Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.     

Programmed death ligand 1 is over-expressed by neutrophils in the blood of patients with active tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world's largest infectious disease problems. Despite decades of intensive study, the immune response to Mtb is incompletely characterised, reflecting the extremely complex interaction between pathogen and host. Pathways that may alter the balance between host protection and pathogenesis are therefore of great interest. One pathway shown to play a role in the pathogenesis of chronic infections, including TB, is the programmed death-1 (PD-1) pathway. We show here that the expression of the programmed death ligand 1 (PD-L1), which interacts with PD-1, is increased in whole blood from active TB patients compared with whole blood from healthy controls or Mtb-exposed individuals, and that expression by neutrophils is largely responsible for this increase.     

The processing and presentation of lipids and glycolipids to the immune system

The recognition of CD1–lipid complexes by T cells was discovered 20 years ago and has since been an emerging and expanding field of investigation. Unlike protein antigens, which are presented on MHC class I and II molecules, lipids can only be presented by CD1 molecules, a unique family of MHC-like proteins whose singularity is a hydrophobic antigen-binding groove. The processing and loading of lipid antigens inside this groove of CD1 molecules require localization to endosomal and lysosomal subcellular compartments and their acidic pHs. This particular environment provides the necessary glycolytic enzymes and lipases that process lipid and glycolipid antigens, as well as a set of lipid transfer proteins that load the final version of the antigen inside the groove of CD1. The overall sequence of events needed for efficient presentation of lipid antigens is now understood and presented in this review. However, a large number of important details have been elusive. This elusiveness is linked to the inherent technical difficulties of studying lipids and the lipid–protein interface in vitro and in vivo. Here, we will expose some of those limitations and describe new approaches to address them during the characterization of lipids and glycolipids antigen presentation.     

Six-month therapy with aerosolized interferon-gamma for refractory multidrug-resistant pulmonary tuberculosis

The aim of this study was to investigate the adjuvant effects of interferon-gamma (IFN-gamma) inhalation therapy for six months in the treatment of refractory multidrug-resistant pulmonary tuberculosis (MDR-TB). Aerosolized IFN-gamma was given to six MDRTB patients with persistent positive smears and cultures despite long-term medical treatment. The patients received aerosolized two million international units of IFN-gamma three times a week for 6 months while they continued on identical antituberculous chemotherapy. Before IFN-gamma inhalation therapy, the patients received a median of 6.5 (range, 4 to 7) antituberculous drugs for median duration of 29 months (range,7 to 76). After IFN-gamma inhalation therapy, sputum smears remained persistently positive in all patients throughout the study period. Sputum cultures were transiently negative at the 4th month in two patients, but became positive again at the end of 6 months of IFN-gamma therapy. Five patients had radiological improvement including three patients who showed a decrease in the size of the cavitary lesions. Resectional surgery could be performed in one patient in whom substantial clinical and radiological improvement was noted after IFN-gamma inhalation therapy. These results suggest that IFN-gamma inhalation therapy may be effective for some cases of refractory MDR-TB who are otherwise not responding to conventional therapy.     

Predictors for tuberculosis co-infection in people living with HIV/AIDs

Background/aimTuberculosis (TB) is one of the most common chronic infectious conditions causing mortality and severe outcomes, particularly in people living with HIV/AIDS (PLWHA). In this study, we aimed to determine the prevalence and predictors of TB among PLWHA.Materials and MethodsWe conducted a retrospective and single-center study of adults (≥18 years) PLWHA registered at our tertiary teaching and research hospital between 2000 and 2016.ResultsA total of 711 PLWHA were included. Of whom, 633 (89.0%) were male. Mean age was 36.53 ±11.55 years (range, 17–79). Thirty-eight (5.3%) patients were diagnosed with active TB. TB development was associated with low CD4+ lymphocyte count (p<0.001), high viral load (p=0.040) and alcohol consumption (p=0.004) but no association with age (p=0.392), gender (p=0.928) and duration since anti-retroviral therapy initiation (p=0.788) was found. Also, a receiver operating characteristic analysis showed that the area under the curves of CD4+ lymphocyte count as a predictor for TB development in PLWHA was 0.717 (p<0.001).ConclusionThere are still clinical challenges to predict TB diagnosis. However, CD4+ lymphocyte count and viral load may be considered as valuable predictors for TB development. Also, community strategies to reduce harmful effect of alcohol use should be developed.     

Dressed not to kill: CD16+ monocytes impair immune defence against tuberculosis

Monocytes are blood leukocytes that can differentiate into several phagocytic cell types, including DCs, which are instrumental to the inflammatory response and host defence against microbes. A study published in this issue of the European Journal of Immunology by Balboa et al. [Eur. J. Immunol. 2013. 43: 335‐347] suggests that a shift of the CD16^? monocyte population toward a CD16⁺ subpopulation may represent an immune evasion strategy that ultimately favors persistence of Mycobacterium tuberculosis. Together with other recent reports, the article by Balboa et al. sheds new light on the function of CD16⁺ monocytes in health and disease; in this commentary, we discuss the implications stemming from these findings.     

