IPSC-Derived Astrocytes to Model Neuroinflammatory and Metabolic Responses in X-linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder caused by pathogenic variants in the ABCD1 gene, leading to accumulation of saturated very long chain fatty acids (VLCFA) in body fluids and tissues including brain and spinal cord. In the absence of a clear genotype-phenotype correlation the molecular mechanisms of the fatal cerebral adrenoleukodystrophy (cALD) and the milder adrenomyeloneuropathy (AMN) phenotypes remain unknown. Given our previous evidence of role of astrocytes in the neuroinflammatory response in X-ALD we investigated the metabolic and molecular profiles of astrocytes derived from induced pluripotent stem cells (iPSC). The iPSCs were in turn generated from skin fibroblasts of healthy controls and patients with AMN or cALD. AMN and cALD astrocytes exhibited lack of ABCD1 and accumulation of VLCFA, a biochemical hallmark of X-ALD disease. Accumulation of VLCFA was significantly higher in cALD astrocytes. Mitochondrial function analysis by Seahorse extracellular flux identified increased oxygen consumption and extracellular acidification rates in cALD astrocytes, yet the ATP levels were decreased. Molecular signaling identified increased phosphorylation of STAT3 in cALD astrocytes, and higher proinflammatory cytokine and Toll like receptor (TLR) expression. CRISPR-Cas9 knock-in of functional ABCD1 gene expression differentially affected the expression of key molecular and metabolic targets in AMN and cALD astrocytes. AMN and cALD iPSC-derived astrocytes and their isogenic controls demonstrate differential aspects of X-ALD metabolic and inflammatory response to ABCD1 mutation and can be further utilized for exploring the contribution of iPSC-derived astrocytes to differential X-ALD disease pathology.

System-based integrated metabolomics and microRNA analysis identifies potential molecular alterations in human X-linked cerebral adrenoleukodystrophy brain.

X-linked adrenoleukodystrophy is a severe demyelinating neurodegenerative disease mainly affecting males. The severe cerebral adrenoleukodystrophy (cALD) phenotype has a poor prognosis and underlying mechanism of onset and progression of neuropathology remains poorly understood. In this study we aim to integrate metabolomic and microRNA (miRNA) datasets to identify variances associated with cALD. Postmortem brain tissue samples from five healthy controls (CTL) and five cALD patients were utilized in this study. White matter from ALD patients was obtained from normal-appearing areas, away from lesions (NLA) and from the periphery of lesions- plaque shadow (PLS). Metabolomics was performed by gas chromatography coupled with time-of-flight mass spectrometry and miRNA expression analysis was performed by next generation sequencing (RNAseq). Principal component analysis revealed that among the three sample groups (CTL, NLA and PLS) there were 19 miRNA, including several novel miRNA, of which 17 were increased with disease severity and 2 were decreased. Untargeted metabolomics revealed 13 metabolites with disease severity-related patterns with 7 increased and 6 decreased with disease severity. Ingenuity pathway analysis of differentially altered metabolites and miRNA comparing CTL with NLA and NLA with PLS, identified several hubs of metabolite and signaling molecules and their upstream regulation by miRNA. The transomic approach to map the crosstalk between miRNA and metabolomics suggests involvement of specific molecular and metabolic pathways in cALD and offers opportunity to understand the complex underlying mechanism of disease severity in cALD.

