Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.
Nirupa D. Jayaraj, Bula J. Bhattacharyya, Abdelhak A. Belmadani, Dongjun Ren, Craig A. Rathwell, Sandra Hackelberg, Brittany E. Hopkins, Herschel R. Gupta, Richard J. Miller, Daniela M. Menichella
Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of a particular aldolase, aldolase B, results in the accumulation of intracellular phosphorylated fructose thus leading to phosphate sequestration and depletion, increased ATP turnover and a plethora of conditions leading to clinical manifestations including fatty liver, hyperuricemia, Fanconi syndrome and severe hypoglycemia. Unfortunately, to date, there is no treatment for HFI and avoiding sugar and fructose in our society has become quite challenging. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI using aldolase B knockout mice. We thus provide evidence for the first time of a potential therapeutic approach for this condition. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.
Miguel A. Lanaspa, Ana Andres-Hernando, David J. Orlicky, Christina Cicerchi, Cholsoon Jang, Nanxing Li, Tamara Milagres, Masanari Kuwabara, Michael F. Wempe, Joshua D. Rabinowitz, Richard J. Johnson, Dean R. Tolan
Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatments of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We utilize the Fah–/– mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We employ translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncover novel upstream regulators and important signaling pathways to be highly enriched in genes changed in regenerating hepatocytes. Specifically, we identify glutathione metabolism — particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT) — to be massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type-specific expression profiling in repopulating hepatocytes and suggests xCT as a potential therapeutic target for supporting antioxidant responses during liver regeneration.
Amber W. Wang, Kirk J. Wangensteen, Yue J. Wang, Adam M. Zahm, Nicholas G. Moss, Noam Erez, Klaus H. Kaestner
BACKGROUND. Poly(ADP-ribose) polymerase (PARP) inhibitors are effective in a broad population of ovarian cancer patients, however resistance caused by low enzyme expression of the drug target, poly(ADP-ribose) polymerase 1 (PARP-1), remains to be clinically evaluated in this context. We hypothesize that PARP-1 expression is variable in ovarian cancer and can be quantified in primary and metastatic disease using a novel positron emitting tomography (PET) imaging agent. METHODS. We used a translational approach to describe the significance of PET imaging of PARP-1 in ovarian cancer. First, we produced PARP1 KO ovarian cancer cell lines using CRISPR/Cas9 gene editing to test loss of PARP-1 as a resistance mechanism to all clinically used PARP inhibitors. Next, we performed pre-clinical microPET imaging studies using ovarian cancer patient derived xenografts in mouse models. Finally, in a phase 1 PET imaging clinical trial we explored PET imaging as a regional marker of PARP-1 expression in primary and metastatic disease through correlative tissue histology. RESULTS. We found deletion of PARP1 causes resistance to all PARP inhibitors in vitro and microPET imaging provides proof of concept as an approach to quantify PARP-1 in vivo. Clinically, we observed a spectrum of standard uptake values (SUVs) for PARP-1 in tumors ranging from 2-12. In addition, we found a positive correlation between PET SUVs and fluorescent immunohistochemistry for PARP-1 (r2: 0.60). CONCLUSIONS. This work confirms the translational potential of a PARP-1 PET imaging agent and supports future clinical trials to test PARP-1 expression as a method to stratify patients for PARP inhibitor therapy. Clinicaltrials.gov: NCT02637934.
Mehran Makvandi, Austin Pantel, Lauren Schwartz, Erin Schubert, Kuiying Xu, Chia-Ju Hsieh, Catherine Hou, Hyoung Kim, Chi-Chang Weng, Harrison Winters, Robert Doot, Michael D. Farwell, Daniel A. Pryma, Roger A. Greenberg, David A. Mankoff, Fiona Simpkins, Robert H. Mach, Lilie L. Lin
Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber (PF)-forming cerebellar granule cells (Cgc). Postnatal Pkn1–/– animals showed a defective PF-Purkinje cell (PC) synapse formation. In vitro, Pkn1–/– Cgc exhibited deregulated axonal outgrowth, elevated AKT phosphorylation and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly Pkn1–/– Cgc had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels and the reduced density of presynaptic specifications in Pkn1–/– Cgc. In line with our in vitro data, Pkn1–/– mice showed AKT hyperactivation, elevated NeuroD2 levels and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgc and subsequently the correct PF-PC synapse formation.