The immunopathogenesis of the HIV tuberculosis immune reconstitution inflammatory syndrome

HIV‐1 patients co‐infected with some pathogens are at risk of developing the immune reconstitution inflammatory syndrome (IRIS) when initiating antiretroviral therapy (ART). IRIS is characterized by inflammation leading to the clinical worsening of a treated infection or the unmasking of a previously undiagnosed condition or infection. It is commonly associated with tuberculosis (TB), 8–43% of the HIV‐TB co‐infected patients prescribed with antitubercular treatment and ART develop TB‐IRIS. Although IRIS has been recognized for over 20 years, relatively little was known until recently about its pathogenesis. Despite these advances in understanding IRIS, there remains no immune biomarker for diagnostic or prognostic purposes. Here, we review the risk factors associated with TB‐IRIS, the challenges in studying this syndrome, and how T lymphocytes, dysregulated cytokine responses, and innate immunity may contribute to the development of TB‐IRIS.     

Targeting glucose uptake with siRNA-based nanomedicine for cancer therapy

Targeting cancer metabolism is emerging as a successful strategy for cancer therapy. However, most of the marketed anti-metabolism drugs in cancer therapy do not distinguish normal cells from cancer cells, leading to severe side effects. In this study, we report an effective strategy for cancer therapy through targeting glucose transporter 3 (GLUT3) with siRNA-based nanomedicine to simultaneously inhibit the self-renewal of glioma stem cells and bulk glioma cells in a glucose restricted tumor micro-environment. We have demonstrated that cationic lipid-assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles can efficiently deliver siRNA into U87MG and U251 glioma stem cells and bulk glioma cells. Nanoparticles carrying specific siRNA targeting GLUT3 (NP_siGLUT3) were able to significantly reduce the expression of GLUT3 in glioma stem cells and bulk glioma cells, while GLUT3 knockdown results in obvious cell metabolism and proliferation inhibition, and further glioma stem cells percentage down-regulation. Moreover, systemic delivery of NP_siGLUT3, via intravenous injection, significantly inhibited tumor growth in a U87MG xenograft model, due to the reduced expression of GLUT3 and down-regulated stemness of glioma cells.     

The expression of early resistance to an infection with Mycobacterium tuberculosis by old mice is dependent on IFN type II (IFN-gamma) but not IFN type I

Old mice can express a transient early resistance to infection with M. tuberculosis that requires the presence of CD8 T cells within the lungs. Further characterization of those CD8 T cells within the aged lung established that the majority of CD8 T cells from old mice expressed the IL-15 receptor (CD122) in combination with bright expression of CD44 (CD44(hi)), and were capable of producing IFN-gamma after T cell receptor cross-linking. It has been previously described that CD8 CD44(hi) T cells proliferate in response to IFN-I, acting via IL-15, and therefore we determined whether IFN-I signaling could be a participant in the response of CD8 T cells within the lungs of old mice infected with M. tuberculosis. We demonstrate here that IFN-I signaling was required for the expansion of CD8 T cells within the aging lung in response to infection with M. tuberculosis, but that IFN-I signaling had no influence on the capacity of old mice to express early resistance to an infection with M. tuberculosis. Resident CD8 T cells were still however capable of producing IFN-gamma, which we demonstrate here to be critical in the expression of early resistance, suggesting that the expression of early resistance requires the participation, but not expansion, of the CD8 T cell pool within the aging lung.     

Differential Interleukin-8 thresholds for chemotaxis and netosis in human neutrophils

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca⁺² chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.     

