Sequential infection of Epstein-Barr virus and cryptococcal encephalitis after umbilical cord blood transplantation in a child with X-linked adrenoleukodystrophy.

Dual infection with two pathogens can be found in few cases of encephalitis. Cases of sequential infection with EBV and cryptococcal encephalitis in post-transplant patients are rare. We describe a 5-year-old boy with X-linked adrenoleukodystrophy who presented sequential infection with EBV and cryptococcal encephalitis after umbilical cord blood transplant. The patient showed fever, vomiting and emotional agitation with EBV DNA detected in CSF on day 100. The child underwent 3 doses of intravenous rituximab with a good response. However, the child presented with right facial paralysis, headache, and fever on day 130 after 2 weeks of clinical stability. Brain MRI demonstrated chronic granuloma formed with ring enhancement. FilmArray ME PCR confirmed the existence of Cryptococcus neoformans/gattii in the CSF. The child underwent sequential treatment with amphotericin liposome B and flucytosine. Maintenance treatment with fluconazole was administered for 1 year. Facial paralysis was on longer present on day 260. Cryptococcus neoformans/gattii was not detected on day 310. The biochemistry and cell count of the CSF were completely normal on day 520. Follow-up 2.5 years after presentation, brain MRI changes showed near complete resolution of the lesions. The child survived for 3 years to the last following-up. Invasive cryptococcal encephalitis is rare and life-threatening complication of transplantation. It is important to recognize dual infections, and perform treatment quickly to improve the prognosis of encephalitis after transplantation.

Targeting foam cell formation in inflammatory brain diseases by the histone modifier MS-275.

OBJECTIVE: To assess class I-histone deacetylase (HDAC) inhibition on formation of lipid-accumulating, disease-promoting phagocytes upon myelin load in vitro, relevant for neuroinflammatory disorders like multiple sclerosis (MS) and cerebral X-linked adrenoleukodystrophy (X-ALD). METHODS: Immunohistochemistry on postmortem brain tissue of acute MS (n = 6) and cerebral ALD (n = 4) cases to analyze activation and foam cell state of phagocytes. RNA-Seq of in vitro differentiated healthy macrophages (n = 8) after sustained myelin-loading to assess the metabolic shift associated with foam cell formation. RNA-Seq analysis of genes linked to lipid degradation and export in MS-275-treated human HAP1 cells and RT-qPCR analysis of HAP1 cells knocked out for individual members of class I HDACs or the corresponding enzymatically inactive knock-in mutants. Investigation of intracellular lipid/myelin content after MS-275 treatment of myelin-laden human foam cells. Analysis of disease characteristic very long-chain fatty acid (VLCFA) metabolism and inflammatory state in MS-275-treated X-ALD macrophages. RESULTS: Enlarged foam cells coincided with a pro-inflammatory, lesion-promoting phenotype in postmortem tissue of MS and cerebral ALD patients. Healthy in vitro myelin laden foam cells upregulated genes linked to LXRα/PPARγ pathways and mimicked a program associated with tissue repair. Treating these cells with MS-275, amplified this gene transcription program and significantly reduced lipid and cholesterol accumulation and, thus, foam cell formation. In macrophages derived from X-ALD patients, MS-275 improved the disease-associated alterations of VLCFA metabolism and reduced the pro-inflammatory status of these cells. INTERPRETATION: These findings identify class I-HDAC inhibition as a potential novel strategy to prevent disease promoting foam cell formation in CNS inflammation.

Cerebral adrenoleukodystrophy is associated with loss of tolerance to profilin.

