Concise Communication

Abstract

Cantu Syndrome (CS) is a complex disorder caused by gain-of-function (GoF) mutations in ABCC9 and KCNJ8, which encode the SUR2 and Kir6.1 subunits, respectively, of vascular smooth muscle (VSM) KATP channels. CS includes dilated vasculature, marked cardiac hypertrophy, and other cardiovascular abnormalities. There is currently no targeted therapy, and it is unknown whether cardiovascular features can be reversed once manifest. Using combined transgenic and pharmacological approaches in a knock-in mouse model of CS, we have shown that reversal of vascular and cardiac phenotypes can be achieved (1) by genetic downregulation of KATP channel activity specifically in VSM, and (2) by chronic administration of the clinically-used KATP channel inhibitor, glibenclamide. These findings demonstrate (i) that VSM KATP channel GoF underlies CS cardiac enlargement, (ii) reversibility of CS-associated abnormalities and (iii) evidence of in vivo efficacy of glibenclamide as a therapeutic agent in CS.

Authors

Conor McClenaghan, Yan Huang, Zihan Yan, Theresa Harter, Carmen M. Halabi, Rod Chalk, Attila Kovacs, Gijs van Haaften, Maria S. Remedi, Colin G. Nichols

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Abstract

Cancer cachexia is a major cause of patient morbidity and mortality, with no efficacious treatment or management strategy. Despite sharing pathophysiological features with a number of neuromuscular wasting conditions, including age-related sarcopenia, the mechanisms underlying cachexia remain poorly understood. Studies of related conditions suggest that pathological targeting of the neuromuscular junction (NMJ) may play a key role in cachexia, but this has yet to be investigated in human patients. Here, high-resolution morphological analyses were undertaken on NMJs of rectus abdominis obtained from patients undergoing upper GI cancer surgery compared with controls (N=30; n=1,165 NMJs). Cancer patients included those with cachexia and weight-stable disease. Despite the low skeletal muscle index and significant muscle fibre atrophy in patients with cachexia, NMJ morphology was fully conserved. No significant differences were observed in any of the pre- and post-synaptic variables measured. We conclude that NMJs remain structurally intact in rectus abdominis in both cancer and cachexia, suggesting that denervation of skeletal muscle is not a major driver of pathogenesis. The absence of NMJ pathology is in stark contrast to related conditions, such as age-related sarcopenia, and supports the hypothesis that intrinsic changes within skeletal muscle, independent of any changes in motor neurons, represent the primary locus of neuromuscular pathology in cancer cachexia.

Authors

Ines Boehm, Janice Miller, Thomas M. Wishart, Stephen J. Wigmore, Richard J.E. Skipworth, Ross A. Jones, Thomas H. Gillingwater

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Abstract

Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from Non-Ketotic Hyperglycinemia (NKH), a neurometabolic disorder characterised by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the sub-commissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived one-carbon units to the folate cycle. We showed that inadequate one-carbon supply, as opposed to excess glycine is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at pre-natal stages, and post-natal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of one-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to pre-natal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a one-carbon donor.

Authors

Chloe Santos, Yun Jin Pai, M. Raasib Mahmood, Kit-Yi Leung, Dawn Savery, Simon N. Waddington, Andrew J. Copp, Nicholas D.E. Greene

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Abstract

Omalizumab is an anti-IgE monoclonal antibody (mAb) approved for the treatment of severe asthma and chronic spontaneous urticaria. Use of Omalizumab is associated with reported side effects, ranging from local skin inflammation at the injection site to systemic anaphylaxis. To date, the mechanisms through which Omalizumab induces adverse reactions are still unknown. Here, we demonstrated that immune complexes formed between Omalizumab and IgE can induce both skin inflammation and anaphylaxis through engagement of IgG receptors (FcγRs) in FcγR-humanized mice. We further developed an Fc-engineered mutant version of Omalizumab, and demonstrated that this mAb is equally potent as Omalizumab at blocking IgE-mediated allergic reactions, but does not induce FcγR-dependent adverse reactions. Overall, our data indicate that Omalizumab can induce skin inflammation and anaphylaxis by engaging FcγRs, and demonstrate that Fc-engineered versions of the mAb could be used to reduce such adverse reactions.

