Innate Immune Activation in the Pathogenesis of a Murine Model of Globoid Cell Leukodystrophy

Globoid cell leukodystrophy is a lysosomal storage disease characterized by the loss of galactocerebrosidase. Galactocerebrosidase loss leads to the accumulation of psychosine and subsequent oligodendrocyte cell death, demyelination, macrophage recruitment, and astroglial activation and proliferation. To date, no studies have elucidated the mechanism of glial cell activation and cytokine and chemokine up-regulation and release. We explored a novel explanation for the development of the pathological changes in the early stages of globoid cell leukodystrophy associated with toll-like receptor (TLR) 2 up-regulation in the hindbrain and cerebellum as a response to dying oligodendrocytes. TLR2 up-regulation on microglia/macrophages coincided with morphological changes consistent with activation at 2 and 3 weeks of age. TLR2 up-regulation on activated microglia/macrophages resulted in astrocyte activation and marked up-regulation of cytokines/chemokines. Because oligodendrocyte cell death is an important feature of globoid cell leukodystrophy, we tested the ability of TLR2 reporter cells to respond to oligodendrocyte cell death. These reporter cells responded in vitro to medium conditioned by psychosine-treated oligodendrocytes, indicating the likelihood that oligodendrocytes release a TLR2 ligand during apoptosis. TLRs are a member of the innate immune system and initiate immune and inflammatory events; therefore, the identification of TLR2 as a potential driver in the activation of central nervous system glial activity in globoid cell leukodystrophy may provide important insight into its pathogenesis.Globoid cell leukodystrophy (GLD; Krabbe’s disease) is an autosomal recessive lysosomal storage disorder. It affects approximately 1 in 100,000 children born each year and often leads to mortality by 2 years of age. GLD results from the lack of metabolic enzyme galactocerebrosidase (GALC).¹ In the absence of GALC, galactocerebroside (GalCer) undergoes alternative catabolism into galactosylsphingosine (alias psychosine) and a fatty acid instead of normal catabolism into its component parts, galactose and ceramide.² Galactosylsphingosine has been shown to cause the terminal pathological changes in the central nervous system (CNS) of individuals affected by GLD: globoid cell formation,^2,3 astroglial cytokine and chemokine secretion,^4–8 and oligodendrocyte death and demyelination.^9–14 In addition to these pathological changes, it is established that monocytes/macrophages are recruited to the CNS early in the disease process and continue to mobilize to the brain until the late stages of disease.^15,16Little is understood, however, about the mechanisms underlying these events, or which cells are involved in eliciting the initial pathological changes.^7,17,18 This study investigated the mechanisms involved in monocyte/macrophage recruitment and cytokine/chemokine up-regulation and secretion, as well as which cell types are the earliest to undergo inflammatory cascades leading to terminal disease.Inflammatory cytokines are up-regulated as a consequence of activation of the innate immune response.^19–22 Given the connection, it is logical to link the innate immune response to inflammation in globoid cell leukodystrophy. Psychosine is a derivative of β-galactosylceramide and a ganglioside; therefore, it could potentially serve as a toll-like receptor (TLR) ligand and induce the up-regulation of cytokine/chemokine secretion and monocyte/macrophage recruitment through the initial activation of a TLR.TLRs are most commonly associated with recognition of specific, exogenously derived recognition of microbial patterns. Examples of these patterns are lipopolysaccharide, peptidoglycan, lipoarabinomannan, and double-stranded RNA.²³ However, TLRs also have known roles in exacerbating inflammation and inflammatory profiles in the brains of other neurodegenerative and demyelinating diseases, such as experimental autoimmune encephalitis (murine model for multiple sclerosis),^24,25 amyotrophic lateral sclerosis,²⁶ and Alzheimer disease.^27,28 Investigating whether TLRs play a role in the initiation of inflammatory signaling pathways in the brains of twitcher mice (the murine model for globoid cell leukodystrophy) is important to understanding how inflammation in GLD is initiated. Elucidating how inflammation is induced in the course of disease will lead to more complete knowledge of the pathogenesis of GLD and the identification of novel therapeutic targets.To date, few studies have been performed that investigate the early events in GLD that lead to the terminal changes associated with the disease. It is known that globoid cell appearance and demyelination occur caudally to rostrally in the twitcher brain,²⁹ but the mechanism has not been studied. Our hypothesis was that TLR up-regulation on perivascular macrophages and/or microglia early in the disease initiates cytokine production and monocyte/macrophage recruitment to brains of affected individuals. After activation, microglia and macrophages activate neighboring astrocytes, which markedly exacerbate cytokine/chemokine secretion and inflammation. This study identifies early morphological changes in resident CNS glial cells and the innate immune system that explain, at least in part, how cells become activated, up-regulate cytokines/chemokines, and recruit monocytes/macrophages.     