CD147单抗介导的基因治疗纳米颗粒的肺癌细胞靶向性研究

目的:本研究采用靶向CD147的单克隆抗体对纳米基因载体颗粒进行靶向修饰后,进行针对肺癌细胞的蛋白激酶Cε(protein kinase Cε,PKCε)小干扰RNA基因治疗,观察其对肺癌细胞增殖和迁移能力的抑制效果。方法:制作可靶向CD147蛋白的磁性纳米基因载体。激光扫描共聚焦显微镜观察肺癌细胞CD147表达量。分别设立CP组、CN组和LP组复合物,按每6孔板孔质粒总量250 ng进行细胞转染。另设CD147靶向载体对照CA组和未转染细胞的对照(control)组。激光扫描共聚焦显微镜观察纳米造影剂的细胞内吞效果。实时荧光定量PCR检测PKCε的mRNA表达。Western blot法检测PKCε、Ki67、MMP3、Wnt1和GAPDH的蛋白表达。平板克隆形成实验检测细胞的增殖能力。Transwell法检测细胞的迁移能力。结果:免疫荧光法染色观察证实,人肺癌A549细胞的胞膜高表达CD147蛋白。CP组细胞中siRNA高效进入A549细胞,质粒内吞效率大于CN组和LP组。CP组、CN组、LP组和CA组的A549细胞中PKCε的mRNA相对表达量分别为control组的(9.76±0.18)%、(98.51±0.32)%、(99.17±0.16)%和(99.68±0.11)%,CP组与control组间的差异有统计学显著性(P0.05),CN组、LP组与control组间的差异无统计学显著性。CP组PKCε、Ki-67、MMP3及Wnt1蛋白的表达量明显降低,CN组和LP组与对照组之间的蛋白表达量的差异无统计学显著性。CP组的克隆形成数量明显少于control组,差异具有统计学显著性(P0.05)。CN组、LP组和CA组的有效克隆数量与control组相比差异没有统计学显著性。CP组的过膜细胞数量明显少于control组,差异具有统计学显著性(P0.05)。CN组、LP组和CA组的数量与control组相比差异没有统计学显著性。结论:靶向CD147修饰的纳米基因载体,可以对肺癌细胞进行高效的PKCε-siRNA基因治疗,实现对肺癌细胞增殖和迁移能力的高效抑制。     

Accelerating the secondary immune response by inactivating CD4(+)CD25(+) T regulatory cells prior to BCG vaccination does not enhance protection against tuberculosis

CD4(+)CD25(+) natural T regulatory cells (Tregs) have been shown to suppress protective immune responses in several different vaccination models. Since the effect of Tregs on vaccination against tuberculosis (Tb) was unknown, we used a murine model to investigate whether natural Tregs suppress the development of protective immunity following Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. Using a monoclonal antibody against CD25, natural Tregs were inactivated prior to vaccination with BCG. The primary immune response was evaluated after BCG vaccination and the secondary immune response was assessed after an intranasal BCG challenge 42 days after vaccination. Inactivation of natural Tregs prior to vaccination led to an increased immune response 14 days after vaccination, increased numbers of antigen-responsive lymphocytes immediately prior to secondary challenge and the earlier appearance of IFN-gamma-producing CD4(+) and CD8(+) lymphocytes in the draining lymph nodes and lungs after challenge. Despite this, protection from virulent Mycobacterium tuberculosis or M. bovis aerosol challenge was unaffected by natural Treg inactivation prior to BCG vaccination. This suggests that increasing the primary and accelerating the secondary immune responses by inactivating natural Tregs at the time of vaccination, does not affect the development of protective immunity to Tb.     

Emerging targets for glioblastoma stem cell therapy

Glioblastoma multiforme (GBM), designated as World Health Organization (WHO) grade IV astrocytoma, is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations, including GBM stem cells (GSCs) which are believed to contribute to tumor recurrence following initial response to therapies. Emerging evidence demonstrates that GBM tumors are initiated from GSCs. The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of GSCs, immunotherapy, and non-coding microRNAs may provide better means of treating GBM. Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs. Several signaling pathways including mTOR, AKT, maternal embryonic leucine zipper kinase (MELK), NOTCH1 and Wnt/b-catenin as well as expression of cancer stem cell markers CD133, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain GSC properties. Moreover, the data published in the Cancer Genome Atlas (TCGA) specifically demonstrated the activated PI3K/AKT/mTOR pathway in GBM tumorigenesis. Studying such pathways may help to understand GSC biology and lead to the development of potential therapeutic interventions to render them more sensitive to chemotherapy and radiation therapy. Furthemore, recent demonstration of dedifferentiation of GBM cell lines into CSC-like cells prove that any successful therapeutic agent or combination of drugs for GBM therapy must eliminate not only GSCs, but the differentiated GBM cells and the entire bulk of tumor cells.     

NRAMP1基因3'UTR多态性与新疆维吾尔族人群结核病易感性的相关性研究

目的 研究人类自然抵抗相关巨噬细胞蛋白1(NRAMP1)基因3'UTR多态性与新疆维吾尔族人群结核病易感性的相关性.方法 选取新疆维吾尔族人群活动性结核病患者224例,正常对照225例,用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)的方法对NRAMP1基因3'UTR进行基因分型,根据基因型对样本分组,经统计学处理,研究NRAMP1基因3'UTR多态性与结核病易感性的相关性.结果 在新疆维吾尔族人群活动性结核病患者中3'UTR TGTG/TGTG基因型159例(71.1%),TGTG/TGTG缺失基因型56例(24.9%),TGTG缺失/TGTG缺失基因型9例(4.0%);正常对照TGTG/TGTG基因型则为185例(82.2%),TGTG/TGTG缺失基因型36例(16.0%),TGTG缺失/TGTG缺失基因型4例(1.8%).正常对照组TGTG/TGTG基因型频率明显高于结核患者(X2=7.94,P<0.01).研究发现TGTG的等位基因频率为0.87,TGTG缺失的等位基因频率为0.13.结论 在新疆维吾尔族人群中NRAMP1基因3'UTR多态性与结核病易感性有明显相关性.     