Childhood cerebral adrenoleukodystrophy (cALD) is a devastating manifestation of ALD accompanied by demyelination, inflammation, and blood brain barrier (BBB) disruption with shared characteristics of an auto-immune disease. We utilized plasma samples pre- and postdevelopment of cALD to determine the presence of specific auto-antibodies. Mass spectrometry of protein specifically bound with post-cALD plasma antibody identified Profilin1 (PFN1) as the target. In a screen of 94 boys with cALD 48 (51%) had anti-PFN1 antibodies, whereas only 2/29 boys with ALD but without cerebral disease, and 0/30 healthy controls showed anti-PFN1 immunoreactivity. Cerebral spinal fluid from those with cALD showed higher levels of PFN1 protein compared with non-cALD samples (324 ± 634 versus 42 ± 23 pg/mL, p = 0.04). Boys that were anti-PFN positive had a significant increase in the amount of gadolinium signal observed on MRI when compared to boys that were anti-PFN1 negative (p = 0.04) possibly indicating increased BBB disruption. Anti-PFN1 positivity was also associated with elevated levels of very long chain fatty acids (C26 of 1.12 ± 0.41 versus 0.97 ± 0.30 mg/dL, p = 0.03) and increased plasma BAFF (973 ± 277 versus 733 ± 269 pg/mL, p = 0.03). In conclusion, anti-PFN may be a novel biomarker associated with the development of cALD in boys with ALD.

PPAR-δ and erucic acid in multiple sclerosis and Alzheimer's Disease. Likely benefits in terms of immunity and metabolism.

The transcription factor, PPARδ is involved in suppressing inflammation, stimulating oligodendroglial biogenesis and myelination. Furthermore, activation of PPARδ directly protects mitochondria against noxious stimuli and stimulates biogenesis of new mitochondria. PPARδ activation directly inhibits neuronal cell death and reduces both the level and neurotoxicity of Amyloid-ß fibers in Alzheimer's Disease (AD) models. Among the important ligands of PPARδ is erucic acid (EA, 22:1 n9), an edible omega-9 fatty acid and a component of Lorenzo's oil, which is used in the treatment of adrenoleukodystrophy (ALD). Nonetheless, the feature of PPARδ-erucic acid interaction has not been extensively studied. EA can also be converted to nervonic acid, an important component of myelin. Hence, EA may act as an anti-inflammatory and remyelinating agent, which might be important in the management of another demyelinating disease, multiple sclerosis (MS). Oxidative injury and mitochondrial damage are among the features of ALD. Direct inhibitory effects of EA was observed on lipid peroxidation and inflammatory enzymes, neutrophil elastase and thrombin. EA also induces catalase, a potent antioxidant peroxisomal enzyme. However, EA is claimed to be a cardiotoxic molecule, yet these studies were mostly performed on rats, which do not efficiently metabolize EA. Further, EA is largely consumed by Asian population and Greenland Eskimos with no signs of cardiac damage. In this review, we shed light on the potential theraputic role of EA in MS and AD by blocking neural cell death, mitigating neuroinflammation and/or inducing myelination.

Impaired plasticity of macrophages in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy is caused by ATP-binding cassette transporter D1 (ABCD1) mutations and manifests by default as slowly progressive spinal cord axonopathy with associated demyelination (adrenomyloneuropathy). In 60% of male cases, however, X-linked adrenoleukodystrophy converts to devastating cerebral inflammation and demyelination (cerebral adrenoleukodystrophy) with infiltrating blood-derived monocytes and macrophages and cytotoxic T cells that can only be stopped by allogeneic haematopoietic stem cell transplantation or gene therapy at an early stage of the disease. Recently, we identified monocytes/macrophages but not T cells to be severely affected metabolically by ABCD1 deficiency. Here we found by whole transcriptome analysis that, although monocytes of patients with X-linked adrenoleukodystrophy have normal capacity for macrophage differentiation and phagocytosis, they are pro-inflammatory skewed also in patients with adrenomyloneuropathy in the absence of cerebral inflammation. Following lipopolysaccharide activation, the ingestion of myelin debris, normally triggering anti-inflammatory polarization, did not fully reverse the pro-inflammatory status of X-linked adrenoleukodystrophy macrophages. Immunohistochemistry on post-mortem cerebral adrenoleukodystrophy lesions reflected the activation pattern by prominent presence of enlarged lipid-laden macrophages strongly positive for the pro-inflammatory marker co-stimulatory molecule CD86. Comparative analyses of lesions with matching macrophage density in cases of cerebral adrenoleukodystrophy and acute multiple sclerosis showed a similar extent of pro-inflammatory activation but a striking reduction of anti-inflammatory mannose receptor (CD206) and haemoglobin-haptoglobin receptor (CD163) expression on cerebral adrenoleukodystrophy macrophages. Accordingly, ABCD1-deficiency leads to an impaired plasticity of macrophages that is reflected in incomplete establishment of anti-inflammatory responses, thus possibly contributing to the devastating rapidly progressive demyelination in cerebral adrenoleukodystrophy that only in rare cases arrests spontaneously. These findings emphasize monocytes/macrophages as crucial therapeutic targets for preventing or stopping myelin destruction in patients with X-linked adrenoleukodystrophy.

