Background: Proglucagon can be processed to Glucagon-Like Peptide-1 (GLP-1) within the islet but its contribution to islet function in humans remains unknown. We sought to understand whether ‘pancreatic’ GLP-1 alters islet function in humans and whether this is affected by type 2 diabetes.Methods: We therefore studied individuals with and without type 2 diabetes on 2 occasions in random order. On one occasion exendin 9-39, a competitive antagonist of the GLP-1 Receptor (GLP1R), was infused, while on the other saline was infused. The tracer dilution technique ([3-3H] glucose) was used to measure glucose turnover during fasting and during a hyperglycemic clamp.Results: Exendin 9-39 increased fasting glucose concentrations; fasting islet hormone concentrations were unchanged, but inappropriate for the higher fasting glucose observed. In people with type 2 diabetes fasting glucagon concentrations were markedly elevated and persisted despite hyperglycemia. This impaired suppression of endogenous glucose production by hyperglycemia. These data show that GLP1R blockade impairs islet function, implying that intra-islet GLP1R activation alters islet responses to glucose and does so to a greater degree in people with type 2 diabetes.
Andrew A. Welch, Rahele A. Farahani, Aoife M. Egan, Marcello C. Laurenti, Maya Zeini, Max Vella, Kent R. Bailey, Claudio Cobelli, Chiara Dalla Dalla Man, Aleksey Matveyenko, Adrian Vella
BACKGROUND Severe, early-onset fetal growth restriction (FGR) causes significant fetal and neonatal mortality and morbidity. Predicting the outcome of affected pregnancies at the time of diagnosis is difficult, thus preventing accurate patient counseling. We investigated the use of maternal serum protein and ultrasound measurements at diagnosis to predict fetal or neonatal death and 3 secondary outcomes: fetal death or delivery at or before 28+0 weeks, development of abnormal umbilical artery (UmA) Doppler velocimetry, and slow fetal growth.METHODS Women with singleton pregnancies (n = 142, estimated fetal weights [EFWs] below the third centile, less than 600 g, 20+0 to 26+6 weeks of gestation, no known chromosomal, genetic, or major structural abnormalities) were recruited from 4 European centers. Maternal serum from the discovery set (n = 63) was analyzed for 7 proteins linked to angiogenesis, 90 additional proteins associated with cardiovascular disease, and 5 proteins identified through pooled liquid chromatography and tandem mass spectrometry. Patient and clinician stakeholder priorities were used to select models tested in the validation set (n = 60), with final models calculated from combined data.RESULTS The most discriminative model for fetal or neonatal death included the EFW z score (Hadlock 3 formula/Marsal chart), gestational age, and UmA Doppler category (AUC, 0.91; 95% CI, 0.86–0.97) but was less well calibrated than the model containing only the EFW z score (Hadlock 3/Marsal). The most discriminative model for fetal death or delivery at or before 28+0 weeks included maternal serum placental growth factor (PlGF) concentration and UmA Doppler category (AUC, 0.89; 95% CI, 0.83–0.94).CONCLUSION Ultrasound measurements and maternal serum PlGF concentration at diagnosis of severe, early-onset FGR predicted pregnancy outcomes of importance to patients and clinicians.TRIAL REGISTRATION ClinicalTrials.gov NCT02097667.FUNDING The European Union, Rosetrees Trust, Mitchell Charitable Trust.
Rebecca Spencer, Kasia Maksym, Kurt Hecher, Karel Maršál, Francesc Figueras, Gareth Ambler, Harry Whitwell, Nuno Rocha Nené, Neil J. Sebire, Stefan R. Hansson, Anke Diemert, Jana Brodszki, Eduard Gratacós, Yuval Ginsberg, Tal Weissbach, Donald M. Peebles, Ian Zachary, Neil Marlow, Angela Huertas-Ceballos, Anna L. David
BACKGROUND. HIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (EC) and people on long-term therapy suggests that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing. METHODS. In this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells. RESULTS. The proviral landscape was dominated by two large clones with replication-competent proviruses integrated into Zinc Finger genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-folds less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene. CONCLUSION. We provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.