In Vitro Expression of Immunoglobulin M and G Subclasses by Murine B Lymphocytes in Response to a Polyclonal Activator from Actinomyces

A cell wall extract from the gram-positive bacterium Actinomyces viscosus contains the mitogen AVIS, a potent polyclonal B-cell activator for murine B lymphocytes. Cultures of splenocytes from heterozygous nude mice in the presence of an optimal concentration of AVIS responded by a deoxyribonucleic acid synthesis response, and proliferaction reached maximal levels after 3 to 4 days. There was no requirement for T cells in the deoxyribonucleic acid synthesis, proliferactive, immunoglobulin M (IgM), or IgG responses. Significant numbers of IgM-producing cells were present as early as day 2 of culture, whereas later in the culture periods (days 3 to 6) IgG-producing plasmablasts and plasma cell were observed. In cultures of splenocytes from nude mice stimulated with AVIS for 4 to 5 days, 20 to 25% of the recoverable cells synthesized IgM, and 10% contained only IgG2 or IgG3; 5 to 8% of the cells stained for both IgM and IgG2 or both IgM and IgG3. Fine-structure analysis of AVIS-stimulated splenocytes from heterozygous nude mice after 3 days of culture demonstrated that 20 to 25% of the cells were activated to various degrees. Of most importance, all of the activated cells had the characteristic of B lymphoblasts, plasmablasts, or plasma cells. This is the first demonstration of a polyclonal B-cell activator other than lipopolysaccharide which induces IgG3 synthesis. We suggest that AVIS may be a useful probe for the exploration of the functional activities of subpopulations of B cells.     

Use of Multivariate Immune Reconstitution Patterns to Describe Immune Reconstitution after Allogeneic Stem Cell Transplantation in Children

Immune reconstitution after hematopoietic stem cell transplantation (HSCT) is a complex process. Impacts of the reconstitution of different immune cells over time are complex and difficult to understand. New mathematical models are needed to better understand this process. In this study, we used principal component analysis to better analyze the process of immune reconstitution after HSCT. Forty-six consecutive patients receiving HSCT for malignant and nonmalignant disorders were included in the study. All patients were followed for at least 24 months after transplantation with regular blood sampling for analysis of lymphocyte subset numbers and function. Exponentially transformed lymphocyte subset counts and lymphocyte functional markers were analyzed to identify major trends in the reconstitution process. Using our multivariate model for mapping immune reconstitution after HSCT, we showed that dysfunctional reconstitution patterns precede severe complications, such as chronic graft-versus-host disease, relapse, and death.     

Immunoglobulin Expression in Human Lymphoblastoid Cell Lines with Early B Cell Features

Immunoglobulin expression was studied by direct immunofluorescence and by biosynthesis experiments in two human cell lines Raji and T 5.1. The basic phenotype of these cells was close to that of pre-B cells: large cells with intracytoplasmic IgM with a predominance of mu chains over light chains and no detectable surface immunoglobulins. The apparent molecular weight of heavy and light chains was abnormally large. Immunoglobulins were secreted at a low rate as pentameric IgM in the T 5.1 line and as subunits and free light chains in the Raji line. Spontaneous variations of this phenotype were observed: the cultured cells acquired mu and lambda chains, then additionally delta chains while they progressively lost detectable cytoplasmic mu chains, thus leading to a mature B cell phenotype. Subsequently, the cells had no detectable surface and cytoplasmic immunoglobulins and then they displayed a pre-B cell phenotype again. Attempts to induce further maturation using various potential inducers were unsuccessful.     