Inflammatory profile in X-linked adrenoleukodystrophy patients: Understanding disease progression.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disease characterized by progressive inflammatory demyelization in the brain, adrenal insufficiency, and an abnormal accumulation of very long chain fatty acids (VLCFA) in tissue and body fluids. Considering that inflammation might be involved in pathophysiology of X-ALD, we aimed to investigate pro- and anti-inflammatory cytokines in plasma from three different male phenotypes (CCER, AMN, and asymptomatic individuals). Our results showed that asymptomatic patients presented increased levels of pro-inflammatory cytokines IL-1ß, IL-2, IL-8, and TNF-α and the last one was also higher in AMN phenotype. Besides, asymptomatic patients presented higher levels of anti-inflammatory cytokines IL-4 and IL-10. AMN patients presented higher levels of IL-2, IL-5, and IL-4. We might hypothesize that inflammation in X-ALD is related to plasmatic VLCFA concentration, since there were positive correlations between C26:0 plasmatic levels and pro-inflammatory cytokines in asymptomatic and AMN patients and negative correlation between anti-inflammatory cytokine and C24:0/C22:0 ratio in AMN patients. The present work yields experimental evidence that there is an inflammatory imbalance associated Th1, (IL-2, IL-6, and IFN-γ), Th2 (IL-4 and IL-10), and macrophages response (TNF-α and IL-1ß) in the periphery of asymptomatic and AMN patients, and there is correlation between VLCFA plasmatic levels and inflammatory mediators in X-ALD. Furthermore, we might also speculate that the increase of plasmatic cytokines in asymptomatic patients could be considered an early biomarker of brain damage and maybe also a predictor of disease progression.

Loss of AMP-activated protein kinase induces mitochondrial dysfunction and proinflammatory response in unstimulated Abcd1-knockout mice mixed glial cells.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPKα1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice) mimics the human AMN phenotype and does not develop the cerebral inflammation characteristic of human ALD. In this study we document that AMPKα1 levels in vivo (in brain cortex and spinal cord) and in vitro in Abcd1-KO mixed glial cells are similar to that of wild type mice. Deletion of AMPKα1 in the mixed glial cells of Abcd1-KO mice induced spontaneous mitochondrial dysfunction (lower oxygen consumption rate and ATP levels). Mitochondrial dysfunction in ALD patient-derived cells and in AMPKα1-deleted Abcd1-KO mice mixed glial cells was accompanied by lower levels of mitochondrial complex (1-V) subunits. More importantly, AMPKα1 deletion induced proinflammatory inducible nitric oxide synthase levels in the unstimulated Abcd1-KO mice mixed glial cells. Taken together, this study provides novel direct evidence for a causal role for AMPK loss in the development of mitochondrial dysfunction and proinflammatory response in X-ALD.

Oxidative stress regulates the ubiquitin-proteasome system and immunoproteasome functioning in a mouse model of X-adrenoleukodystrophy.

Oxidative damage is a pivotal aetiopathogenic factor in X-linked adrenoleukodystrophy. This is a neurometabolic disease characterized by the accumulation of very-long-chain fatty acids owing to the loss of function of the peroxisomal transporter Abcd1. Here, we used the X-linked adrenoleukodystrophy mouse model and patient's fibroblasts to detect malfunctioning of the ubiquitin-proteasome system resulting from the accumulation of oxidatively modified proteins, some involved in bioenergetic metabolism. Furthermore, the immunoproteasome machinery appears upregulated in response to oxidative stress, in the absence of overt inflammation. i-Proteasomes are recruited to mitochondria when fibroblasts are exposed to an excess of very-long-chain fatty acids in response to oxidative stress. Antioxidant treatment regulates proteasome expression, prevents i-proteasome induction and translocation of i-proteasomes to mitochondria. Our findings support a key role of i-proteasomes in quality control in mitochondria during oxidative damage in X-linked adrenoleukodystrophy, and perhaps in other neurodegenerative conditions with similar pathogeneses.

