Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid α-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, α-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.

Structural requirements for antigen presentation by mouse CD1

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid αgalactosylceramide (α-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the α helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and α-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that α-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A′ pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) α chains and different β chains respond differently to α-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR β in defining natural killer T cell specificity, despite the more restricted diversity of the α chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR α and β chains survey a surface composed of both mCD1.1 and the carbohydrate portion of α-GalCer.

Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography

CD1 is an MHC class I-like antigen-presenting molecule consisting of a heavy chain and β2-microglobulin light chain. The in vitro refolding of synthetic MHC class I molecules has always required the presence of ligand. We report here the use of a folding method using an immobilized chaperone fragment, a protein disulphide isomerase, and a peptidyl-prolyl cis-trans isomerase (oxidative refolding chromatography) for the fast and efficient assembly of ligand-free and ligand-associated CD1a and CD1b, starting with material synthesized in Escherichia coli. The results suggest that “empty” MHC class I-like molecules can assemble and remain stable at physiological temperatures in the absence of ligand. The use of oxidative refolding chromatography thus is extended to encompass complex multisubunit proteins and specifically to members of the extensive, functionally diverse and important immunoglobulin supergene family of proteins, including those for which a ligand has yet to be identified.

Germ line deletion of the CD1 locus exacerbates diabetes in the NOD mouse

Quantitative and qualitative defects in CD1-restricted natural killer T cells have been reported in several autoimmune-prone strains of mice, including the nonobese diabetic (NOD) mouse. These defects are believed to be associated with the emergence of spontaneous autoimmunity. Here we demonstrate that both CD1d-null NOD and CD1d-null NOD/BDC2.5 T cell receptor transgenic mice have an accelerated onset and increased incidence of diabetes when compared with CD1d+/− and CD1d+/+ littermates. The acceleration of disease did not seem to result from changes in the T helper (Th)1/Th2 balance because lymphocytes purified from lymphoid organs and pancreatic islets of wild-type and CD1d-null mice secreted equivalent amounts of IFN-γ and IL-4 after stimulation. In contrast, the pancreata of CD1d-null mice harbored significantly higher numbers of activated memory T cells expressing the chemokine receptor CCR4. Notably, the presence of these T cells was associated with immunohistochemical evidence of increased destructive insulitis. Thus, CD1d-restricted T cells are critically important for regulation of the spontaneous disease process in NOD mice.

Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris

The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.

SERR spectroelectrochemical study of cytochrome cd1 nitrite reductase co-immobilized with physiological redox partner cytochrome c552 on biocompatible metal electrodes

© 2015 Silveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Circular polarization of excitonic luminescence in CdTe quantum wells with excess electrons of different densities

The circular polarization of excitonic luminescence is studied in CdTe/Cd1-xMgxTe quantum wells with excess electrons of low density in an external magnetic field. It is observed that the circular polarization of X and X- emissions has opposite signs and is influenced by the excess electron density. If the electron density is relatively high so that the emission intensity of the negatively charged excitons X- is much stronger than that of the neutral excitons X, a stronger circular polarization degree of both X and X- emissions is observed. We find that the circular polarization of both X- and X emissions is caused by the spin polarization of the excess electrons due to the electron-spin-dependent nature of the formation of X-. If the electron density is relatively low and the emission intensity of X- is comparable to that of X, the circular polarization degree of X and X- emissions is considerably smaller. This fact is interpreted as due to a depolarization of the excess electron spins, which is induced by the spin relaxation of X-.

A novel white light emitting long-lasting phosphor

A novel white light emitting long-lasting phosphor Cd1-xDyxSiO3 is reported in this letter. The Dy3+ doped CdSiO3 phosphor emits white light. The phosphorescence can be seen with the naked eye in the dark clearly even after the 254 nm UV irradiation have been removed for about 30 min. In the emission spectrum of 5% Dy3+ doped CdSiO3 phosphor, there are two emission peaks of Dy3+, 580 mn (F-4(9/2)-->H-6(13/2)) and 486 nm (F-4(9/2)-->H-6(15/2)), as well as a broad band emission located at about 410 nm. All the three emissions form a white light with CIE chromaticity coordinates x=0.3874, y=0.3760 and the color temperature is 4000 K under 254 mn excitation. It indicated that this phosphor is a promising new luminescent material for practice application.

