Chlorphentermine Suppresses the Phosphatidylinositol Pathway in Concanavalin A-Activated Mouse Splenic Lymphocytes

We have previously demonstrated that the chlorphentermine (CP)¹-induced impairment in lymphocyte blastogenesis involves drug-induced inhibition of an event which occurs very early during lymphocyte activation. An early event, which is associated with mitogen-induced lymphocyte activation, involves the hydrolysis of phosphatidylinositol by phospholipase C to yield inositol phosphates and diacylglycerol as products. Inositol phosphates and diacylglycerol then function as mediators of a trans-membrane signal for the continuation of the cellular response. It was the purpose of the present study to determine the effects of CP on this phosphatidylinositol pathway. We demonstrated that formation of inositol phosphates in lymphocytes increases progressively above control over a 2 hour period following concanavalin A (Con A)-stimulation. In contrast, lymphocytes pre-incubated with 10^-5M CP for 60 min, then stimulated with Con A for 2 hours in the presence of 10^-5M CP, exhibit a significantly depressed inositol phosphate formation. In addition, CP also inhibited the activity of phospholipase C (IC₅₀ = 0.58 mM), the enzyme responsible for the formation of inositol phosphates during lymphocyte activation. Further, lymphocytes activated in a manner that bypasses the phosphatidylinositol pathway are not inhibited by 10^-7M or 10^-9M CP as are cells activated with Con A. These results suggest that the suppression of the phosphatidylinositol pathway may be involved in the inhibition by CP of lymphocyte blastogenesis induced by Con A.     

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.     

Anatomical and affinity state comparisons between dopamine D1 and D2 receptors in the rat central nervous system

The anatomical distributions and affinity states of dopamine D₁ and D₂ receptors were compared in the rat central nervous system using quantitative autoradiography. [³H]SCH23390 and [³H]spiperone (in the presence of 100 nM mianserin) were used to label the D₁, and D₂ receptors, respectively. The densities of D₁ and D₂ receptors displayed a positive correlation among 21 brain regions (Pearson correlation coefficient, r = 0.80, P < 0.001).

The affinity states for the D₁ and D₂ receptors were found to be quite different from each other, and different from the results obtained by others using homogenate preparations. Both the D₁ and D₂ receptors were best modeled using a two-state model. In the absence of exogenous guanine nucleotides and using the nonselective agonist dopamine as the competitor, the D₁ receptor was primarily in a low affinity agonist state (R_H = 21 ± 6%), whereas the D₂ receptor was primarily in the high affinity agonist state (R_H = 77 ± 3%). In the presence of 10 μM guanylyl-imidodiphosphate orguanosine-5'-O-(2-thiophosphate) both the D₁ and the D₂ receptor were completely in a low affinity agonist state (R_L = 100%). These affinity states were found both in the nucleus accumbens and olfactory tubercle using dopamine as the competitor and in the striatum using selective D₁ or D₂ agonists as competitors.

Receptor occupancy of the D₂ receptor with either an agonist or antagonist did not alter the affinity states of the D₁ receptor, and conversely, receptor occupancy of the D₁ receptor did not alter the affinity states of the D₂ receptor.

The correlation between densities of D₁ and D₂ receptors provides an anatomical framework for evaluating behavioral and electrophysiological evidence of an interaction between the two dopamine receptor subtypes. This interaction does not appear to be due to a sharing or coupling of G-proteins in such a way that binding to one dopamine receptor subtype alters the affinity state of the other receptor subtype. The differences between dopamine receptor distributions described by labeled agonists and antagonists may be due in part to differences in their affinity states. The low proportion of high affinity state D₁ receptors may explain some of the difficulties in assigning specific behavioral roles to the D₁ receptor.     

