Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.     

a-Tocopherol Content and a-Tocopherol Transfer Protein Expression in Leukocytes of Children with Acute Leukemia

α-Tocopherol (a form of vitamin E) is a fat-soluble vitamin that can prevent lipid peroxidation of cell membranes. This antioxidant activity of α-tocopherol can help to prevent cardiovascular disease, atherosclerosis and cancer. We investigated the α-tocopherol level and the expression of α-tocopherol transfer protein (α-TTP) in the leukocytes of children with leukemia. The plasma and erythrocyte α-tocopherol levels did not differ between children with leukemia and the control group. However, lymphocytes from children with leukemia had significantly lower α-tocopherol levels than lymphocytes from the controls (58.4±39.0 ng/mg protein versus 188.9±133.6, respectively; p<0.05), despite the higher plasma α-tocopherol/cholesterol ratio in the leukemia group (5.83±1.64 μmol/mmol versus 4.34±0.96, respectively; p<0.05). No significant differences in the plasma and leukocyte levels of isoprostanes (the oxidative metabolites of arachidonic acid) were seen between the leukemia patients and controls. The plasma level of acrolein, a marker of oxidative stress, was also similar in the two groups. Investigation of α-TTP expression by leukocytes using real-time PCR showed no difference between the two groups. These findings suggest that there may be comparable levels of lipid peroxidation in children with untreated leukemia and controls, despite the reduced α-tocopherol level in leukemic leukocytes.     

Dynamic characteristics of the fluorescence of human peripheral blood neutrophilic polymorphonuclear leukocytes and lymphocytes intravitally fluorochrome-stained with acridine orange]

Neutrophil segmentonuclear leukocytes and lymphocytes of the human peripheral blood vitally stained with Acridine Orange (AO) in concentrations of 250 and 330 mcg/ml show different fluorescence dynamics. The number of neutrophil segmentonuclear granulocytes with green fluorescence of nuclei decreases, whereas the number of cells with red fluorescence of nuclei increases. As a criterion of this process, time T1/2 is taken during which the number of green-fluorescent cells decreases twofold. With AO concentrations of 250 and 330 mcg/ml, T1/2 is equal to 40 or 5 minutes, resp. The nuclei of lymphocytes within a 60 minutes observation show green fluorescence. This effect is likely to be due to structural-functional peculiarities of neutrophil segmentonuclear granulocytes and lymphocytes.     

α-Aminoadipic Acid and α,ɛ-Diaminopimelic Acid in Inoculated Pea Plants (Pisum sativum) and Root Nodule Bacteria (Rhizobium leguminosarum)

Observations were made on the content of α-aminoadipic acid and α-aminophimelic acid (DAP) in pea plants, nodules and Rhizobium leguminosarum, strain HT3. The preparations were purified by ion exchange chromatography, Qualitative analyses were made by paper chromatography, and quantitative analyses by means of an automatic amino acid analysator. In the whole plant and seeds the content of α-aminoadipic acid soluble in 70% ethanol varied between 10 and 80 μg/g dry weight. The shoot and red nitrogen fixing nodules contained more of this acid than roots and green inactive nodules. In the insoluble fraction of the shoot its concentration was 0.4-0.6 mg/g dry weight. α-Aminoadipic acid was not found in free living rhizobia, which again contained a considerable amount of α-aiaminopimelic acid, about 0.5 mg/g dry weight. The synthesis of DAP was intensive also in root nodules. In red nodules, which fixed molecular nitrogen, the content of DAP was 2.1 mg/g dry weight and in green inactive nodules 1.3 mg/g dry weight. It was shown that in the nodules DAP is closely connected with cell wall peptides of bacteroids. DAP could not be found in pea plants outside the nodules.     

Human mononuclear phagocyte-associated antigens: I. Identification and characterization

Rabbit antisera were produced against a lymphokine-activated human macrophage cell line, U937 (αU937), and human peritoneal macrophages (αPEMØ). After absorption with AB erythrocytes, pooled platelets, and B-lymphoblastoid cell lines, both antisera reacted by microcytotoxicity, indirect immunofluorescence (IF), and radioimmunoassay (RIA) with adherence-purified human peripheral blood monocytes, splenic and peritoneal macrophages, and leukemic myelomonoblasts. A panel of normal human T lymphocytes, B lymphocytes, and erythroid-myeloid or lymphoblastoid cell lines failed to react with both αU937 and αPEMØ. Although both heteroantisera reacted against polymorphonuclear leukocytes (PMNs), after absorption with PMNs specific reactivity against mononuclear phagocytes remained. Absorption of αU937 and αPEMØ with myelomonoblastic leukemia cells (AMML) removed IF and RIA activity against both PMNs and monocytes but not against splenic and peritoneal macrophages. In contrast, absorptions of both heteroantisera preparations with splenic macrophages abolished their IF and RIA reactivity not only to splenic and peritoneal macrophages but also to peripheral blood monocytes and leukemic myelomonoblasts. These results are consistent with (1) both antisera defining specific monocyte/macrophage-associated antigens(s) which are distinct from MHC-coded HLA-A,B,C, and DR antigens, and (2) expression of common monocyte/macrophage-associated antigen(s) and uniquely associated antigen(s) selectively expressed on tissue macrophages. These reagents will be useful in delineating human monocyte/macrophage differentiation as well as the immunological functions of mononuclear phagocytes.     

