The mGlu2/3 agonist LY379268 reduces sucrose taking, seeking, and motivation in male and female rats.

OBJECTIVES: The mGlu2/3 receptor agonist LY379268 reduces sucrose-seeking, but not sucrose-taking, in male rats. This study explored the generality of this effect across the sexes. In addition, the effect of the drug on motivation to receive sucrose was assessed. METHODS: Adult male and female Long-Evans rats ( N = 91) were challenged with LY379268 in three experiments: (1) a fixed ratio (FR) schedule of reinforcement (taking), (2) extinction of responding previously reinforced on the FR (seeking) or (3) responding reinforced on a progressive ratio (PR) schedule of reinforcement (motivation). For each experiment, rats first responded to 10% liquid sucrose on an FR in 10 daily 2-h sessions. For the PR study, this was followed by training on a PR for 7 daily 3-h sessions. Rats were then challenged in a counterbalanced order with LY379268 (0, 1.5, 3 and 6 mg/kg; IP; 30-min pretreatment) on test days, followed by either three reacquisition days of FR (experiments 1 and 2) or PR (experiment 3) responding. RESULTS: Female rats responded more to sucrose on the FR and PR. LY379268 reduced responding in all three experiments. LY379268 challenge to sucrose taking on the FR produced an inverted U-shaped function while extinction responding and responding for sucrose on the PR were decreased dose-dependently, with PR responding insensitive to the 1.5 mg/kg dose. There were no sex-dependent effects of the drug on sucrose-directed responding. CONCLUSIONS: The sucrose anti-taking, -seeking, and -motivation effects of LY379268 across male and female rats support further evaluation of glutamate modulation as an antiaddiction pharmacotherapy.

RG/RGG repeats in the C. elegans homologs of Nucleolin and GAR1 contribute to sub-nucleolar phase separation.

The intrinsically disordered RG/RGG repeat domain is found in several nucleolar and P-granule proteins, but how it influences their phase separation into biomolecular condensates is unclear. We survey all RG/RGG repeats in C. elegans and uncover nucleolar and P-granule-specific RG/RGG motifs. An uncharacterized protein, K07H8.10, contains the longest nucleolar-like RG/RGG domain in C. elegans. Domain and sequence similarity, as well as nucleolar localization, reveals K07H8.10 (NUCL-1) to be the homolog of Nucleolin, a protein conserved across animals, plants, and fungi, but previously thought to be absent in nematodes. Deleting the RG/RGG repeats within endogenous NUCL-1 and a second nucleolar protein, GARR-1 (GAR1), demonstrates these domains are dispensable for nucleolar accumulation. Instead, their RG/RGG repeats contribute to the phase separation of proteins into nucleolar sub-compartments. Despite this common RG/RGG repeat function, only removal of the GARR-1 RG/RGG domain affects worm fertility and development, decoupling precise sub-nucleolar structure from nucleolar function.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

A longitudinal and cross-sectional study of plasma neurofilament light chain concentration in Charcot-Marie-Tooth disease.

Advances in genetic technology and small molecule drug development have paved the way for clinical trials in Charcot-Marie-Tooth disease (CMT); however, the current FDA-approved clinical trial outcome measures are insensitive to detect a meaningful clinical response. There is, therefore, a need to identify sensitive outcome measures or clinically relevant biomarkers. The aim of this study was to further evaluate plasma neurofilament light chain (NFL) as a disease biomarker in CMT. Plasma NFL was measured using SIMOA technology in both a cross-sectional study of a US cohort of CMT patients and longitudinally over 6 years in a UK CMT cohort. In addition, plasma NFL was measured longitudinally in two mouse models of CMT2D. Plasma concentrations of NFL were increased in a US cohort of patients with CMT1B, CMT1X and CMT2A but not CMT2E compared with controls. In a separate UK cohort, over a 6-year interval, there was no significant change in plasma NFL concentration in CMT1A or HSN1, but a small but significant reduction in patients with CMT1X. Plasma NFL was increased in wild type compared to GARSC201R mice. There was no significant difference in plasma NFL in GARSP278KY compared to wild type mice. In patients with CMT1A, the small difference in cross-sectional NFL concentration vs healthy controls and the lack of change over time suggests that plasma NFL may lack sufficient sensitivity to detect a clinically meaningful treatment response in adulthood.