Modulating Effects of Sensory and Autonomic Neuropeptides on Murine Splenocyte Proliferation and Cytokine Secretion Indd by Leishmania Major

The intimate, bidirectional link between neuroendocrine and immune systems is now accepted. A modulating effect of the nervous system on immune and inflammatory responses has been corroborated by identification of neuropeptide receptors on immunocompetent cells and the finding that neuropeptides can regulate leukocyte functions. The present study was undertaken to investigate the possible immunomodulatory role of sensory (SOM, CGRP and SP) and autonomic (VIP and NPY) neuropeptides in a murine model of cutaneous leishmaniasis, using two genetically different inbred mouse strains, BALB/c and C57BL/6, respectively susceptible and resistant to Leishmania (L.) major infection. The parameters studied were extent of splenocyte proliferation, as measured by thymidine uptake, and the ability of these cells to secrete IFN-γ and IL-4 by using a two-site ELISA, upon in vitro challenge with L. major parasites and addition of the neuropeptides. The resistant mouse splenocyte proliferation was enhanced by SOM, CGRP, and VIP at 10^-5', 10^-6 and 10^-9 M concentration, respectively, but was inhibited by NPY at 10^-5M. Proliferation of the splenocytes from the susceptible strain was inhibited by SOM (10^-11 M) and CGRP(10^-5 M). Somatostatin, at various concentrations, stimulated IFN-γ secretion in both mouse strain splenocytes, and IL-4 production in the susceptible mouse. Calcitonin gene-related peptide enhanced IFN-γ secretion in susceptible mouse splenocytes at 10^-6 10^-7 and 10^-9 M, as did VIP at 10^-10 M and NPY at 10^-7 M. Vasuactive intestinal peptide also stimulated IL-4 production in BALB/c splenocytes at all concentrations used Substance P had no effect on either cell proliferation or cytokine sccrction in eithcr of the two mouse strains.

These findings indicatc that the nervous system, represented by sensory and autonomic nerve terminals and their content of neuroinediators, may be involved in the pathophysiology of cutaneous leishmaniasis.     

Characterization of a neuronal interleukin-1 receptor and the corresponding mRNA in the mouse anterior pituitary cell line AtT-20.

The serotonin (5-HT)_1A agonist, LY 165,163 (1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine), also known as PAPP, has been suggested to exert effects via an interaction with dopamine receptors. Thus, in this study, we examined its ability to induce rotation in rats sustaining unilateral 6-hydroxy-dopamine lesions of the substantia nigra, an in vivo model of dopaminergic activity. In analogy to the direct dopamine (mixed D₁/D₂) agonist, apomorphine, (0.01–0.63 mg/kg), LY 165,163 (0.16–10.0 mg/kg) dose-dependently elicited robust and substained contralateral rotation. Its maximal effect was comparable to that of apomorphine and its duration of action more extended. Rotation elicited by LY 165,163 (10.0 mg/kg) was resistant to the 5-HT_1A antagonist, (−)-alprenolol. It was also unaffected by the selective D₁ antagonist, SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5,tetraphydro-1H-3- benzazepine) (2.5 mg/kg) or the selective D₂ antagonist, raclopride (10.0 mg/kg) when each was administered alone. However, upon joint administration they clearly diminished the effect of LY 165,163. The dopamine antagonist, haloperidol (D₂ > D₁) also reduced the action of LY 165,163. This profile of partial antagonism by mixed D₁ and D₂ receptor blockade has been reported previously for apomorphine and contrasts to that seen with selective D₁ or D₂ agonists, the actions of which are completely blocked by D₁ or D₂ antagonists, respectively. In conclusion, the present data demonstrate that LY 165,163 exerts pronounced rotation in nigral-lesioned rats: this reflects a mixed D₁/D₂ action rather than an activation of 5-HT_1A sites. Thus, in addition to an agonist action at 5-HT_1A receptors, dopaminergic effects contribute to the pharmacological profile of LY 165,163.     

Chemiluminescence in Human Whole Blood: Modulation by the Cocarcinogens Phorbol Diester and Polychlorobiphenyls