Graves' orbitopathy in a patient with adrenoleukodystrophy after bone marrow transplantation.

OBJECTIVE: For many years, the treatment of X-linked childhood cerebral adrenoleukodystrophy (XALD) consisted of hydrocortisone replacement and a mixture of short chain-fatty acids, known as 'Lorenzo's oil'. Recently, bone marrow transplantation (BMT) has also been used. CASE REPORT: We report the case of a patient affected by XALD who developed Graves' hyperthyroidism (GH) and Graves' orbitopathy (GO) after BMT and who we could follow-up for 6.5 years afterwards. EVIDENCE SYNTHESIS: A boy affected by XALD was treated at the age of 6 years, with a whole BMT from his sister. One year after BMT, the transplanted patient presented TSH at the lower normal value and 3 years later he developed thyrotoxicosis. After a further 2 years, the patient developed GO, which showed clinical evidence of reactivation 5 years after its onset as a consequence of an attempt to treat thyrotoxicosis by means of I(131) (300 MBq). Seven years after BMT, the donor showed alterations of thyroid autoimmunity and 1 year thereafter she developed GH. She never presented GO during a subsequent 5 year follow-up. CONCLUSIONS: This case illustrates that autoimmunity originating from a pre-symptomatic donor can be transferred into the host during allogeneic stem cell transplantation. In cases where autoimmune phenomena are recognized in the donor prior to donation, alternative donors or T-cell manipulation of the graft might be considered.

Successful allogeneic unrelated bone marrow transplantation using reduced-intensity conditioning for the treatment of X-linked adrenoleukodystrophy in a one-yr-old boy.

The childhood cerebral form of X-linked ALD is a demyelinating disorder of the central nervous system, which rapidly leads to total disability and death. Allogeneic stem cell transplantation benefits patients who show early evidence of the demyelination. We report here a one-yr-old boy with ALD who received HLA-matched unrelated BMT in an early stage of the disease after careful planning and observation since his birth. BMT was performed when MRI began to show slight signal intensity changes in the white matter of the brain. Pretransplant conditioning consisted of fludarabine, l-PAM and TBI (2 Gy). GVHD prophylaxis consisted of cyclosporine A and short-course methotrexate. The patient showed an uneventful BMT course with fast and stable engraftment. Following BMT, the plasma levels of VLCFA decreased gradually and MRI changes improved. The patient did not have any evidence of further neurological deterioration 22 months following the transplant. Although this is still a short follow-up, it has been shown that BMT should be considered when a child has a biochemical diagnosis and MRI findings of ALD without any neurological signs. RIST should be considered as a pretransplant conditioning for ALD.

Immune response in leukodystrophies.

Although the genetics and biochemistry of leukodystrophies have been extensively explored, the immune response in these disorders has received relatively little attention. Both the disease course and its response to treatment may be highly dependent on the immune system. In this review, we compare three common leukodystrophies, each with a different immune response: (1) X-linked adrenoleukodystrophy, which demonstrates a severe, lymphocytic inflammatory response; (2) metachromatic leukodystrophy, which yields a histiocytic response; and (3) vanishing white-matter disease, in which no inflammation is typically seen. We highlight the biochemical, pathologic, and clinical differences, while focusing on the immune response in each disease. We also review the response of leukodystrophies to immunomodulatory therapies and interventions such as hematopoietic stem-cell transplantation. Future studies may delineate specific inflammatory markers as possible candidates for therapeutic intervention.

Immunopathogenesis of adrenoleukodystrophy: current understanding.