Authors

Bianca Balbino, Pauline Herviou, Ophélie Godon, Julien Stackowicz, Odile Richard-Le Goff, Bruno Iannascoli, Delphine Sterlin, Sébastien Brûlé, Gael A. Millot, Faith M. Harris, Vera A. Voronina, Kari C. Nadeau, Lynn E. Macdonald, Andrew J. Murphy, Pierre Bruhns, Laurent L. Reber

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Abstract

Cancer immune evasion is achieved through multiple layers of immune tolerance mechanisms including immune editing, recruitment of tolerogenic immune cells, and secretion of immune suppressive cytokines. Recent success with immune checkpoint inhibitors in cancer immunotherapy suggests a dysfunctional immune synapse as a pivotal tolerogenic mechanism. Tumor cells express immune synapse proteins to suppress the immune system, which is often modulated by epigenetic mechanisms. When the methylation status of key immune synapse genes was interrogated, we observed disproportionately hyper-methylated co-stimulatory genes and hypo-methylation of immune checkpoint genes, which were negatively associated with functional T-cell recruitment to the tumor microenvironment. Therefore, the methylation status of immune synapse genes reflects tumor immunogenicity and correlates with survival.

Authors

Anders Berglund, Matthew Mills, Ryan M. Putney, Imène Hamaidi, James Mulé, Sungjune Kim

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Abstract

In patients with acute myeloid leukemia (AML), 10% to 30% with the normal karyotype express mutations in regulators of DNA methylation, such as TET2 or DNMT3A, in conjunction with activating mutation in the receptor tyrosine kinase FLT3. These patients have a poor prognosis because they do not respond well to established therapies. Here, utilizing mouse models of AML that recapitulate cardinal features of the human disease and bear a combination of loss-of-function mutations in either Tet2 or Dnmt3a along with expression of Flt3ITD, we show that inhibition of the protein tyrosine phosphatase SHP2, which is essential for cytokine receptor signaling (including FLT3), by the small molecule allosteric inhibitor SHP099 impairs growth and induces differentiation of leukemic cells without impacting normal hematopoietic cells. We also show that SHP099 normalizes the gene expression program associated with increased cell proliferation and self-renewal in leukemic cells by downregulating the Myc signature. Our results provide a new and more effective target for treating a subset of patients with AML who bear a combination of genetic and epigenetic mutations.

Authors

Ruchi Pandey, Baskar Ramdas, Changlin Wan, George Sandusky, Morvarid Mohseni, Chi Zhang, Reuben Kapur

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Abstract

Tuberculosis (TB) remains a major infectious disease worldwide. TB treatment displays a bi-phasic bacterial clearance, in which the majority of bacteria clear within the first month of treatment, but residual bacteria remains non-responsive to treatment and eventually may become resistant. Here, we have shown that Mycobacterium tuberculosis (M.tb) is taken up by mesenchymal stem cells (MSCs), where it established dormancy and became highly non-responsive to isoniazid, a major constituent of Directly Observed Treatment Short-course (DOTS). Dormant M.tb induced quiescence in MSCs and promoted their long-term survival. Unlike macrophages, where M.tb resides in early-phagosomal compartments, in MSCs the majority of bacilli were found in the cytosol, where they promoted rapid lipid-synthesis, hiding within lipid-droplets. Inhibition of lipid-synthesis prevented dormancy and sensitized the organisms to isoniazid. Thus, we have established that M.tb gains dormancy in MSCs, which thus serve as a long-term natural-reservoir of dormant M.tb. Interestingly, in the murine-model of TB, induction of autophagy eliminated M.tb from MSCs and consequently, the addition of rapamycin to an isoniazid treatment regimen successfully attained sterile clearance and prevented disease reactivation.