A human whole blood chemiluminescence (CL) assay was established using zymosan as cell activator. Aroclor 1254 was found to inhibit this CL response in a direct linear relation to its concentration, (50% inhibitory dose, (ID₅₀) equal to 5 × 10^-4 M) in diluted blood samples of 10 normal human subjects. In comparison the ID₅₀ of other inhibitors was 1.3 × 10^-3 M for ethylenediamine tetraacetic acid, 3.3 × 10^-3 M for ascorbic acid, 4 × 10^-3 M for reduced glutathione, 1.2 × 10^-3M for ethanol, 2.5 × 10^-1 for methanol and 3.7 × 10^-1 M for dimethyl sulfoxide. Using 12-o-tetradecanoyl-phorbol-13-acetate (TPA) as cell activator the CL response was likewise inhibited by Aroclor 1254 with an ID₅₀ of 4.5 × 10^-4 M. However, it was found that Aroclor 1254 alone has a stimulatory CL effect on otherwise unactivated cells. To compare the mechanisms involved in the CL elicited by the three stimulants zymosan, TPA and Aroclor 1254, the CL signal was measured in the presence of cytochalasin B. Cytochalasin B inhibited zymosan-induced CL, had a smaller inhibitory effect on TPA-induced CL but it could augment the CL response initiated by Aroclor 1254. This pattern of responses implicates Aroclor 1254 in the activation of eicosanoid metabolism as it matches the differential responses reported for arachidonic acid.     

NEUROPEPTIDES MODULATE A MURINE MONOCYTE/MACROPHAGE CELL LINE CAPACITY FOR PHAGOCYTOSIS AND KILLING OF LEISHMANIA MAJOR PARASITES

Host-parasite interactions and their outcome constitute a critical and challenging step in disease establishment in cutaneous leishmaniasis. In the present in vitro study we investigated the possible modulating effects of both sensory and autonomic neuropeptides that normally exist in human and mouse skin, on the uptake and leishmanicidal capacity of macrophages on Leishmania (L.) major parasites, using a monocyte/macrophage murine cell line (Raw 264.7). The sensory neuropeptides somatostatin (SOM), calcitonin gene-related peptide (CGRP) and substance P (SP) suppressed the macrophage capacity for phagocytosing L. major promastigotes at different concentrations, 10^-10-10^-5M, however, the suppressive effect of SP does not reach a significant level. CGRP and SP enhanced the leishmanicidal capacity of macrophages at 10^-7M, and 10^-5 M, respectively, whereas SOM was without effect.

The autonomic neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) both suppressed the phagocytic and leishmanicidal capacities of macrophages at various concentrations, 10^-10-10^-5M.

The findings indicate that neuropeptides have modulating effects on macrophage-L. major interactions. These effects might be exerted by a direct action on macrophages or indirectly through induction of other mediators.     

The Effects of Cytochalasins on Lymphocytes: V. Interaction of Trifluoperazine and Cytochalasin B in Inhibition of Human Lymphocyte Proliferation

Trifluoperazine (TFP), a phenothiazine derivative, is known to inhibit calmodulin-mediated phenomena. We report here that TFP reversibly inhibited lymphocyte proliferative responses to mitogenic lectins. This inhibition was observed only when TFP was added during the early stages of exposure of lymphocytes to the stimulus. Furthermore, at sub optimally inhibitory concentrations of each compound, effects of TFP on lymphocyte proliferation were additive to those of cytochalasin B (CB). Incubation of lymphocytes in TFP (10^-5 -10^-4 M) markedly inhibited cytochalasin B binding to the actin associated, low affinity binding site without affecting its binding to the high affinity site or to the medium affinity site. This effect developed gradually during incubation with TFP, becoming demonstrable after 30 minutes reaching maximum after 30-60 min of incubation at 37.

The findings suggest the occurrence of an interaction of TFP with the lymphocyte cytoskeleton, which may play a role in the impairment in the transmission of the mitogenic signal.     