Beneficial Effects of the Direct AMP-Kinase Activator PXL770 in In Vitro and In Vivo Models of X-Linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a severe orphan disease caused by mutations in the peroxisomal ABCD1 transporter gene, leading to toxic accumulation of Very Long-Chain Fatty Acids (VLCFA - in particular C26:0) resulting in inflammation, mitochondrial dysfunction and demyelination. AMP-activated protein kinase (AMPK) is downregulated in ALD, and its activation is implicated as a therapeutic target. PXL770 is the first direct allosteric AMPK activator with established clinical efficacy and tolerability. Methods: We investigated its effects in ALD patient-derived fibroblasts/lymphocytes and Abcd1 KO mouse glial cells. Readouts included VLCFA levels, mitochondrial function and mRNA levels of proinflammatory genes and compensatory transporters (ABCD2-3). After PXL770 treatment in Abcd1 KO mice, we assessed VLCFA levels in tissues, sciatic nerve axonal morphology by electronic microscopy and locomotor function by open-field/balance-beam tests. Results: In patients' cells and Abcd1 KO glial cells, PXL770 substantially decreased C26:0 levels (by ∼90%), improved mitochondrial respiration, reduced expression of multiple inflammatory genes and induced expression of ABCD2-3 In Abcd1 KO mice, PXL770 treatment normalized VLCFA in plasma and significantly reduced elevated levels in brain (-25%) and spinal cord (-32%) versus untreated (P < 0.001). Abnormal sciatic nerve axonal morphology was also improved along with amelioration of locomotor function. Conclusion: Direct AMPK activation exerts beneficial effects on several hallmarks of pathology in multiple ALD models in vitro and in vivo, supporting clinical development of PXL770 for this disease. Further studies would be needed to overcome limitations including small sample size for some parameters, lack of additional in vivo biomarkers and incomplete pharmacokinetic characterization. SIGNIFICANCE STATEMENT: Adrenoleukodystrophy is a rare and debilitating condition with no approved therapies, caused by accumulation of very long-chain fatty acids. AMPK is downregulated in the disease and has been implicated as a potential therapeutic target. PXL770 is a novel clinical stage direct AMPK activator. In these studies, we used PXL770 to achieve preclinical validation of direct AMPK activation for this disease - based on correction of key biochemical and functional readouts in vitro and in vivo, thus supporting clinical development.

Therapeutic potential of deuterium-stabilized (R)-pioglitazone-PXL065-for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) results from ABCD1 gene mutations which impair Very Long Chain Fatty Acids (VLCFA; C26:0 and C24:0) peroxisomal import and ß-oxidation, leading to accumulation in plasma and tissues. Excess VLCFA drives impaired cellular functions (e.g. disrupted mitochondrial function), inflammation, and neurodegeneration. Major disease phenotypes include: adrenomyeloneuropathy (AMN), progressive spinal cord axonal degeneration, and cerebral ALD (C-ALD), inflammatory white matter demyelination and degeneration. No pharmacological treatment is available to-date for ALD. Pioglitazone, an anti-diabetic thiazolidinedione, exerts potential benefits in ALD models. Its mechanisms are genomic (PPARγ agonism) and nongenomic (mitochondrial pyruvate carrier-MPC, long-chain acyl-CoA synthetase 4-ACSL4, inhibition). However, its use is limited by PPARγ-driven side effects (e.g. weight gain, edema). PXL065 is a clinical-stage deuterium-stabilized (R)-enantiomer of pioglitazone which lacks PPARγ agonism but retains MPC activity. Here, we show that incubation of ALD patient-derived cells (both AMN and C-ALD) and glial cells from Abcd1-null mice with PXL065 resulted in: normalization of elevated VLCFA, improved mitochondrial function, and attenuated indices of inflammation. Compensatory peroxisomal transporter gene expression was also induced. Additionally, chronic treatment of Abcd1-null mice lowered VLCFA in plasma, brain and spinal cord and improved both neural histology (sciatic nerve) and neurobehavioral test performance. Several in vivo effects of PXL065 exceeded those achieved with pioglitazone. PXL065 was confirmed to lack PPARγ agonism but retained ACSL4 activity of pioglitazone. PXL065 has novel actions and mechanisms and exhibits a range of potential benefits in ALD models; further testing of this molecule in ALD patients is warranted.

Endogenous antibody responsiveness to epidermal growth factor receptor is associated with immunoglobulin allotypes and overall survival of patients with glioblastoma.

BACKGROUND: Immunoglobulin γ marker (GM) and κ marker (KM) allotypes, hereditary antigenic determinants of γ and κ chains, respectively, have been shown to be associated with immunity to a variety of self and nonself antigens, but their possible contribution to immunity to the tumor-associated antigens epidermal growth factor receptor (EGFR) and EGFR variant (v)III has not been evaluated. The aim of the present investigation was to determine whether the interindividual variation in endogenous antibody responsiveness to EGFR and EGFRvIII is associated with particular GM, KM, and Fcγ receptor (FcγR) genotypes and whether antibody levels were associated with the overall survival of patients with glioblastoma. METHODS: A total of 126 Caucasian participants with glioblastoma were genotyped for several GM, KM, and FcγR alleles and characterized for IgG antibodies to EGFR and EGFRvIII antigens. RESULTS: The anti-EGFR antibody levels associated with GM 3/3 homozygotes and GM 3/17 heterozygotes were similar (15.9 vs 16.4 arbitrary units [AU]/µL) and significantly lower than those associated with GM 17/17 homozygotes (19.6 AU/µL; nominal P = .007). Participants homozygous for the GM 21 allele also had significantly higher levels of anti-EGFR antibodies than GM 5/5 homozygotes and GM 5/21 heterozygotes (20.1 vs 16.0 and 16.3 AU/µL; nominal P = .005). Similar associations were found with immune responsiveness to EGFRvIII. Higher anti-EGFR and anti-EGFRvIII antibody levels were associated with enhanced overall survival (16 vs 11 mo, nominal P = .038 and 20 vs 11 mo, nominal P = .004, respectively). CONCLUSIONS: GM allotypes contribute to humoral immunity to EGFR in glioblastoma.