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. We found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, we found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. Our findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Blockade of LAG-3 in PD-L1-Deficient Mice Enhances Clearance of Blood Stage Malaria Independent of Humoral Responses.

T cells expressing high levels of inhibitory receptors such as PD-1 and LAG-3 are a hallmark of chronic infections and cancer. Checkpoint blockade therapies targeting these receptors have been largely validated as promising strategies to restore exhausted T cell functions and clearance of chronic infections and tumors. The inability to develop long-term natural immunity in malaria-infected patients has been proposed to be at least partially accounted for by sustained expression of high levels of inhibitory receptors on T and B lymphocytes. While blockade or lack of PD-1/PD-L1 and/or LAG-3 was reported to promote better clearance of Plasmodium parasites in various mouse models, how exactly blockade of these pathways contributes to enhanced protection is not known. Herein, using the mouse model of non-lethal P. yoelii (Py) infection, we reveal that the kinetics of blood parasitemia as well as CD4+ T follicular helper (TFH) and germinal center (GC) B cell responses are indistinguishable between PD-1-/-, PD-L1-/- and WT mice. Yet, we also report that monoclonal antibody (mAb) blockade of LAG-3 in PD-L1-/- mice promotes accelerated control of blood parasite growth and clearance, consistent with prior therapeutic blockade experiments. However, neither CD4+ TFH and GC B cell responses, nor parasite-specific Ab serum titers and capacity to transfer protection differed. We also found that i) the majority of LAG-3+ cells are T cells, ii) selective depletion of CD4+ but not CD8+ T cells prevents anti-LAG-3-mediated protection, and iii) production of effector cytokines by CD4+ T cells is increased in anti-LAG-3-treated versus control mice. Thus, taken together, these results are consistent with a model in which blockade and/or deficiency of PD-L1 and LAG-3 on parasite-specific CD4+ T cells unleashes their ability to effectively clear blood parasites, independently from humoral responses.

Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models.

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.

Accumulating Evidence for Axonal Translation in Neuronal Homeostasis.

The specialized structure of the neuron requires that homeostasis is sustained over the meter or more that may separate a cell body from its axonal terminus. Given this impressive distance and an axonal volume that is many times that of the cell body, how is such a compartment grown during development, re-grown after injury, and maintained throughout adulthood? While early answers to these questions focused on the local environment or the cell soma as supplying the needs of the axon, it is now well-established that the axon has some unique needs that can only be met from within. Decades of research have revealed local translation as an indispensable mechanism of axonal homeostasis during development and regeneration in both invertebrates and vertebrates. In contrast, the extent to which the adult, mammalian axonal proteome is maintained through local translation remains unclear and controversial. This mini-review aims to highlight important experiments that have helped to shape the field of axonal translation, to discuss conceptual arguments and recent evidence that supports local translation as important to the maintenance of adult axons, and to suggest experimental approaches that have the potential to further illuminate the role of axonal translation in neuronal homeostasis.

Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.

Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system of CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behavior concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. Through in vitro experiments, mutant, but not wild-type, GlyRS was shown to aberrantly interact with the Trk receptors and cause misactivation of Trk signaling, which is essential for sensory neuron differentiation and development. Together, this work suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.

Severity of Demyelinating and Axonal Neuropathy Mouse Models Is Modified by Genes Affecting Structure and Function of Peripheral Nodes.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited polyneuropathies. Mutations in 80 genetic loci can cause forms of CMT, resulting in demyelination and axonal dysfunction. The clinical presentation, including sensory deficits, distal muscle weakness, and atrophy, can vary greatly in severity and progression. Here, we used mouse models of CMT to demonstrate genetic interactions that result in a more severe neuropathy phenotype. The cell adhesion molecule Nrcam and the Na+ channel Scn8a (NaV1.6) are important components of nodes. Homozygous Nrcam and heterozygous Scn8a mutations synergized with both an Sh3tc2 mutation, modeling recessive demyelinating Charcot-Marie-Tooth type 4C, and mutations in Gars, modeling dominant axonal Charcot-Marie-Tooth type 2D. We conclude that genetic variants perturbing the structure and function of nodes interact with mutations affecting the cable properties of axons by thinning myelin or reducing axon diameter. Therefore, genes integral to peripheral nodes are candidate modifiers of peripheral neuropathy.