组胺对星形胶质细胞Egr-1表达的调节作用

 目的: 探讨组胺对星形胶质细胞早期反应生长因子-1(Egr-1)表达是否具有调节作用。方法: 将野生型(WT)和组氨酸脱羧酶敲除(HDC-KO)小鼠及组胺处理的HDC-KO小鼠取脑,并提取皮层组织总RNA。将原代培养的大鼠皮层星形胶质细胞分别给予不同浓度的组胺(10^-8、10^-7、10^-6、10^-5或10^-4 mol/L)处理15、30、60、120或240 min。组胺H₁、H₂受体拮抗剂分别于组胺给药前15 min加入。组胺处理完毕后,提取细胞总RNA或蛋白。利用real-time PCR和Western blot测定Egr-1的表达。结果: 与WT小鼠相比,HDC-KO小鼠大脑皮层Egr-1的mRNA表达量显著降低,而外源性给予组胺则能促进其Egr-1的mRNA表达。在培养的星形胶质细胞上,组胺可促进Egr-1的mRNA表达,其中10^-5 mol/L的组胺作用最强,而组胺(10^-5 mol/L)处理30 min时Egr-1的mRNA表达量达到峰值,相应的Egr-1蛋白表达于60 min时显著增高,该作用可被组胺H₁受体拮抗剂而非H₂受体拮抗剂显著抑制。结论: 组胺对大脑皮层组织及培养的星形胶质细胞Egr-1表达具有上调作用,该作用与激动组胺H₁受体有关。     

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。     

Serotonin and Serotoninergic Agents Affect Proliferation of Normal and Transformed Lymphoid Cells

Blastogenic transformation of murine spleen cells elicited with concanavalin A was suppressed by serotonin 10^--¹² to 10^--⁶ M, and marginally stimulated by its antagonists ketanserin and propranolol in low concentrations (10^--¹⁵ to 10^--¹¹ M). Ketanserin (5-HT₂ receptor ligand) and propranolol (5-HT_1A and beta-adrenergic ligand) did not block the suppressive effect of serotonin if used along with it in equimolar concentrations (10^--⁹ M). Ergot-alkaloid dihydroergosine suppressed, whereas dihydroergotoxin stimulated the bldstogenic transformation. Opposite effects of the agents were obtained in experiments with mouse myeloma X63/Ag 8.653 and hybridoma SHV 125 cell lines, which unlike normal lymphoid cells, are homologous cell populations and proliferate spontaneously. The data indicate that serotonin and its antagonists interfere directly with mitosis and/or autocrine stimulation of target cells.     

体素内不相干运动弥散加权成像在受压腰骶神经根诊断中的应用

目的 探讨MR体素内不相干运动(IVIM)弥散加权成像(DWI)在受压腰骶神经根诊断中应用的可行性。方法 前瞻性对照研究。纳入2017年4—10月30例腰椎间盘突出致神经根受压患者(观察组)的常规腰椎MR序列及3D-Fiesta序列、IVIM-DWI序列图像;另按性别、年龄匹配纳入30名健康志愿者作为对照组。在GE ADW 4.6工作站,使用MADC软件包测量对照组双侧L₄、L₅、S₁神经节的扩散系数(D)、灌注相关扩散系数(D*)、灌注分数(f)和表观扩散系数(ADC)值,以及观察组患者受压侧及其对侧神经根的D、D*、f、ADC值。比较对照组同节段左右两侧神经节和不同节段神经节各观察项目测量值,以及观察组受压侧神经根与对侧正常神经根各观察项目测量值。绘制受压神经根D值和ADC值的ROC曲线,评价诊断效果。结果 对照组L₄、L₅、S₁神经根的D值分别为(0.603±0.064)×10^-3 mm²/s、(0.624±0.079)×10^-3 mm²/s、(0.628±0.088) ×10^-3 mm²/s, D*值分别为(3.815±0.541) ×10^-3 mm²/s、(3.862±0.414)×10^-3 mm²/s、(3.915±0.611) ×10^-3 mm²/s; f值分别为0.454%±0.076%、0.484%±0.101%、0.445%±0.094%; ADC值分别为(0.934±0.085)×10^-3 mm²/s、(0.945±0.051)×10^-3 mm²/s、(0.953±0.064)×10^-3 mm²/s。观察组神经根受压侧D、D*、f、ADC值分别为(0.669±0.081)×10^-3 mm²/s、(3.852±0.776)×10^-3 mm²/s、0.528%±0.115%、(1.096±0.087)×10^-3 mm²/s,健侧D、D*、f、ADC值分别为(0.617±0.080)×10^-3 mm²/s、(3.961±0.684)×10^-3 mm²/s、0.479%±0.083%、(0.938±0.074)×10^-3 mm²/s。对照组同节段左右两侧神经节和不同节段神经节所测D、D*、f、ADC值,差异均无统计学意义(P值均＞0.05)。观察组受压侧神经根与对侧正常神经根比较:D和ADC值均升高,差异均有统计学意义(P值均＜0.01);D*、f值差异均无统计学意义(P值均＞0.05)。绘制并分析ROC曲线,D值对诊断神经根受压具有较高效能,其次是ADC值,D值的AUC为0.923(95%CI 0.803~0.987),ADC值的 AUC为0.895(95%CI 0.865~0.999)。结论 IVIM模型的MR DWI技术可用于腰骶神经根检查,且与单指数模型的MR DWI相比能更详细、准确地反映神经根受压后的病理改变。     