Promoting thiol expression increases the durability of antitumor T-cell functions.

Ex vivo-expanded CD8(+) T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced antitumor efficacy, relative to CD8(+) T cells with a central memory-like phenotype (TCM cells). Furthermore, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM cells possess relatively greater antioxidative capacity than TEM cells. Here, we report that TCM cells exhibit a relative increase compared with TEM cells in the expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity, and glycolytic enzyme levels. Notably, T-cell receptor-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, upregulated thiol levels and antioxidant genes. A comparison of antitumor CD8(+) T-cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth and that profiling this biomarker may have benefits to adoptive T-cell immunotherapy protocols.

Immunoglobulin genes implicated in glioma risk.

Both genetic and environmental factors are thought to be causal in gliomagenesis. Several genes have been implicated in glioma development, but the putative role of a major immunity-related gene complex member, immunoglobulin heavy chain γ (IGHG) has not been evaluated. Prior observations that IGHG-encoded γ marker (GM) allotypes exhibit differential sensitivity to an immunoevasion strategy of cytomegalovirus, a pathogen implicated as a promoter of gliomagenesis, has lead us to hypothesize that these determinants are risk factors for glioma. To test this hypothesis, we genotyped the IGHG locus comprising the GM alleles, specifically GM alleles 3 and 17, of 120 glioma patients and 133 controls via TaqMan® genotyping assay. To assess the associations between GM genotypes and the risk of glioma, we applied an unconditional multivariate logistic regression analysis adjusted for potential confounding variables. In comparison to subjects who were homozygous for the GM 17 allele, the GM 3 homozygotes were over twice as likely, and the GM 3/17 heterozygotes were over three times as likely, to develop glioma. Similar results were achieved when analyzed by combining the data corresponding to alleles GM 3 and GM 3/17 in a dominant model. The GM 3/17 genotype and the combination of GM 3 and GM 3/17 were found to be further associated with over 3 times increased risk for high-grade astrocytoma (grades III-IV). Allele frequency analyses also showed an increased risk for gliomas and high-grade astrocytoma in association with GM 3. Our findings support the premise that the GM 3 allele may present risk for the development of glioma, possibly by modulating immunity to cytomegalovirus.

Immunoglobulin genes influence the magnitude of humoral immunity to cytomegalovirus glycoprotein B.

Human cytomegalovirus (HCMV) is a risk factor for many human diseases, but among exposed individuals, not everyone is equally likely to develop HCMV-spurred diseases, implying the presence of host genetic factors that might modulate immunity to this virus. Here, we show that antibody responsiveness to HCMV glycoprotein B (gB) is significantly associated with particular immunoglobulin GM (γ marker) genotypes. Anti-HCMV gB antibody levels were highest in GM 17/17 homozygotes, intermediate in GM 3/17 heterozygotes, and lowest in GM 3/3 homozygotes (28.2, 19.0, and 8.1 µg/mL, respectively; P=.014). These findings provide mechanistic insights in the etiopathogenesis of HCMV-spurred diseases.

A quantitative increase in regulatory T cells controls development of vitiligo.