云南澜沧老厂大型银多金属矿床成矿年代及地球化学

老厂矿床位于三江成矿带南段昌宁—孟连裂谷的次级澜沧断陷盆地中，昌宁—孟连裂谷是三江成矿带南段的重要成矿段之一，其大地构造位于保山―掸邦微陆块东缘，兰坪—思茅盆地、临沧地块西缘，属东特提斯构造域，是冈瓦纳古陆与欧亚大陆巨型缝合带的组成部分。 矿床开采历史悠久，始于明朝永乐二年（1404年），至今已605年。古时炼银弃铅，最高年产白银30万两。解放后主要开采古人废弃的高铅炉渣，并对深部原生矿体进行了初步勘探，90年代至今对深部银铅矿体进行了详细勘查，深部矿体是矿床主要的开采对象。它以独特的成矿地质特征、富银（铅锌矿石中平均含量为629×10-6，方铅矿中平均2069×10-6）、伴生元素多（In、Se、Te、Ga、Cd、Bi）、规模大（In、Se、Te、Cd已达大型规模）、含Sn等特征而受到广大地质工作者的关注。它是“三江”成矿带南段最具代表性的铅银矿床类型之一，也是昌宁—孟连裂谷内目前探明的唯一大型银铅锌多金属矿床，具有十分重要的研究价值。 虽然前人已从矿床地质、成矿条件、控矿因素等方面对老厂大型银铅锌多金属矿床进行过研究，但研究工作较为零散，在成矿物质与成矿流体来源、矿床成因、花岗斑岩与成矿等方面还存在较大争议。本文在深入细致的野外地质工作基础上，利用多种现代分析测试技术，对矿床进行了较为系统的矿物学、岩石学、年代学和矿床地球化学研究，进而查明了矿床成矿物质与成矿流体来源、揭示了成矿地球动力学背景、探讨了矿床的成因、初步建立了矿床成因模式。论文取得的主要成果如下： 1．查明了硫化物的物质组分及形成阶段。闪锌矿以高铁（早期平均11.51%，晚期平均8.41%）为基本特征，并伴生多种特征的微量元素，早期闪锌矿是Fe、In的主要载体，Cu、Cd、Mn则主要富集在晚阶段闪锌矿中；方铅矿是Ag的主要载体，其早期富Ag、Te、Bi，含Se、Cu，中期主要富Ag，晚期以含As、Bi为特征；其它硫化物成分较单一。 2．揭示了伴生元素赋存状态和富集规律。矿床伴生元素含量高，闪锌矿中Cd平均4293.19×10-6，In平均555.37×10-6，方铅矿中Te平均143.81×10-6，As、Se、Bi、Ga含量也很高。初步估算金属储（万吨）量分别为： Ga 0.097，Cd1.84， In0.13，Se0.06 ，Te0.15，Bi1.28，As4.31。闪锌矿是Ga、In、Cd主要载体，Te、Bi主要赋存在方铅矿中，Se则主要在铅锌、黄铁矿石中富集。 3．首次获得了精确的成矿年代学数据。单颗粒闪锌矿—黄铁矿Rb-Sr法获得矿床成矿年龄t=（45±3.6）Ma，（87Sr/86Sr）i=0.70977±0.00034。证实了矿床成矿与隐伏花岗斑岩关系密切，矿床形成是三江成矿带南段对喜马拉雅碰撞造山成矿运动的响应。 4．首次系统研究了花岗斑岩的地质、地球化学特征。矿床花岗斑岩具高硅、超钾、富碱、贫钠和低镁、钙、铁、磷的特征，岩石为过铝质岩石；其轻稀土富集、Eu为弱负异常、基本无Ce异常、岩石富集大离子亲石元素，具有较高的锶初始值和较低εNd及高的Nd模式年龄，与三江地区富碱斑岩具有相似的地球化学特征。岩浆主要来源于加厚下地壳重熔，成岩过程中有地幔物质的加入，为同碰撞构造环境下形成。 5．首次探讨了容矿沉积建造及矿石、矿物的地球化学特征。矿床碳酸盐岩轻稀土富集、Eu正异常及Ce异常和其微量元素特征与热水沉积岩石相似，表明了碳酸盐岩的热水沉积成因，同时矿石及其硫化物微量和稀土元素特征也指示了在早石炭世矿区发生了广泛的火山喷流热水沉积成矿作用。 6．查明了成矿物质及成矿流体来源。成矿元素研究显示矿床成矿物质具有多来源的特征，Pb同位素研究表明矿床Pb可能来自不同地层岩石的淋滤， S同位素组成显示其可能主要来源于海水对下伏火山岩地层的淋滤与海水硫酸盐的还原，花岗斑岩岩浆也可能提供了部分S。C-O、H-O同位素和矿石及其硫化物稀土元素反映矿床早期成矿流体主要源于深部岩浆，晚期主要来源于喜山期花岗斑岩岩浆热液。 7．初步建立了成矿模式。老厂矿床是长期以来多种地质作用下的综合产物，具有成矿物质多来源，成矿阶段多期次特点。经历了早石炭世火山喷流热水沉积成矿和喜山期花岗斑岩岩浆热液叠加改造成矿作用，热水成矿作用下形成了以黄铁矿为主的块状硫化物矿床，喜山期富碱花岗斑岩岩浆热液流体携带了大量的Cu、Mo、Ag、Pb等成矿元素，在它的叠加改造作用下形成了老厂银多金属矿床。总之矿床为火山喷流沉积—岩浆热液叠加改造成因。

