈实验表明浸泡10^-2 mol/L AgNO₃中制备的载银人工骨材料对金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为(13.00±1.52)mm 及(12.30±1.65)mm;浸泡10^-3 mol/L AgNO₃中制备的载银人工骨材料分别为(11.50±0.73) mm及(11.00±0.46) mm。浸泡10^-4,10^-5 mol/L AgNO₃载银人工骨材料对两种细菌均无抑菌圈产生。细胞毒性试验结果表明100%浸泡10^-2,10^-3,10^-4,10^-5 mol/L AgNO₃的载银珊瑚羟基磷灰石人工骨材料的细胞毒性分别为3级、1级、0级、0级。急性全身毒性试验表明浸泡10^-3 mol/L AgNO₃中的人工骨浸提液对小鼠无明显的急性毒性反应,具有良好的安全性。结果表明浸泡10^-3 mol/L AgNO₃中的载银珊瑚羟基磷灰石人工骨在体外对金黄色葡萄球菌及大肠杆菌有明显抗菌作用,且具有良好的生物相容性及安全性。     

Effects of lipoxin A4 on chemotaxis and degranulation of human eosinophils stimulated by platelet-activating factor and N-formyl-L-methionyl-L-leucyl-L-phenylalanine

Lipoxins are trihydroxytetraene metabolites derived through a double lipoxygenation of arachidonic acid. Lipoxin A₄ (LXA₄) was prepared by total chemical synthesis, and its capacity to modulate eosinophil migration has been evaluated. LXA₄ is a weak and partial chemotactic agent; at 10⁻⁶ M, it achieved about 20% of the response of 10⁻⁶ M platelet-activating factor (PAF). Preincubation of eosinophils with increasing doses of LXA₄ (10⁻¹⁰−10⁻⁵ M) resulted in a concentration-dependent inhibition of cell migration induced by 10^-6 M formyl-methionyl-leucyl-phenylalanine (FMLP) and 10^-6 M PAF. The concentration of LXA₄ which produced 50% inhibition (IC₅₀) of eosinophil migration was approximately 10^-6 M. LXA₄ (10^-10-10^-6 M) did not elicit ECP release or modulate ECP release induced by 10^-6 M FMLP. LXA₄ may have antiallergic properties in preventing eosinophilic migration.     

Leukotrienes (LTC4, LTD4) confer glass non-adherence on leukocytes of asthmatic individuals. Dependency on cyclooxygenase products and calcium ion

It has been suggested that cysteinyl-containing leukotrienes (LT) are important mediators in bronchial asthma. Since leukotrienes have been shown to mediate the leukocyte adherence inhibition (LAI) phenomenon observed in cancer-bearing host we have devised a modified LAI assay which determines the acquisition of non-adherence properties of leukocytes following a challenge with pure synthetic LT. Our results demonstrate that peripheral blood leukocytes of asthmatic individuals acquire non-adherence properties when challenged with pure synthetic leukotriene C₄ and D₄, a property not shared by peripheral blood leukocytes of control healthy individuals. Furthermore, we demonstrate that LT activity as manifested by the LAI assay is dependent on cycloxygenase products, since 2×10⁻⁶ M Indomethacin abrogated the LT-induced LAI and is restored by the addition of 2×10⁻⁶ M prostaglandin E₂ which is also synergistic to LT activity. Our results further suggest the possibility that leukotriene activity is dependent on calcium ions since it was negated by known calcium antagonists. It is thus suggested that the LT-induced LAI may serve as a tool for the study of the interrelationship between the metabolic pathways of arachidonic acid and calcium ion homeostasis.     