Adrenoleukodystrophy is a neurometabolic disease with a decreased ability to degrade very long chain fatty acids (VLCFA) and significant phenotypic variation. Unlike most neurometabolic diseases, the success of hematopoietic stem cell transplantation (HSCT) is based on acquiring a new immune system rather than enzyme replacement. VLCFA accumulation appears necessary but not sufficient for pathogenesis. Evidence for the involvement of different components of the immune system in the pathogenesis of the cerebral lesions (cellular, cytokines, humoral, and complement) is reviewed, along with both HSCT and non-HSCT immunologic approaches to treatment and future directions.

State of the art. Four easy pieces: interconnections between tissue injury, intermediary metabolism, autoimmunity, and chronic degeneration.

Four questions are posed: (1) Can tissue damage itself provoke autoimmunity? (2) Can genetic mutations of key structures produce tissue pathology and thus provoke autoimmunity? (3) Can acute immune damage produce tissue degeneration without further hallmarks of an immune response? (4) Can intermediary metabolism modulate immune damage to tissues? Four answers are given: (1) Tissue injury itself may lead to autoimmunity. Both innate and adaptive immunity may arise as a response to tissue injury, and the immune attack can further damage tissue. (2) Genetic mutations can lead to an immune response indistinguishable from autoimmunity, exemplified from Duchenne's Muscular Dystrophy and X-linked adrenoleukodystrophy. (3) Chronic immune damage may lead to tissue degeneration, with or without further hallmarks of an immune response. Variations on this theme, including inverse scenarios, are also possible: Inborn errors of metabolism may lead to tissue damage that may provoke an adaptive and or innate immune response. The immune response might further damage tissue. (4) Finally, perturbations of intermediary metabolism may modulate the immune response, controlling the extent of immune-mediated damage. Examples are taken from perturbations in the cholesterol pathway that influence the characteristics of the immune response, and with tryptophan metabolites that modulate autoimmunity and graft rejection. Inflammatory, degenerative, and autoimmune neurological disease will be discussed in terms of their implications for pathogenic mechanisms underlying chronic obstructive pulmonary disease.

T-cell receptor Vbeta gene usage in CSF lymphocytes in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder with impaired very-long-chain fatty acid (VLCFA) metabolism that produces a neurological disease with significant variability of clinical phenotypes even within kindred. The two most common forms are the cerebral form (CALD) with an important inflammatory reaction at the active edge of demyelinating lesions, resembling some aspects of multiple sclerosis pathology, and adrenomyeloneuropathy (AMN), which involves the spinal cord and in which the inflammatory reaction is mild or absent. One hypothesis is that the phenotypic variability is related to T cell-mediated immune mechanisms playing a primary role in the demyelinating pathogenic process of CALD. The present study aims to test the hypothesis that CSF of patients with the CALD form contains highly restricted T cell populations. The variable regions of the T cell receptor beta chains (TCR Vbeta) were studied in CSF from 29 ALD patients with different phenotypes. RNA was extracted and cDNA synthesized from CSF lymphocytes; TCR Vbeta gene segments were amplified from the cDNA by polymerase chain reaction (PCR) using 20 family-specific primers. PCR products were analyzed by Southern blot. Some amplified Vbeta products were sequenced. The majority of ALD patients (21/29), whatever their phenotype, exhibited oligoclonal T cell expansion. However the overexpression of some TCR Vbeta families was heterogeneous among the different patients without any preponderance of specific Vbeta families or any clustering according to clinical phenotype. In particular a dominant TCR Vbeta utilization was not found in patients with CALD.

Genetic variations and humoral immune responses to myelin oligodendroglia glycoprotein in adult phenotypes of X-linked adrenoleukodystrophy.