Rinderpest and peste de petits ruminants viruses exhibit neurovirulence in mice

Members of the morbillivirus genus, canine distemper (CDV), phocine distemper virus (PDV), and the cetacean viruses of dolphins and porpoises exhibit high levels of CNS infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, it is possible that all morbilliviruses infect the CNS but in some hosts are rapidly cleared by the immune response. In this study, we assessed whether RPV and PPRV have the potential to be neurovirulent. We describe the outcome of infection, of selected mouse strains, with isolates of RPV, PPRV, PDV, porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), and a wild-type strain of MV. In the case of RPV virus, strains with different passage histories have been examined. The results of experiments with these viruses were compared with those using neuroadapted and vaccine strains of MV, which acted as positive and negative controls respectively. Intracerebral inoculation with RPV (Saudi/81) and PPRV (Nigeria75/1) strains produced infection in Balb/C and Cd1, but not C57 suckling mice, whereas the CAM/RB rodent-adapted strain of MV infected all three strains of mice. Weanling mice were only infected by CAM/RB. Intranasal and intraperitoneal inoculation failed to produce infection with any virus strains. We have shown that, both RPV and PPRV, in common with other morbilliviruses are neurovirulent in a permissive system. Transient infection of the CNS of cattle and goats with RPV and PPRV, respectively, remains a possibility, which could provide relevant models for the initial stages of MV infection in humans.

Micro-environmental conditions modulate protein secretion and infectivity of the Trichinella spiralis L1 larva

After digestion of infected meat the free L1 of Trichinella spp. penetrate the intestinal mucosa where they moult to the mature adult stage. We have used proteomics to identify changes in protein secretion during in vitro culture of free T. spiralis muscle larvae under different environmental conditions, and to correlate these changes with their infectivity in mice. Muscle larvae were cultured in different media (RPMI-1640, C-199 and HBSS) under conditions of anaerobiosis, microaerobiosis and in 5% CO(2) at 37 degrees C. Following incubation the larval excretory/secretory proteins were analysed by two-dimensional gel electrophoresis and the larvae were used to orally infect naïve CD1 mice. For all culture media tested, infectivity of the L1 was preserved following incubation in anaerobic conditions. In contrast, the infectivity of worms cultured in nutrient-rich media was almost completely abolished in both microaerobiosis and in the presence of 5% CO(2). Some infectivity was retained in poor or reduced culture media. Comparative analysis of larval infectivity and protein secretion showed that loss of infectivity correlated with the appearance of non-tyvelosylated proteins that in turn may be related to the onset of moulting.