自体外周血造血干细胞移植后造血重建中CD226分子在NK细胞上表达的变化

本文应用双重免疫荧光染色和流式细胞术分析,观察自体外周血造血干细胞移植患者造血重建中,CD226分子在CD56bright和CD56dimNK细胞亚群表达的变化.结果显示,在自体外周血造血干细胞移植造血重建中,于干细胞回输第12天,CD56 NK细胞占外周血淋巴细胞百分率为26.6%,其中CD56bright亚群,占CD56 NK细胞87.3%;CD56 CD226 细胞占CD56 NK细胞92.1%,CD56brightCD226 细胞占CD56 CD226 细胞89.9%.在自体外周血造血干细胞移植造血重建中,CD56bright亚群是NK细胞造血重建最早出现并占优势的一个亚群, CD226分子可能作为一种分化标记主要表达在CD56bright亚群上.     

Aberrant Immunoglobulin Kappa Locus Rearrangement in a Patient with CARD11-Related B Cell Lymphocytosis

Journal of Clinical Immunology -     

Multiple Antigen Peptide Containing B and T Cell Epitopes of F1 Antigen of Yersinia pestis Showed Enhanced Th1 Immune Response in Murine Model

Yersinia pestis is a facultative bacterium that can survive and proliferate inside host macrophages and cause bubonic, pneumonic and systemic infection. Apart from humoral response, cell‐mediated protection plays a major role in combating the disease. Fraction 1 capsular antigen (F1‐Ag) of Y. pestis has long been exploited as a vaccine candidate. In this study, F1‐multiple antigenic peptide (F1‐MAP or MAP)‐specific cell‐mediated and cytokine responses were studied in murine model. MAP consisting of three B and one T cell epitopes of F1‐antigen with one palmitoyl residue was synthesized using Fmoc chemistry. Mice were immunized with different formulations of MAP in poly DL‐lactide‐co‐glycolide (PLGA) microspheres. F1‐MAP with CpG oligodeoxynucleotide (CpG‐ODN) as an adjuvant showed enhanced in vitro T cell proliferation and Th1 (IL‐2, IFN‐γ and TNF‐α) and Th17 (IL‐17A) cytokine secretion. Similar formulation also showed significantly higher numbers of cytokine (IL‐2, IFN‐γ)‐secreting cells. Moreover, F1‐MAP with CpG formulation showed significantly high (P < 0.001) percentage of CD4⁺ IFN‐γ⁺ cells as compared to CD8⁺ IFN‐γ⁺ cells, and also more (CD4‐ IFN‐γ)⁺ cells secrete perforin and granzyme as compared to (CD8‐ IFN‐γ)⁺ showing Th1 response. Thus, the study highlights the importance of Th1 cytokine and existence of CD4⁺ and CD8⁺ immune response. This study proposes a new perspective for the development of vaccination strategies for Y. pestis that trigger T cell immune response.     

Immune Reconstitution Following Autologous Stem Cell Transplantation in Patients with High-Risk Neuroblastoma at the Time of Immunotherapy

Outcomes for patients with high-risk neuroblastoma (HR-NBL) are significantly improved with the addition of immunotherapy (dinutuximab?+?cytokines) following autologous hematopoietic stem cell transplantation (auto-HSCT). We hypothesized that the immune system is not fully reconstituted at the initiation of immunotherapy. To test this hypothesis, we evaluated hematologic and immune subsets in 34 patients with HR-NBL before and after auto-HSCT. We found that absolute T, B, and NK cell counts at the time of immunotherapy were below normal in 80% of patients. Patients with residual disease at the time of transplantation had significantly lower absolute lymphocyte counts (ALC; P?=?.008), lower CD16⁺ cell counts (P?=?.009), and an abnormal ratio of cytokine-releasing to cytotoxic NK cells at the time of dinutuximab treatment. In addition, the preparative regimen used for auto-HSCT predicted immune recovery. Finally, higher total white blood cell count (P?=?.013) and ALC (P?=?.013) at 3 months after completion of therapy were measured in patients who remained in remission compared with those who relapsed. Our results indicate that most patients with HR-NBL do not have full immune reconstitution at the time of dinutuximab treatment after auto-HSCT, and that immune recovery may correlate with disease-related outcomes in patients with high-risk disease.     