STING-Licensed Macrophages Prime Type I IFN Production by Plasmacytoid Dendritic Cells in the Bone Marrow during Severe Plasmodium yoelii Malaria.

Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14+ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the Plasmodium yoelii 17X YM surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of Plasmodium parasites. This robust type I IFN production required priming of pDCs by CD169+ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation in vivo and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes.

Paclitaxel-induced epithelial damage and ectopic MMP-13 expression promotes neurotoxicity in zebrafish.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxel-induced peripheral neuropathy remain elusive, and therapies that prevent or alleviate this condition are not available. We established a zebrafish in vivo model to study the underlying mechanisms and to identify pharmacological agents that may be developed into therapeutics. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin before induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and up-regulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Thus, our studies provide evidence that the epidermis plays a critical role in this condition, and we provide a previously unidentified candidate for therapeutic interventions.

Synaptic Deficits at Neuromuscular Junctions in Two Mouse Models of Charcot-Marie-Tooth Type 2d.

Patients with Charcot-Marie-Tooth Type 2D (CMT2D), caused by dominant mutations in Glycl tRNA synthetase (GARS), present with progressive weakness, consistently in the hands, but often in the feet also. Electromyography shows denervation, and patients often report that early symptoms include cramps brought on by cold or exertion. Based on reported clinical observations, and studies of mouse models of CMT2D, we sought to determine whether weakened synaptic transmission at the neuromuscular junction (NMJ) is an aspect of CMT2D. Quantal analysis of NMJs in two different mouse models of CMT2D (Gars(P278KY), Gars(C201R)), found synaptic deficits that correlated with disease severity and progressed with age. Results of voltage-clamp studies revealed presynaptic defects characterized by: (1) decreased frequency of spontaneous release without any change in quantal amplitude (miniature endplate current), (2) reduced amplitude of evoked release (endplate current) and quantal content, (3) age-dependent changes in the extent of depression in response to repetitive stimulation, and (4) release failures at some NMJs with high-frequency, long-duration stimulation. Drugs that modify synaptic efficacy were tested to see whether neuromuscular performance improved. The presynaptic action of 3,4 diaminopyridine was not beneficial, whereas postsynaptic-acting physostigmine did improve performance. Smaller mutant NMJs with correspondingly fewer vesicles and partial denervation that eliminates some release sites also contribute to the reduction of release at a proportion of mutant NMJs. Together, these voltage-clamp data suggest that a number of release processes, while essentially intact, likely operate suboptimally at most NMJs of CMT2D mice. SIGNIFICANCE STATEMENT: We have uncovered a previously unrecognized aspect of axonal Charcot-Marie-Tooth disease in mouse models of CMT2D. Synaptic dysfunction contributes to impaired neuromuscular performance and disease progression. This suggests that drugs which improve synaptic efficacy at the NMJ could be considered in treating the pathophysiology of CMT2D patients.

The T-Cell Inhibitory Molecule Butyrophilin-Like 2 Is Up-regulated in Mild Plasmodium falciparum Infection and Is Protective During Experimental Cerebral Malaria.

Plasmodium falciparum infection can result in severe disease that is associated with elevated inflammation and vital organ dysfunction; however, malaria-endemic residents gain protection from lethal outcomes and manifest only mild symptoms during infection. To characterize host responses associated with this more effective antimalarial response, we characterized whole-blood transcriptional profiles in Rwandan adults during a mild malaria episode and compared them with findings from a convalescence sample. We observed transcriptional up-regulation in many pathways, including type I interferon, interferon γ, complement activation, and nitric oxide during malaria infection, which provide benchmarks of mild disease physiology. Transcripts encoding negative regulators of T-cell activation, such as programmed death ligand 1 (PD-L1), programmed death 1 ligand 2 (PD-L2), and the butyrophilin family member butyrophilin-like 2 (BTNL2) were also increased. To support an important functional role for BTNL2 during malaria infection, we studied chimeric mice reconstituted with BTNL2(-/-) or wild-type hematopoietic cells that were inoculated with Plasmodium berghei ANKA, a murine model of cerebral malaria. We found that BTNL2(-/-) chimeric mice had a significant decrease in survival compared with wild-type counterparts. Collectively these data characterize the immune responses associated with mild malaria and uncover a novel role for BTNL2 in the host response to malaria.