Influence of Arecoline on Immune System: I. Short Term Effects on General Parameters and on the Adrenal and Lymphoid Organs

Arecoline, a suspected carcinogenic/cocarcinogenic alkaloid was screened to explore in detail its immunomodulatory influence in murine model system. The oral LD₅₀ value for male mice was 371 mg/kg bw whereas it was 309 mg/kg bw for female mice. The subcutaneous LD₅₀ value for both sexes was 97 mg/kg bw. Only a marginal difference was observed in intraperitoneal LD₅₀ values between male (120 mg/kg bw) and female (109 mg/kg bw) mice. Arecoline was administered subcutaneously to male mice at subtoxic dose levels (5, 10, and 20 mg/kg bw) for 1, 2 and 3 weeks on a daily basis. In groups where significant decreases in body weight were present (at 20 mg/kg bw for both sexes), reductions in thymus weight were also noted. Spleen, mesenteric lymph nodes (MLN), liver, and kidney showed moderate reductions in their weights. Histopathological effects at 20 mg/kg bw included lymphocyte depletion of the thymic cortex, and the B and T lymphocyte areas in spleen and MLN. In concordance with the zona fasciculate hypertrophy of adrenals, corticosterone concentration in serum increased depending on the dose with a significant elevation at 20 mg/kg bw. While total protein, albumin, glucose, acid phosphatase and hemoglobin concentrations were not altered, increases in SG0T and SGPT levels were observed at the high dose. The white and red blood cell counts decreased in a dose-dependent manner. Marked reduction in cell number of thymus, and moderate effect on cellularity of spleen and MLN, were observed at 20 mg/kg bw. In vitro exposure of rat thymocytes to arecoline resulted in a biphasic oxygen consumption response with progressive increase in oxygen consumption, reaching a maximum value at 10^-5 M and decreasing sharply at 10^-3 M, Exogenously added substrates such as glucose, pyruvic acid and lactic acid retarded the fall in the oxygen consumption induced at 10^-3 M arecoline. These observations demonstrate the effects of arecoline on lymphoid organs, which may be due to its direct action or through the elevation of corticosterone.     

Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca²⁺]_i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca²⁺]_i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca²⁺]_i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca²⁺]_i showed La³⁺-sensitive, temporal changes in the form of [Ca²⁺]_i oscillations with a baseline [Ca²⁺]_i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca²⁺]_i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca²⁺]_i oscillations, VIP dose-dependendy (10^-12 to 10^-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca²⁺]_i in these cells, was attenuated in the presence of La³⁺ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10^-4 to 10^-3 M) and forskolin (10^-4M) had no effect on [Ca²⁺]_i oscillations, or on [Ca²⁺]_i in cells without oscillations. In cell suspensions, baseline [Ca²⁺]_i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca²⁺]_i. These findings suggest that: a) VIP modulates the amplitude of [Ca²⁺]_i oscillations generated by a cytosolic [Ca²⁺] oscillator in a subset of cells at a concentration of 10^-12M, a thousand-fold below the K_D for the VIP receptor; b) baseline [Ca²⁺] values may be related to both the ability of cells to generate spontaneous [Ca²⁺] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca²⁺]_i changes are not detectable when studied in cell suspensions.     

Effect of sodium fluoride on the murine splenic immune response to Porphyromonas gingivalis in vitro

Spleen cells from saline- and Porphyromonas gingivalis-primed mice were cultured and stimulated with or without P. gingivalis and added with or without various concentration of sodium fluoride (NaF). Cell proliferation, antigen-specific IgG antibodies and both IFN-γ and IL-10 levels were determined by a colorimetric assay, ELISA and commercial ELISA kits respectively. The results showed that NaF at concentration of 1×10^-6 M enhanced but at concentration of 1×10^-1 M abolished the immune response to P. gingivalis, suggesting that NaF at low concentration may act as an adjuvant but at high concentration may be toxic to the P. gingivalis-induced murine splenic immune response in vitro.     