淋巴细胞合成的内源性儿茶酚胺对T细胞增殖功能的影响

目的：提供淋巴细胞合成儿茶酚胺（CAs）的证据,并探讨淋巴细胞合成的内源性CAs对淋巴细胞自身功能的影响及其受体介导机制。方法：用RT-PCR技术检测CAs合成的限速酶酪氨酸羟化酶（TH）mRNA在大鼠肠系膜淋巴结细胞内的表达。用单胺氧化酶（CAs降解酶）的抑制剂优降宁及α1、α2、β1和β2肾上腺素受体（AR）的拮抗剂作用于淋巴细胞．然后用四唑蓝比色法测定淋巴细胞对刀豆蛋白A（ConA）刺激的增殖反应。结果：大鼠肠系膜淋巴结细胞具有,TH mRNA的表达,并且淋巴细胞在用ConA刺激活化后,其TH mRNA的表达明显上调。10^-6和10^-5mol／L优降宁能显著抑制ConA诱导的T淋巴细胞增殖,而10^-7mol／L优降宁不能明显降低T淋巴细胞的增殖反应。β2-AR拮抗剂ICI 118551（10^-7和10^-6mol／L）可完全阻断优降宁（10^-5mol／L）对T细胞增殖的抑制作用;α1-AR拮抗剂柯喃因和α2-AR拮抗剂育亨宾部分阻断优降宁抑制T细胞增殖的作用;而β1-AR拮抗剂阿替洛尔不能阻断优降宁的抑制作用。结论：淋巴细胞具有合成CAs的能力,这种合成能力随着淋巴细胞的激活而明显增强。淋巴细胞合成的内源性CAs可能通过自分泌或／和旁分泌路径主要激活淋巴细胞上的β2-AR,从而抑制T细胞的增殖反应。     

Role of α-fetoprotein in differentiation of regulatory T lymphocytes

The effect of native α-fetoprotein (AFP) on the expression of T-regulatory lymphocyte (Treg) markers by activated CD4⁺ lymphocytes with different proliferative status was studied. α-Fetoprotein did not affect the ratio of proliferating and non-proliferating activated CD4⁺ cells. In the study of Treg differentiation, it was found that AFP at concentrations of 50 and 100 μg/mL significantly inhibited the number of nonproliferating CD4⁺FOXP3⁺ and CD4⁺FOXP3⁺HELIOS⁺ lymphocytes without affecting the expression of Treg markers by proliferating CD4⁺ lymphocytes.     

The differential requirement of mushroom body α/β subdivisions in long-term memory retrieval in Drosophila

The mushroom body (MB), a bilateral brain structure possessing about 2000-2500 neurons per hemisphere, plays a central role in olfactory learning and memory in Drosophila melanogaster. Extensive studies have demonstrated that three major types of MB neurons (α/β, α’/β’ and γ) exhibit distinct functions in memory processing, including the critical role of approximately 1000 MB α/β neurons in retrieving long-term memory. Inspired by recent findings that MB α/β neurons can be further divided into three subdivisions (surface, posterior and core) and wherein the α/β core neurons play an permissive role in long-term memory consolidation, we examined the functional differences of all the three morphological subdivisions of MB α/β by temporally precise manipulation of their synaptic outputs during long-term memory retrieval. We found the normal neurotransmission from a combination of MB α/β surface and posterior neurons is necessary for retrieving both aversive and appetitive long-term memory, whereas output from MB α/β posterior or core subdivision alone is dispensable. These results imply a specific requirement of about 500 MB α/β neurons in supporting long-term memory retrieval and a further functional partitioning for memory processing within the MB α/β region.     

Lymphocyte populations of Callithrix jacchus marmosets.