Receptor expression in orbital inflammatory syndromes and implications for targeted therapy

To investigate the expression of multiple therapeutic targets in tissue specimens from patients with orbital inflammatory syndromes, the clinical records of 16 patients treated for orbital inflammation between January 2003 and November 2005 for whom tissue blocks were available were reviewed retrospectively. Immunohistochemical staining was performed on archived specimens using commercially available monoclonal antibodies against CD3, CD20, CD22, CD23, CD25, and CD52 antigens. The histologic diagnoses were confirmed, and the immunohistochemical staining patterns were agreed upon by both collaborating pathologists (JLJ and PC-B). The study included 13 women and 3 men who ranged in age from 4 to 79 years (mean, 46 years). The histologic diagnoses were as follows: orbital pseudotumor in six patients; sarcoidosis, three; eosinophilic granuloma, one; necrobiotic xanthogranuloma, one; nonspecified granulomatous inflammation, one; Graves' ophthalmopathy, one; Wegener's granulomatosis, one; and reactive lymphoid hyperplasia, two. One orbital lymphoma specimen and one foreign body reaction specimen were used as controls. CD20 was strongly expressed in all specimens except three (Wegener's granulomatosis, eosinophilic granuloma, and nonspecified granulomatous inflammation specimens), and CD25 was strongly expressed in all specimens except the Wegener's granulomatosis specimen, in which this antigen was only moderately expressed. CD20 and CD25 were strongly or moderately expressed in most of the tested specimens of orbital inflammation. If our findings are confirmed in a larger study, rituximab, which targets CD20, and denileukin diftitox (ONTAK), which targets CD25, should be considered for future clinical trials for orbital inflammatory syndromes.     

T cells and adaptive immunity to Mycobacterium tuberculosis in humans

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4⁺ and CD8⁺ T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.     

Requirement of the serine at residue 329 for lipid raft recruitment of DNAM-1 (CD226)

Upon antigen recognition by the TCR, leukocyte function-associated antigen-1 (LFA-1) physically associates with the leukocyte adhesion molecule DNAM-1 (CD226), for which the serine phosphorylation at residue 329 (S329) of DNAM-1 plays a critical role. The TCR-mediated signal also induces the formation of the immunological synapse (IS), in which lipid raft-associated molecules, including LFA-1, DNAM-1, protein kinase C, Fyn and others, are recruited, resulting in efficient signal transduction for T cell activation. However, the molecular mechanisms of lipid raft recruitment of many associated molecules have remained unclear. Here, we demonstrate that, while both wild-type (WT) and mutant DNAM-1 at S329 were polarized at the IS, the WT, but not mutant, DNAM-1 associated with lipid rafts at the peripheral supra-molecular activation clusters. We also demonstrate that the association of DNAM-1 with lipid rafts was necessary for the tyrosine phosphorylation of DNAM-1, which is essential for LFA-1-mediated co-stimulatory signaling for naive T cell proliferation and differentiation.     

Chemokine receptor genotype and response to interleukin-2 therapy in HIV-1-infected individuals

Interleukin-2 therapy is an immune-based treatment for HIV-1-infected individuals with declining CD4(+) T cell counts. Intravenous IL-2 produces an elevation of circulating CD4(+) T cells, but with a varying degree of effectiveness in individual patients. IL-2 is also known to increase the expression of chemokine receptors, coreceptors for HIV-1. Allelic variation in chemokine receptor genes can markedly affect the course of HIV disease; consequently, we analyzed CCR5 and CCR2B genotypes among a cohort of HIV-1-infected individuals that received IL-2 therapy. DNA was extracted from treated individuals and genotyping was performed using PCR followed by allele-specific detection or cleavage of the amplified product. Samples from 47 trial participants (25 CIV-IL-2 group; 22 placebo group) were analyzed for CCR5 and CCR2B genotype. We report that CCR5 Delta 32 heterozygous individuals had a greater CD4(+) T cell response to continuous intravenous IL-2 (CIV-IL-2) treatment than those homozygous for the wild-type allele (median = 427 vs 237 cells/mm(3); P = 0.03). This study highlights the importance of interactions between IL-2 and CCR5; at the clinical level, it argues for assessment of chemokine receptor genotype in IL-2 and perhaps other immune-based therapy trials.