Mycoplasma contamination and viral immunomodulatory activity: dendritic cells open Pandora's box

During in vitro investigations on the interaction of classical swine fever virus (CSFV) – an immunosuppressive viral pathogen – with monocyte-derived dendritic cells (MoDC) a soluble factor with a strong anti-proliferative activity for T lymphocytes was found. This activity, with an inhibitory dilution 50% (ID₅₀) of 10³–10⁷, was induced after virus infection of monocytes differentiating into DC. UV-inactivation of the supernatants and blocking experiments with a monoclonal antibody against the E2 envelope protein of CSFV initially indicated a virus-dependency. However, further investigations including filtration and centrifugation experiments as well as antibiotic treatment demonstrated the involvement of mycoplasma. This was confirmed by a Hoechst 33258 staining, PCR and mycoplasma cultures—Mycoplasma hyorhinis was identified as the contaminant. Elucidation of a mycoplasma presence occurred under conditions in which the original virus stocks prepared in SK6 cells were negative for mycoplasma using the above tests. Moreover, conventional passage of the virus on the SK6 cells used for this purpose did not reveal any mycoplasma. It was the passage of virus in MoDC rather than SK6 cells that was required to expose the contamination. Three passages of the anti-proliferative supernatants on MoDC cultures increased the ID₅₀ 10³-fold; only when these MoDC-derived supernatants were employed was the mycoplasma contaminant also detectable on SK6 cells. In conclusion, these data demonstrate that regular testing of cell lines and virus stocks for mycoplasma does not necessarily identify their presence, and that application of passage in MoDC cultures could prove an aid for identifying initially undetectable levels of mycoplasma contamination.     

Modulation of Cytoskeleton Assembly Capacity and Oxidative Response in Aged Neutrophils

Several reports have emphasized that aged polymorphonuclear cells (PMN) exhibit an impairment of superoxide anion (O^-₂) generation when triggered with formyl-methionyl-leucine-phenylalanine (FMLP) in comparison to the younger counterpart. Since microfilaments and microtubules are involved in PMN-mediated functions, in a group of old donors we assessed the effects of either actin stabilizing and disrupting agents, i.e. phalloidin and cytochalasin B, or microtubule stabilization or disruption by taxol and colchicine, respectively, on FMLP-triggered neutrophil oxidative responsiveness. Results show that phalloidin treatment, at a concentration ranging from 10^-6 to 10^-8 M, gave rise to an inhibition of O^-₂ release by aged PMN, while the same effect was seen in similarly treated young cells at a concentration of 10^-7 M only. On the contrary, cytochalasin B pretreatment led to an enhancement of O^-₂ generation in both young and aged neutrophils, even if to a lower extent in the latter group. At the same time, taxol at 10^-8 M strength inhibited young cell responsiveness, while no effects were induced by colchicine treatment. Quite interestingly, elderly neutrophil function was negatively modulated by both microtubule affecting compounds.

Alltogether, these findings suggest the possible relevance of cytoskeletal affecting compounds in the modulation of FMLP-stimulated O^-₂ release during senescence.     

Regulation of Human Cytotoxic Responses by Complement: C3, C3b and C3d Preparations Enhance Human Allogeneic Cytotoxic Responses

Complement components and complement. breakdown products have been found to participate in the regulation of the immune response. In the present study we investigated the effect of C3 and its fragments, C3b, C3c and C3d on human allogeneic cell mediated lympholysis (CML). C3 and C3b at a concentration of 275 M × 10^-9 and C3d at a concentration of 330 M × 10^-9 enhanced human allogeneic CML by at least two fold. In contrast C3C did not affect CML responses. Both C3b and C3d had to be present at the initiation of the cultures in order to exert their effect. Similar doses of C3b and C3d did not affect the mixed lymphocyte responses (3_H-thymidine uptake) while higher doses were clearly inhibitory. None of the preparations induced proliferative or cytotoxic responses in the absence of allogeneic stimulating cells. C3b and C3d added to the mixed lymphocyte cultures caused increased production of interleukin 2. We conclude that C3b and C3d facilitate allogeneic cytotoxic responses through increased production of interleukin 2.