A lymphocyte population of common marmosets (Callithrix jacchus) was identified by rosette formation with African green monkey erythrocytes; the rosette-forming cells appeared to be T lymphocytes, as approximately 62% of circulating lymphocytes and 85% of thymus cells formed rosettes with African green monkey erythrocytes. In addition, common marmoset lymphoid cells carrying T-lymphotropic Herpesvirus saimiri or Herpesvirus ateles formed rosettes with African green monkey erythrocytes and treatment of common marmoset circulating lymphocytes with an anti-T cell serum and complement (C′) eliminated rosette-forming cells. Common marmoset T lymphocytes apparently carry a surface receptor for African green monkey erythrocytes, but unlike humans and other closely related nonhuman primates, T lymphocytes of common marmosets fail to form rosettes with sheep erythrocytes.     

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (⁶⁰Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

     

Identification, characterization, and epitope mapping of human monoclonal antibody J19 that specifically recognizes activated integrin α4β7

Integrin α(4)β(7) is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing. The rolling and firm adhesions of lymphocytes rely on the dynamic shift between the inactive and active states of integrin α(4)β(7), which is associated with the conformational rearrangement of integrin molecules. Activation-specific antibodies, which specifically recognize the activated integrins, have been used as powerful tools in integrin studies, whereas there is no well characterized activation-specific antibody to integrin α(4)β(7). Here, we report the identification, characterization, and epitope mapping of an activation-specific human mAb J19 against integrin α(4)β(7). J19 was discovered by screening a human single-chain variable fragment phage library using an activated α(4)β(7) mutant as target. J19 IgG specifically bound to the high affinity α(4)β(7) induced by Mn(2+), DTT, ADP, or CXCL12, but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α(4)β(7)-MAdCAM-1 interaction. The epitope of J19 IgG was mapped to Ser-331, Ala-332, and Ala-333 of β(7) I domain and a seven-residue segment from 184 to 190 of α(4) β-propeller domain, which are buried in low affinity integrin with bent conformation and only exposed in the high affinity extended conformation. Taken together, J19 is a potentially powerful tool for both studies on α(4)β(7) activation mechanism and development of novel therapeutics targeting the activated lymphocyte expressing high affinity α(4)β(7).     

Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression

Three-color immunofluorescence has been used to determine the co-expression of cell surface antigens on human peripheral blood lymphocytes. Monoclonal antibodies or avidin were coupled to either FITC (green), phycoerythrin (orange), or Texas Red (red) fluorochromes. These three fluorochromes could be independently measured by using a dual laser FACS IV system equipped with an argon ion laser (488 nm) and a dye laser (600 nm). Human peripheral blood lymphocytes were stained with the following combinations of reagents: (1) FITC anti-Leu-11a + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; (2) FITC anti-Leu-11a + PE anti-Leu-3a + TR avidin/biotin anti-Leu-7; (3) FITC anti-Leu-8 + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; and (4) FITC anti-Leu-11a + PE anti-Leu-2 + TR avidin/biotin anti-Leu-8. The light scatter, green fluorescence, orange fluorescence, and red fluorescence signals for each sample were stored by a Consort 40 PDP/11 computer in list mode files. Sequential reanalysis of the data directly demonstrated the existence of several unrecognized subpopulations of lymphocytes. Previously, we reported that the anti-Leu-7 and anti-Leu-11 antibodies can be used to identify discrete subsets of human NK cells with distinct functional capacities. In this report, we show that these subsets can be further subdivided on the basis of Leu-8 and Leu-2 expression. Thus, these studies illustrate how multicolor and multiparameter flow cytometry can further our understanding of cellular heterogeneity within this group of lymphocytes.     

Structure, function, and chemical synthesis of Vaejovis mexicanus peptide 24: a novel potent blocker of Kv1.3 potassium channels of human T lymphocytes

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ～ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/β motif consisting of a single-turn α-helix and a three-stranded antiparallel β-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.     

EGCG has Dual and Opposing Effects on the N-terminal Region of Self-associating α-synuclein Oligomers

Oligomers of the protein α-synuclein (α-syn) are thought to be a major toxic species in Parkinson’s disease, particularly through their ability to permeabilize cell membranes. The green tea polyphenol epigallocatechin gallate (EGCG) has been found to reduce this ability. We have analyzed α-syn oligomer dynamics and interconversion by H/D exchange monitored by mass spectrometry (HDX-MS). Our results show that the two oligomers OI and OII co-exist in equilibrium; OI is a multimer of OII and its dissociation can be followed by HDX-MS by virtue of the correlated exchange of the N-terminal region. Urea destabilizes the α-syn oligomers, dissociating OI to OII and monomers. Oligomers exposed to EGCG undergo Met oxidation. Intriguingly, EGCG induces an oxidation-dependent effect on the structure of the N-terminal region. For the non-oxidized N-terminal region, EGCG increases the stability of the folded structure as measured by a higher level of protection against H/D exchange. In contrast, protection is clearly abrogated in the Met oxidized N-terminal region. Having a non-oxidized and disordered N-terminal region is known to be essential for efficient membrane binding. Therefore, our results suggest that the combined effect of a structural stabilization of the non-oxidized N-terminal region and the presence of a disordered oxidized N-terminal region renders the oligomers less cytotoxic by decreasing the ability of the N-terminal region to bind to cell membranes and facilitate their permeabilization.     