Stephanie zur Nedden, Rafaela Eith, Christoph Schwarzer, Lucia Zanetti, Hartwig Seitter, Friedrich Fresser, Alexandra Koschak, Angus J.M. Cameron, Peter J. Parker, Gottfried Baier, Gabriele Baier-Bitterlich
Non-antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalised neo-epitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen-anti-CLEC-9A mAb conjugates target cross-presenting DCs, adjuvant must be co-delivered for cytotoxic T-cell (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating ovalbumin (OVA) antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation, CTL and antibody responses. OVA-Clec9A-TNE-induced DC activation required CD4 and CD8 epitopes, CD40 and IFN-α. Clec9A-TNE encapsulating human papillomavirus (HPV) E6-E7 significantly suppressed HPV-associated tumor growth while E6-E7-CpG did not. Clec9A-TNE loaded with pooled B16F10 melanoma neo-epitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neo-epitopes. Clec9A-TNE encapsulating six neo-epitopes significantly suppressed B16-F10 melanoma growth in a CD4 T cell-dependent manner. Thus, cross-presenting DCs targeted with antigen-Clec9A-TNE stimulate therapeutically-effective tumor-specific immunity, dependent on T cell help.
Bijun Zeng, Anton P.J. Middelberg, Adrian Gemiarto, Kelli MacDonald, Alan G. Baxter, Meghna Talekar, Davide Moi, Kirsteen M. Tullett, Irina Caminschi, Mireille H. Lahoud, Roberta Mazzieri, Riccardo Dolcetti, Ranjeny Thomas
A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that re-directing T cells to specialized niches in the bone marrow (BM) that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration towards vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis and a heightened capacity to generate poly-functional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, re-directed homing of T cells to the BM confers increased memory differentiation and anti-tumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.
Anjum B. Khan, Ben Carpenter, Pedro Santos e Sousa, Constandina Pospori, Reema Khorshed, James Griffin, Pedro Veliça, Mathias Zech, Sara Ghorashian, Calum Forrest, Sharyn Thomas, Sara Gonzalez Anton, Maryam Ahmadi, Angelika Holler, Barry Flutter, Zaida Ramirez-Ortiz, Terry K. Means, Clare L. Bennett, Hans Stauss, Emma Morris, Cristina Lo Celso, Ronjon Chakraverty
The lack of defined correlates of protection hampers development of vaccines against tuberculosis (TB). In vitro mycobacterial outgrowth assays are thought to better capture the complexity of the human host/Mycobacterium tuberculosis (Mtb) interaction. We used a PBMC-based “mycobacterial-growth-inhibition-assay” (MGIA) to investigate the capacity to control outgrowth of Bacille Calmette-Guérin (BCG). Interestingly, strong control of BCG outgrowth was observed almost exclusively in individuals with recent exposure to Mtb, but not in (long-term) latent TB infection, and only modestly in BCG vaccinees. Mechanistically, control of mycobacterial outgrowth strongly correlated with the presence of a CD14dim monocyte population, but also required the presence of T cells. The nonclassical monocytes produced CXCL10, and CXCR3-receptor blockade inhibited the capacity to control BCG outgrowth. Expression of CXCR3 splice variants was altered in recently Mtb exposed individuals. Cytokines previously associated with trained immunity were detected in MGIA supernatants, and CXCL9, CXCL10, and CXCL11 represent new markers of trained immunity. These data indicate that CXCR3-ligands are associated with trained immunity and critical factors in controlling mycobacterial outgrowth.In conclusion, control of mycobacterial outgrowth early after exposure to Mtb is the result of trained immunity mediated by a CXCL10-producing non-classical CD14dim monocyte subset.