Observation of the Effect of Substance P on Human T and B Lymphocyte Proliferation

In the present experiment we attempted to confirm the specific stimulation of substance P (SP) on human T lymphocyte proliferation as reported by Payan et al. and to observe its effect on human B lymphocyte proliferation. As a result, no definite mitogenic effect of SP (10^-11 to 10^-7 M) was found on both human T and B cells, no significant influence of SP on the blastogenic response of T cells to Con A or PHA stimulation in a wide range of concentrations and no effect on proliferative response of B cells to Staphylococcus aureus Cowen strain I stimulation were shown. Payan's conclusion was based on percentage increase of ³H-TdR incorporation. But as the background cpm is generally very low, even 100 per cent increase would only indicate a very small cpm increase, so in this situation percentage increase might give false phenomenon, and only cpm could tell whether the effect was strong or weak. Thus, we believe that is the reason for our disagreement with Payan's conclusion.     

Effects of dopamine D3 receptor agonists on pilocarpine-induced limbic seizures in the rat

The discrete localization of D₃ receptors in the nucleus accumbens and subjacent islands of Calleja bears a close resemblance to the dopamine-sensitive anticonvulsant site in the anteroventral striatum. To determine if these D₃ receptors were capable of attenuating limbic motor seizures induced by pilocarpine, dopamine agonists with preferential or non-selective D₃ affinity were injected stereotaxically into these limbic brain regions of the rat via indwelling cannulae prior to pilocarpine challenge. Reliable clonic seizures were obtained by administering the proconvulsive dopamine D₁ agonist SKF 38393 (10mg/kg i.p.) followed by a subconvulsant dose of pilocarpine (280–300 mg/kg i.p.). Bilateral intra-accumbens pretreatment with the D₃ > D₂ agonist RU 24213 (0.2 pmol-7 nmol) significantly delayed the onset of seizures, with a minimum effective dose of 2 pmol, without altering their frequency or severity. The more selective D₃ agonist LY 171555 (0.2 pmol-7.8 nmol) was less potent, and only attenuated pilocarpine-induced seizures at a dose (500 pmol) that would have stimulated accumbens D2 receptors as well. Intra-accumbens injections of the highly potent and selective D₃ agonist 7-OH-DPAT (20 pmol to 7 nmol) afforded no protection against pilocarpine-induced seizures. Apomorphine, a mixed D₁D₂D₃ agonist, delayed seizure onset at 100–500 pmol, but not at higher doses. RU 24213, LY 171555 and 7-OH-DPAT were all modestly anticonvulsant when microinjected into the islands of Calleja at D₂D₃ unselective doses.

These data support the notion that dopamine systems limit seizure propagation through the limbic forebrain, but suggest this protective effect is mediated by D₂ rather than D₃ receptors.     

Interleukin-2 Induced Mitogenesis of Human Peripheral Blood T-Lymphocytes: Role of Accessory Cells

We and others have shown that Interleukin-2 (IL-2) is mitogenic to a subset of unstimulated T lymphocytes in human peripheral blood (1-7). We extend our work here in showing that prolonged continuous exposure of human peripheral blood lymphocytes to exogenous IL-2 throughout the 7-8 day culture is not necessary since mitogenesis occurs reproducibly after short term (2-3 hr) pulse exposure. The mitogenic effect of pulse exposure to IL-2 is not significantly reduced by inclusion of anti-Tac monoclonal antibody in the pulsing medium. However, anti-Tac monoclonal antibody markedly inhibits the response if present continuously throughout the 7 day culture.