Vα14 NKT cells activated by alpha-galactosylceramide augment lipopolysaccharide-induced nitric oxide production in mouse intra-hepatic lymphocytes

Vα14 natural killer T (Vα14 NKT) cells activated by α-galactosylceramide (α-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-γ plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by α-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of α-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by α-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, α-GalCer did not augment LPS-induced NO production in IHLs from IFN-γ knockout mice. In flow cytometry analysis of IHLs from α-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Vα14 NKT cells were activated.     

Determination of the complete order parameter tensor for a lipid methylene group from 1H- and 2H-NMR spin labels

Proton-proton dipolar splittings are obtained as a function of temperature for the α-methylene in a potassium palmitate - (β-ω) - d₂₉ (70 wt.%) / D₂O (30 wt.%) sample above and below the gel to liquid crystal phase transition. These splittings and corresponding deuteron quadrupole splittings are used to specify the complete order parameter tensor for the α-methylene group. Deduction of lipid structural information from the complete-order parameter tensor is discussed.     

The Effect of α-Interferon on Thymidine,Uridine, and Leucine Uptake and Ultrastructure of Peripheral Blood Mononuclear Cells from Multiple Myeloma Patients

The effect of IFN α-2b on thymidine, uridine, and leucine uptake was examined on peripheral blood mononuclear cells (PBMC) of healthy donors and 15 patients with multiple myeloma (MM). In addition, the surface ultrastructure of the cells incubated without or with IFN α-2b was examined with a scanning electron microscope. The results showed that IFN had no effect on thymidine, uridine, or leucine uptake of unstimulated MM and control PBMC. On the other hand IFN inhibited thymidine, and uridine uptake of PWM-stimulated MM PBMC, but had no effect on healthy donor stimulated PBMC. 1FN inhibited also thymidine and uridine uptake in PHA-stimulated healthy donors and MM patients′ PBMC. The cellular surface ultrastructure of MM lymphocytes incubated with 100 u/ml IFN showed disappearance of the microvilli and formation of cellular pits, whereas in healthy donor lymphocytes IFN caused flattening of microvilli.     

The effect of cultivated mixed-species green fodder on intake,milk production and milk composition of housed dairy goats

The majority of New Zealand dairy goat farmers utilise cultivated green-fed fodder dominated by perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.), but evidence from other ruminant species suggests that milk production may be improved when using a more diverse array of species within the green fodder. The aim of this experiment was to determine whether feeding lactating dairy goats a mixed-species green fodder (MF, consisting of perennial ryegrass, timothy (Phleum pratense L.), prairie grass (Bromus willdenowii Kunth), white clover, red clover (Trifolium pratense L.), lucerne (Medicago sativa L.), chicory (Cichorium intybus L.) and plantain (Plantago lanceolata L.) improves dietary intake, milk yield and composition compared with a standard ryegrass and white clover green fodder (SF). Thirty-six mid-lactation goats were housed indoors in pairs and split into two groups (A and B). The trial was split into three periods – firstly a uniformity period of 6 days, in which all goats were fed a combination of both green fodder types, followed by two treatment periods (P1 and P2) of 12 days, respectively. For P1, group A was fed MF and group B was fed SF, and then the group diets were switched for P2. Goats fed MF had 13% greater dry matter intake and 7% greater milk yield than goats fed SF. In addition, the milk protein and fat concentration of goats fed MF were 4% greater than for those fed SF, whereas there was no effect on milk lactose concentration. There was no treatment effect on the levels of protein, glucose, urea or non-esterified fatty acids in the blood of the goats. An effect of green fodder type on milk fat profile was demonstrated, with proportions of pentadecylic acid (C15:0), cis-vaccenic acid (C18:1 c11), linoleic acid (C18:2 n6) and α-linolenic acid (C18:3 n3) being increased in response to MF consumption. In contrast, iso-C15 and iso-C17 proportions were lesser. In summary, this study demonstrated that goats fed MF increased green fodder intake and milk production compared with goats fed SF. The green fodder type affected the fatty acid profile of goat’s milk, with MF increasing the levels of beneficial polyunsaturated omega fatty acids (linoleic and α-linolenic acids).