Simone A. Joosten, Krista E. van Meijgaarden, Sandra M. Arend, Corine Prins, Fredrik Oftung, Gro Ellen Korsvold, Sandra V. Kik, Rob J.W. Arts, Reinout van Crevel, Mihai G. Netea, Tom H.M. Ottenhoff
BACKGROUND. Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited and multi-disciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS. To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimised analysis for detection of low mutant allele frequency. RESULTS. We discovered multiple mosaic activating variants in four genes of the RAS-MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germ-line RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterisation and two transgenic zebrafish AVM models which recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inihibitor, Vemurafinib, restored blood flow in AVM. CONCLUSIONS. Our findings uncover a major cause of sporadic vascular malformations of different clinical types, and thereby offer the potential of personalised medical treatment by repurposing existing licensed cancer therapies. FUNDING. This work was funded or supported by grants from AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, L’Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research (US).
Lara Al-Olabi, Satyamaanasa Polubothu, Katherine Dowsett, Katrina A. Andrews, Paulina Stadnik, Agnel P. Joseph, Rachel Knox, Alan Pittman, Graeme Clark, William Baird, Neil Bulstrode, Mary Glover, Kristiana Gordon, Darren Hargrave, Susan M. Huson, Thomas S. Jacques, Gregory James, Hannah Kondolf, Loshan Kangesu, Kim M. Keppler-Noreuil, Amjad Khan, Marjorie J. Lindhurst, Mark Lipson, Sahar Mansour, Justine O'Hara, Caroline Mahon, Anda Mosica, Celia Moss, Aditi Murthy, Juling Ong, Victoria E. Parker, Jean-Baptiste Rivière, Julie C. Sapp, Neil J. Sebire, Rahul Shah, Branavan Sivakumar, Anna Thomas, Alex Virasami, Regula Waelchli, Zhiqiang Zeng, Leslie G. Biesecker, Alex Barnacle, Maya Topf, Robert K. Semple, E. Elizabeth Patton, Veronica A. Kinsler
The discovery of an HIV-1 cure remains a medical challenge because the virus rebounds quickly after the cessation of combination antiretroviral drug therapy (cART). Here, we investigate the potential of an engineered tandem bi-specific broadly neutralizing antibody (bs-bnAb) as an innovative product for HIV-1 prophylactic and therapeutic interventions. We discovered that by preserving two scFv binding domains of each parental bnAb, a single-gene-encoded tandem bs-bnAb, namely BiIA-SG, displayed significantly improved breadth and potency. BiIA-SG neutralized all 124 HIV-1 pseudotyped viruses tested, including global subtypes/recombinant forms, transmitted/founder viruses, and variants less or not susceptible to parental and many bnAbs, with an average IC50 value of 0.073 µ/ml (range < 0.001 to 1.03 µg/ml). In humanized mice, an injection of BiIA-SG conferred sterile protection when administered prior to challenges with diverse live HIV-1 stains. Moreover, while BiIA-SG delayed viral rebound in a short-term therapeutic setting when combined with cART, a single injection of AAV-transferred BiIA-SG gene resulted dose-dependently in prolonged in vivo expression of BiIA-SG, which was associated with complete viremia control and subsequent elimination of infected cells in humanized mice. These results warrant the clinical development of BiIA-SG as a promising bs-bnAb-based biomedical intervention for prevention and treatment of HIV-1 infections.
Xilin Wu, Jia Guo, Mengyue Niu, Minghui An, Li Liu, Hui Wang, Xia Jin, Qi Zhang, Ka Shing Lam, Tongjin Wu, Hua Wang, Qian Wang, Yanhua Du, Jingjing Li, Lin Cheng, Hang Ying Tang, Hong Shang, Linqi Zhang, Paul Zhou, Zhiwei Chen
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