乙型肝炎患者免疫功能的检测及其临床意义

研究乙型肝炎患者外周血T淋巴细胞亚群、NK细胞和血清免疫球蛋白的变化及临床意义.采用流式细胞仪检测150例乙肝患者和30名健康者(对照组)外周血T淋巴细胞亚群(CD3 CD4 、CD3 CD8 )、NK细胞,免疫散射法检测血清免疫球蛋白(IgG、IgM、IgA)变化.结果表明各临床类型乙肝患者NK细胞降低,与对照组比较有显著性差异(P<0.01);慢性乙型肝炎组、慢性重型乙型肝炎组、肝硬化组外周血CD3 CD4 、CD3 CD8 T细胞均下降,其中慢性重型乙型肝炎组外周血CD3 CD8 与对照组比较有显著性差异(P<0.01),急性乙型肝炎组外周血T细胞亚群变化不明显(P>0.05);各临床类型乙型肝炎患者血清免疫球蛋白IgG、IgA随着病情的进展逐渐升高,与对照组比较有显著性差异(P<0.01).因此认为慢性乙肝患者存在细胞免疫和体液免疫功能紊乱,免疫功能的检测对乙肝的诊断、治疗及预后的判断有着一定临床意义.     

The Effect of Formula-based Nutritional Treatment on Colitis in a Murine Model

BackgroundExclusive enteral nutrition (EEN) induces remission in pediatric Crohn''s disease (CD). The exact mechanism of EEN therapy in CD is unknown, but alteration of the intestinal microflora after EEN is thought to affect mucosal healing. To determine the link between EEN therapy and therapeutic efficacy in CD, we established a murine model of dextran sulfate sodium (DSS)-induced colitis and applied EEN therapy.MethodsEight-week-old C57BL/6 mice were administered DSS for 4 days to induce colitis, and either normal chow (NC) or EEN was administered for the following 4 days. The mice were grouped according to the feeding pattern after DSS administration: DSS/NC and DSS/EEN groups. The clinical course was confirmed via daily observation of the weight and stool. Fecal samples were collected and 16sRNA sequencing was used. The mice were sacrificed to confirm colonic histopathology.ResultsWeight reduction and increase in disease activity were observed as the day progressed for 4 days after DSS administration. There was significant weight recovery and improvement in disease activity in the EEN group compared to that in the NC group. Verrucomicrobia and Proteobacteria abundances tended to increase and Bacteroidetes abundance decreased in the EEN group. In the EEN group, significant changes in the β-diversity of the microbiota were observed. In the analysis of microbiome species, abundances of Akkermansia muciniphila, Clostridium cocleatum, mucin-degrading bacteria, Flintibacter butyricus, and Parabacteroides goldsteinii, which are beneficial microbiota, were significantly increased in the EEN group compared to those in the NC group. More abundant mucins were confirmed in the colonic histopathology of the EEN group. These microbial and histopathological differences suggested that EEN might improve colitis symptoms in a murine colitis model by promoting mucin recycling and subsequently inducing the healing effect of the gut barrier.ConclusionEEN showed clinical efficacy in a murine model of colitis. Based on the increase in mucin-degrading bacteria and the pathological increase in mucin production after EEN administration, it can be observed that mucin plays an important role in the therapeutic effect of EEN.     

Capping and Co-Capping of Membrane Immunoglobulin and Lipid-Conjugated Immunoglobulin Inserted in the Cell Membrane of B Lymphocytes

Human and mouse immunoglobulins (Ig) or F(ab')2 fragments of rabbit Ig were conjugated to lipids and the conjugates inserted into the membrane of mouse spleen cells. It was found that nearly all B cells, but not T cells, became decorated with lipid immunoglobulin. Both endogenous Ig receptors and inserted Ig capped after the addition of cross-linking F(ab')2 antibodies and in both cases capping required energy. Capping of endogenous mouse Ig led to co-capping of inserted human Ig, but the reverse was not true.