T-cell cytolytic activity targeting epidermal melanocytes is shown to cause progressive depigmentation and autoimmune vitiligo. By using the recently developed transgenic mice h3TA2 that carry T cells with a HLA-A2-restricted human tyrosinase peptide (h-Tyr)-reactive TCR and develop spontaneous vitiligo from an early age, we addressed the mechanism regulating autoimmune vitiligo. Depigmentation was significantly impaired only in IFN-γ-knockout h3TA2 mice but not in TNF-α- or perforin-knockout h3TA2 mouse strains, confirming a central role for IFN-γ in vitiligo development. In addition, regulatory T cells (Tregs) were relatively abundant in h3TA2-IFN-γ(-/-) mice, and depletion of the Treg-engaging anti-CD25 antibody fully restored the depigmentation phenotype in h3TA2-IFN-γ(-/-) mice, mediated in part through the upregulation of proinflammatory cytokines such as IL-17 and IL-22. Further therapeutic potential of Treg abundance in preventing progressive depigmentation was evaluated by adoptively transferring purified Treg or using rapamycin. Both the adoptive transfer of Tregs and the use of rapamycin induced a lasting remission of vitiligo in mice treated at the onset of disease, or in mice with established disease. This leads us to conclude that reduced regulatory responses are pivotal to the development of vitiligo in disease-prone mice, and that a quantitative increase in the Treg population may be therapeutic for vitiligo patients with active disease.

T cells expanded in presence of IL-15 exhibit increased antioxidant capacity and innate effector molecules.

Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. Whereas long-term IL-2 exposure has been shown to promote apoptosis and limit CD8(+) memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and helps maintain a memory CD8(+) T-cell population. However, mechanisms for superior outcomes for IL-15 as compared to IL-2 are still under investigation. Our data shows that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules glutathione reductase (GSR), thioredoxin reductase 1 (TXNDR1), peroxiredoxin (PRDX) and superoxide dismutase (SOD). An increased expression of cell-surface thiols, intracellular glutathione, and thioredoxins was also noted in IL-15 cultured T cells. Additionally, IL-15 cultured T cells showed an increase in cytolytic effector molecules. Apart from increased level of Granzyme A and Granzyme B, IL-15 cultured T cells exhibited increased accumulation of reactive oxygen (ROS) and reactive nitrogen species (RNS) as compared to IL-2 cultured T cells. Overall, this study suggests that T cells cultured in IL-15 show increased persistence not only due to levels of anti-apoptotic proteins, but also due to increased anti-oxidant levels, which is complimented by increased cytolytic effector functions.

In vitro propagation of rose.

In vitro propagation of rose is an important tool for rapid multiplication and development of new cultivars with desirable traits. However, successful in vitro propagation requires an understanding of specific requirements and precise manipulation of various factors. Efficient protocols for different stages of micropropagation using apical buds or nodal segments are currently available. Recently, new challenges for refinements of protocols for high rate of shoot multiplication and development of cost effective methods has gained importance. Significance of the liquid static culture for shoot proliferation and root induction for rose has also gained prominence. Other distinct approaches of in vitro propagation include organogenesis and embryogenesis. These approaches are important for the successful implementation of various biotechnological techniques used for rose improvement programmes. Types of explants, media and optimization of conditions are major factors for successful regeneration of rose.

Inhibition of superoxide generation upon T-cell receptor engagement rescues Mart-1(27-35)-reactive T cells from activation-induced cell death.

Cytotoxic T lymphocytes (CTL) may undergo massive expansion upon appropriate antigenic stimulation. Homeostasis is maintained by a subsequent "contraction" of these cells. Activation-induced cell death (AICD) and programmed cell death prevent the untoward side effects, arising from excessive numbers and prolonged persistence of activated CTL, that occur upon uncontrolled and/or continued expansion. However, effector cell persistence has been identified as a hallmark of successful T-cell-mediated adoptive immunotherapy. Thus, prevention of AICD may be critical to achieve more successful clinical results. We have previously shown that treatment with the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 protects human melanoma epitope Mart-1(27-35)-reactive CTL from apoptotic death upon their reencounter with cognate antigen. However, inhibition of JNK also interferes with the functional ability of the CTL to secrete IFN-gamma. Here, we show that reactive oxygen species (ROS) inhibitors, such as the superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), efficiently protected Mart-1(27-35)-reactive primary CTL from AICD without impairing their functional capability. MnTBAP prevented the increase in intracellular ROS, mitochondrial membrane collapse, and DNA fragmentation observed in control-treated cells upon cognate antigen encounter. Furthermore, the mechanism of AICD prevention in primary CTL included blockade of JNK activation. Finally, tumor-reactive in vitro expanded tumor infiltrating lymphocytes, which are used clinically in cancer immunotherapy, also benefit from MnTBAP-mediated antioxidant treatment. Thus, modulation of the redox pathway might improve CTL persistence and lead to better clinical results for T cell-based immunotherapies.