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.

Methodology/Principal Findings: We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.

Conclusions/Significance: Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.

Avaliação do efeito de metais pesados na fertilidade do ratinho

A infertilidade é um problema actual que afecta cerca de 8 a 12% dos casais em idade fértil, sendo 50% dos casos atribuídos ao factor masculino. A exposição ocupacional e/ou ambiental a metais pesados representa uma das suas principais causas. O chumbo, o cádmio e o crómio são metais com elevada utilização industrial e muito persistentes no ambiente, sendo motivo de grande preocupação devido aos seus efeitos na saúde reprodutiva dos trabalhadores e da população em geral. Neste trabalho estudaram-se os efeitos do cloreto de chumbo (PbCl2), cloreto de cádmio (CdCl2) e cromato de potássio (K2CrO4) na fertilidade, usando como modelo ratinhos machos ICR-CD1. Os ratinhos foram injectados subcutaneamente com 74 e 100 mg de PbCl2/kg pc ou com 5 e 10 mg de K2CrO4/kg pc, respectivamente, durante 4 dias consecutivos ou com 1, 2 e 3 mg de CdCl2/kg pc numa única injecção. Nos ensaios com PbCl2 e K2CrO4 os animais foram sacrificados 5 e 35 dias após o início da exposição, enquanto que nos ensaios com CdCl2 os animais foram sacrificados após 24 horas e 35 dias. O cloreto de chumbo não alterou a histologia do testículo nem do epidídimo, mas induziu um aumento da percentagem de células em fase S no testículo. O cloreto de chumbo alterou também alguns parâmetros dos espermatozóides, tais como a motilidade, morfologia e integridade do acrossoma. Contudo, não foram observados efeitos na integridade do DNA ou na estrutura da cromatina. O cloreto de cádmio induziu lesões severas e não reversíveis nos testículos que, após 35 dias, reverteram em necrose testicular. O cloreto de cádmio alterou ainda as subpopulações de células testiculares após 24 horas e induziu IMS no testículo após 35 dias. Em consequência das lesões no testículo, a densidade espermática foi severamente afectada após 35 dias. A exposição ao cloreto de cádmio afectou também a morfologia, a motilidade e a integridade acrossómica nos espermatozóides. O cloreto de cádmio induziu ainda fragmentação do DNA nestas células após 35 dias. O cromato de potássio alterou a morfologia dos espermatozóides e a integridade do acrossoma, sobretudo nos animais sacrificados após 35 dias. Verificou-se ainda uma redução da motilidade dos espermatozóides. Não foram detectados efeitos genotóxicos nos espermatozóides, devido à acção do K2CrO4 nas doses testadas. Dos parâmetros avaliados neste trabalho, destacam-se duas novas abordagens, nomeadamente o programa informático Snakes e a análise do conteúdo em DNA de células de testículo, a partir de material incluído em parafina. O Snakes permitiu fazer medições rigorosas do diâmetro dos tubos seminíferos, enquanto que a fixação de amostras de testículo de ratinhos em formol tamponado e inclusão em parafina resultou numa boa preservação do DNA, possibilitando assim a quantificação das subpopulações de células testiculares por citometria de fluxo. Os resultados obtidos para os diferentes parâmetros testados indicam que a motilidade dos espermatozóides e a integridade do acrossoma sejam parâmetros sensíveis à toxicidade de metais. O acrossoma aparenta ser um dos principais alvos da toxicidade dos metais e cuja reacção acrossómica prematura pode reduzir a capacidade do espermatozóide para fertilizar o oócito. Assim, este trabalho representa uma contribuição para uma melhor compreensão dos efeitos do chumbo, cádmio e crómio na fertilidade masculina.