The mitogenic effect of IL-2 is dependent on the presence of accessory cells (monocytes) but the accessory cell requirement can be replaced by the phorbol ester TPA (10^-8 to 10^-11 M). Purified monocytes subjected to short term pulse exposure to IL-2 can cause proliferative response in unprimed autologous lymphocytes in cocultures. The mitogenic effect of IL-2 pulsed monocytes can not be suppressed by inclusion of anti-Tac antibody in the pulsing medium although the same concentration of the antibody suppresses the effect if present throughout culture. The response of lymphocytes to IL-2 pulsed monocytes is not inhabitable by the continuous presence of a monoclonal antibody to human HLA-DR antigens (OK-Ial) in culture.     

Role of CD4+ regulatory T cells in hyperbaric oxygen-mediated immune nonresponsiveness

We have previously shown that hyperbaric oxygen culture (HOC [95% O₂, 5% CO₂, 25 psi]) is an effective pretransplant tissue-modification technique that results in long-term allograft survival and the induction of systemic immune tolerance in a murine model. Here we address the immune modulatory effects of HOC-treatment of human immune responses using the in vitro mixed lymphocyte reaction (MLR). Pretreatment of allogeneic stimulator cells with HOC results in abrogation of cytotoxic T lymphocyte (CTL) activity, proliferative responses, and IFNγ production in a 7-day MLR. These responses can be restored either by the addition of IFNγ or IL-2 on day 0, or by blocking the activity of IL-4 and IL-10. The addition of IL-2 on day 4 does not restore allospecific CTL activity. The failure of HOC-treated cells to induce allospecific CTL is not due to the induction of anergy, demonstrated by the failure to restore responses after restimulation with allogeneic cells in the presence of IL-2. Removal of CD4⁺ cells prior to restimulation, results in restoration of CTL activity in MLR cultures restimulated with HOC-treated allogeneic cells. These results suggest that HOC-induced immune nonresponsiveness is mediated by the development of CD4⁺ regulatory cells in a Th2-type environment.     

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.     

Effects of Dynorphin on the Pha-Induced Lymphocyte Proliferation in Vitro

The effect of the opioid peptide dynorphin (DYN) on PHA-induced lymphocyte proliferation has been evaluated in the present study.

A significant increase in PHA-induced lymphocyte activation was observed when DYN was added to cultures 48 hr after the mitogenic stimulation. This effect occurred at suboptimal (3.12 and 6.25 ug/ml) PHA concentrations and at DYN doses ranging from 10^-9 to 10^-12 M. Conversely, DYN did not affect the lymphocyte blastogenic process either when added before, or simultaneously or after intense PHA (12.5 ug/ml) stimulation.

Naloxone preincubation did not modify the above described effects.

Results suggest that DYN might play a role in neuroendocrine regulation of the lymphocyte blastogenic process.     

Gastrointestinal Regulatory Peptides Modulate Mouse Lymphocyte Functions Under Serum-Free Conditions In Vitro

Conditions are described for performing mitogen (Concanavalin A, Con A; lipopolysaccharide, LPS) and mixed lymphocyte reaction (MLR) cultures using serum-free medium. The effects of exogenously adding several gastrointestinal regulatory peptides (β-endorphin, substance P, met-enkephalin, vasoactive intestinal peptide, bombesin and somatostatin) on the incorporation of ³H-methyl-thymidine was determined. It was observed that mitogen stimulation of lymph node cells with Con A was inhibited (70% of control) by vasoactive intestinal peptide (VIP) but spleen cells stimulated by LPS were insensitive to immunomodulation (98% of control). The ability of VIP to inhibit Con A induced thymidine incorporation was concentration dependent (10^-6 to 10^-18 M) and was not attributable to kinetic shifts or cell toxicity. None of the other tested neuropeptides affected Con A or LPS induced blastogenesis. MLR cultures were inhibited by VIP, β-endorphin and somatostatin in a biphasic manner with maximal inhibition observed at 10^-8 to 10^-12 M. Both substance P and bombesin exhibited slight immunoenhancing properties at 10^-14 to 10^-18 M. Met-enkephalin was ineffective as an immunomodulator of MLR cultures. The utility of using serum-free medium in identifying neuropeptides with immunomodulatory properties are discussed.