Inflammatory monocyte effector mechanisms.

Monocytes are blood-derived mononuclear phagocytic cells that traffic throughout the body and can provide rapid innate immune effector responses in response to microbial pathogen infections. Among blood monocytes, the most abundant subset in mice is represented by inflammatory Ly6C(+) CCR2(+) monocytes and is the functional equivalent of the CD14(+) monocytes in humans. Herein we focus on published evidence describing the exquisite functional plasticity of these cells, and we extend this overview to their multiples roles in vivo during host immune defenses against microbial pathogen infections, as antigen-presenting cells, inflammatory cells or Trojan horse cells.

Iron overload, hematopoietic cell transplantation, and graft-versus-host disease.

Many patients who undergo hematopoietic cell transplantation (HCT) present with anemia and have received red blood cell transfusions before HCT. As a result, iron overload is frequent and appears to be particularly prominent in patients with myelodysplastic syndromes. There is evidence that peritransplant events contribute to further iron accumulation, although the mechanism that disrupts normal iron homeostasis remains to be determined. Recent studies suggest that iron overload, as determined by ferritin levels, a surrogate marker for iron, is a risk factor for increased non-relapse mortality after HCT. Iron overload is associated with an increased rate of infections, in particular with fungal organisms. Furthermore anecdotal data suggest that increased hepatic iron may mimic the clinical picture of (chronic) graft-versus-host-disease (GVHD). Whether excess iron contributes to GVHD and whether iron depletion, be it by phlebotomy or chelation, reduces the post-transplantation complication rate and improves transplant outcome is yet to be determined.

Stroma-dependent apoptosis in clonal hematopoietic precursors correlates with expression of PYCARD.

The role of the marrow microenvironment in the pathophysiology of myelodysplastic syndromes (MDSs) remains controversial. Using stromal/hematopoietic cell cocultures, we investigated the effects of stroma-derived signals on apoptosis sensitivity in hematopoietic precursors. The leukemia-derived cell line KG1a is resistant to proapoptotic ligands. However, when cocultured with the human stromal cell line HS5 (derived from normal marrow) and exposed to tumor necrosis factor-alpha (TNF-alpha), KG1a cells showed caspase-3 activation and induction of apoptosis. Apoptosis was contact dependent. Identical results were obtained in coculture with primary stroma. Gene-expression profiling of KG1a cells identified coculture-induced up-regulation of various genes involved in apoptosis, including PYCARD. Suppression of PYCARD expression in KG1a by miRNA interfered with apoptosis. Knockdown of the TNF receptor 1 (TNFR1) or TNFR2 in HS5 cells had no effect. However, knockdown of R1 in KG1a cells prevented TNF-alpha-induced apoptosis, while apoptosis was still induced by TNF-alpha-related apoptosis-inducing ligand. Primary CD34(+) cells from MDS marrow, when cocultured with HS5 and TNF-alpha, also underwent apoptosis. In contrast, no apoptosis was observed in CD34(+) cells from the marrow of healthy donors. These data indicate that stroma may convey not only protective effects on hematopoietic cells, but, dependent upon the milieu, may also facilitate apoptosis.

Transplantation of allogeneic T cells alters iron homeostasis in NOD/SCID mice.

Iron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT), but the mechanisms leading to overload are unknown. Here, we determined iron levels and the expression of iron regulatory proteins in the liver and gut of nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice that underwent transplantation with syngeneic (histocompatible) or allogeneic (histoincompatible) T lymphocytes. Infusion of histoincompatible T cells resulted in a significant rise in serum iron levels and liver iron content. Iron deposition was accompanied by hepatocyte injury and intestinal villous damage. Feeding of low- or high-iron diet was associated with appropriate ferroportin 1 and hepcidin responses in mice given histocompatible T cells, whereas mice given histoincompatible T cells showed inappropriate up-regulation of duodenal ferroportin 1 and a loss of expression of hepatic hepcidin. These findings suggest that alloreactive T cell-dependent signals induced dysregulation of intestinal iron absorption, which contributed to liver iron overload after HCT.