Authors

Samreen Fatima, Shashank Shivaji Kamble, Ved Prakash Dwivedi, Debapriya Bhattacharya, Santosh Kumar, Anand Ranganathan, Luc Van Kaer, Sujata Mohanty, Gobardhan Das

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Abstract

Gout is caused by deposition of monosodium urate crystals in joints when plasma uric acid levels are chronically elevated beyond the saturation threshold, mostly due to renal underexcretion of uric acid. Although molecular pathways of this underexcretion have been elucidated, its etiology remains mostly unknown. We demonstrate that gout can be caused by a mutation in LDHD within the putative catalytic site of the encoded d-lactate dehydrogenase, resulting in augmented blood levels of d-lactate, a stereoisomer of l-lactate, which is normally present in human blood in miniscule amounts. Consequent excessive renal secretion of d-lactate in exchange for uric acid reabsorption culminated in hyperuricemia and gout. We showed that LDHD expression is enriched in tissues with a high metabolic rate and abundant mitochondria and that d-lactate dehydrogenase resides in the mitochondria of cells overexpressing the human LDHD gene. Notably, the p.R370W mutation had no effect on protein localization. In line with the human phenotype, injection of d-lactate into naive mice resulted in hyperuricemia. Thus, hyperuricemia and gout can result from the accumulation of metabolites whose renal excretion is coupled to uric acid reabsorption.

Authors

Max Drabkin, Yuval Yogev, Lior Zeller, Raz Zarivach, Ran Zalk, Daniel Halperin, Ohad Wormser, Evgenia Gurevich, Daniel Landau, Rotem Kadir, Yonatan Perez, Ohad S. Birk

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Abstract

Novel approaches for adjunctive therapy are urgently needed for infections complicated by antibiotic-resistant pathogens and for patients with compromised immunity. Necrotizing fasciitis (NF) is a destructive skin and soft tissue infection. Despite treatment with systemic antibiotics and radical debridement of necrotic tissue, lethality remains high. The key iron regulatory hormone hepcidin was originally identified as a cationic antimicrobial peptide (AMP), but its putative expression and role in the skin, a major site of AMP production, has never been investigated. We report here that hepcidin production is induced in the skin of patients with Group A Streptococcal (GAS) NF. In a GAS-induced NF model, mice lacking hepcidin in keratinocytes failed to restrict systemic spread of infection from an initial tissue focus. Unexpectedly, this effect was due its ability to promote production of the CXCL1 chemokine by keratinocytes resulting in neutrophil recruitment. Unlike CXCL1, hepcidin is resistant to degradation by major GAS proteases and could therefore serve as a reservoir to maintain steady state levels of CXCL1 in infected tissue. Finally, injection of synthetic hepcidin at the site of infection can limit or completely prevent systemic spread of GAS infection suggesting that hepcidin agonists could have a therapeutic role in NF.

Authors

Mariangela Malerba, Sabine Louis, Sylvain Cuvellier, Srikanth Mairpady Shambat, Camille Hua, Camille Gomart, Agnès Fouet, Nicolas Ortonne, Jean-Winoc Decousser, Annelies S. Zinkernagel, Jacques R.R. Mathieu, Carole Peyssonnaux

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Abstract

A number of highly potent and broadly neutralizing antibodies (bNAbs) against the human immunodeficiency virus (HIV) have recently been shown to prevent transmission of the virus, suppress viral replication, and delay plasma viral rebound following discontinuation of antiretroviral therapy in animal models and infected humans. However, the degree and extent to which such bNAbs interact with primary lymphocytes have not been fully delineated. Here, we show that certain glycan-dependent bNAbs, such as PGT121 and PGT151, bind to B, activated T, and natural killer (NK) cells of HIV-infected and -uninfected individuals. Binding of these bNAbs, particularly PGT121 and PGT151, to activated CD4+ and CD8+ T cells was mediated by complex-type glycans and was abrogated by enzymatic inhibition of N-linked glycosylation. In addition, a short-term incubation of PGT151 and primary NK cells led to degranulation and cellular death. Our data suggest that the propensity of certain bNAbs to bind uninfected/bystander cells has the potential for unexpected outcomes in passive-transfer studies and underscore the importance of antibody screening against primary lymphocytes.

Authors

Jana Blazkova, Eric W. Refsland, Katherine E. Clarridge, Victoria Shi, J. Shawn Justement, Erin D. Huiting, Kathleen R. Gittens, Xuejun Chen, Stephen D. Schmidt, Cuiping Liu, Nicole Doria-Rose, John R. Mascola, Alonso Heredia, Susan Moir, Tae-Wook Chun

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