The lack of phenotype/genotype association in X-linked adrenoleukodystrophy (X-ALD) has prompted the search for disease modifying factors. We previously demonstrated increased serum antibody responses against myelin oligodendrocyte glycoprotein (MOG) in various clinical phenotypes of X-ALD allowing speculations that myelin specific humoral immune responses might be involved in phenotype generation of X-ALD. In the present study, we investigated the possible association of (1) a naturally occurring variable number tandem repeat (vntr) polymorphism (C allele) in the 3' flanking region of the interleukin-6 gene (IL-6), previously demonstrated to modify the course of Alzheimer's disease, systemic lupus erythematodes and Multiple Sclerosis (MS), (2) a tetranucleotide repeat polymorphism (TAAA)(n) in the 3' flanking region of the MOG gene and (3) HLA class II alleles with adult clinical phenotypes and serum antibody responses to MOG in 70 adult X-ALD patients. HLA class II alleles, (TAAA)(n) MOG gene polymorphisms, and IL-6 C allele were not associated with clinical phenotypes. Anti-MOG antibodies were detectable in 17/54 X-ALD patients (31.5%). Anti-MOG antibodies were associated with the 226 bp (TAAA)(n) MOG gene polymorphism but not with distinct clinical phenotypes.

Potential environmental and host participants in the early white matter lesion of adreno-leukodystrophy: morphologic evidence for CD8 cytotoxic T cells, cytolysis of oligodendrocytes, and CD1-mediated lipid antigen presentation.

The 2 most common forms of X-linked adreno-leukodystrophy (ALD) are the juvenile or childhood cerebral form with inflammatory demyelination and the adult adrenomyeloneuropathy (AMN) involving spinal cord tracts without significant inflammation. Modifier genes or environmental factors may contribute to the phenotypic variability. We performed immunohistochemical, an in situ polymerase chain reaction, and TUNEL analyses to identify several viruses, lymphocyte subpopulations, apoptotic cells, and effector molecules, focusing on morphologically normal white matter, dysmyelinative and acute demyelinative lesions. No distinguishing viral antigens were detected. Most lymphocytes were CD8 cytotoxic T cells (CTLs) with the alpha/beta TCR, and they infiltrated morphologically unaffected white matter. Only a few oligodendrocytes were immunoreactive for caspase-3. MHC class II- and TGF-beta-positive microglia were present. CD44, which can mediate MHC-unrestricted target cell death, was seen on many lymphocytes and white matter elements. CD1 molecules, which play major roles in MHC-unrestricted lipid antigen presentation, were noted. Our data indicate that unconventional CD8 CTLs are operative in the early stages of dysmyelination/demyelination and that cytolysis of oligodendrocytes, rather than apoptosis, appears to be the major mode of oligodendrocytic death. The presentation of lipid antigens may be a key pathogenetic element in ALD and AMN-ALD.

Serum autoantibody responses to myelin oligodendrocyte glycoprotein and myelin basic protein in X-linked adrenoleukodystrophy and multiple sclerosis.

We analyzed the sera of 51 patients with various phenotypes of X-linked adrenoleukodystrophy (X-ALD), 20 patients with multiple sclerosis (MS) and 22 healthy volunteers for the presence of autoantibodies specific for the recombinant extracellular immunoglobulin-like domain of human myelin oligodendrocyte glycoprotein (rhMOG(Igd)) and myelin basic protein (MBP). Anti-rhMOG(Igd) autoantibodies were significantly more frequent in X-ALD and MS patients as opposed to healthy individuals (p<0.05). Anti-MBP autoantibodies were present in about one-fourth of X-ALD and MS patients but in less than 10% of healthy individuals. Anti-rhMOG(Igd) autoantibody responses were not accompanied by increased T cell reactivity against rhMOG(Igd). These findings may have important implications for the understanding of humoral anti-myelin immunoreactivity in demyelinating diseases of the central nervous system such as X-ALD and MS.

Myelin oligodendrocyte glycoprotein (MOG) polymorphisms and adrenoleukodystrophy.

We describe four novel sequence variants in the Myelin Oligodendrocyte Glycoprotein (MOG) gene. A total of six sequence variants of the MOG gene were identified in eleven out of 44 ALD patients investigated: G15A, CTC repeat in exon 1, Val142Leu, Val145Ile, 551+68A-->G and 551+77C-->T. Screening studies demonstrated that all these polymorphisms are present in 50 unaffected control male individuals of the same population and in the different phenotypes of ALD patients, indicating that they do not contribute to phenotype variability in ALD.