Filippo Dragoni, Abena Kwaa, Caroline C.G. Traut, Rebecca T. Veenhuis, Bezawit A. Woldemeskel, Angelica Camilo-Contreras, Hayley E. Raymond, Arbor G. Dykema, Eileen P. Scully, Amanda M. Rosecrans, Kellie N. Smith, Frederic D. Bushman, Francesco R. Simonetti, Joel N. Blankson
BACKGROUND Generally, clinical assessment of gonadal testosterone (T) in human physiology is determined using concentrations measured in peripheral blood. Prostatic T exposure is similarly thought to be determined from peripheral T exposure. Despite the fact that androgens drive prostate cancer, peripheral T has had no role in the clinical evaluation or treatment of men with localized prostate cancer.METHODS To assess the role of local androgen delivery in prostate cancer, we obtained blood from the (periprostatic) prostatic dorsal venous complex in 266 men undergoing radical prostatectomy from July 2014 to August 2021 and compared dorsal T (DT) levels with those in circulating peripheral blood (PT) and prostatic tissue. Comprehensive targeted steroid analysis and unbiased metabolomics analyses were performed. The association between the DT/PT ratio and progression-free survival after prostatectomy was assessed.RESULTS Surprisingly, in some men, DT levels were enriched several-fold compared with PT levels. For example, 20% of men had local T concentrations that were at least 2-fold higher than peripheral T concentrations. Isocaproic acid, a byproduct of androgen biosynthesis, and 17-OH-progesterone, a marker of intratesticular T, were also enriched in the dorsal vein of these men, consistent with testicular shunting. Men with enriched DT had higher rates of prostate cancer recurrence. DT/PT concentration ratios predicted worse outcomes even when accounting for known clinical predictors.CONCLUSIONS These data suggest that a large proportion of men have a previously unappreciated exposure to an undiluted and highly concentrated T supply. Elevated periprostatic T exposure was associated with worse clinical outcomes after radical prostatectomy.FUNDING National Cancer Institute (NCI), NIH grants R01CA172382, R01CA236780, R01CA261995, R01CA249279, and R50CA251961; US Army Medical Research and Development Command grants W81XWH2010137 and W81XWH-22-1-0082.
Mohammad Alyamani, Patrick Michael, Daniel Hettel, Lewis Thomas, Scott D. Lundy, Mike Berk, Mona Patel, Jianbo Li, Hooman Rashidi, Jesse K. McKenney, Eric A. Klein, Nima Sharifi
BACKGROUND. FXLEARN, the first-ever large multi-site trial of effects of disease-targeted pharmacotherapy on learning, was designed to explore a new paradigm for measuring effects of mechanism-targeted treatment in fragile X syndrome (FXS). In FXLEARN, the effects of mGluR5 negative allosteric modulator (NAM) AFQ056 on language learning were evaluated in 3-6 year-old children with FXS, expected to have more learning plasticity than adults, where prior trials of mGluR5 NAMs have failed. METHODS. After a 4-month single-blind placebo lead-in, participants were randomized 1:1 to AFQ056 or placebo, with 2 months of dose optimization to the maximum tolerated dose, then 6 months of treatment during which a language learning intervention was implemented for both groups. The primary outcome was a centrally scored videotaped communication measure, the Weighted Communication Scale (WCS). Secondary outcomes were objective performance-based and parent-report cognitive and language measures. RESULTS. FXLEARN enrolled 110 participants, randomized 99, and 91 completed the placebo-controlled period. Although both groups made language progress and there were no safety issues, the change in WCS score during the placebo-controlled period was not significantly different between the AFQ056 and placebo-treated groups, nor were there any significant between-group differences in change in any secondary measures. CONCLUSION. Despite the large body of evidence supporting use of mGluR5 NAMs in animal models of FXS, this study suggests that this mechanism of action does not translate into benefit for the human FXS population and that better strategies are needed to determine which mechanisms will translate from pre-clinical models to humans in genetic neurodevelopmental disorders. TRIAL REGISTRATION. ClincalTrials.gov NCT02920892 FUNDING. This study was supported by NeuroNEXT network NIH grants U01NS096767, U24NS107200, U24NS107209, U01NS077323, U24NS107183, U24NS107168, U24NS107128, U24NS107199, U24NS107198, U24NS107166, U10NS077368, U01NS077366, U24NS107205, U01NS077179, and U01NS077352, NIH grant P50HD103526 and Novartis IIT grant AFQ056X2201T for provision of AFQ056.
Elizabeth Berry-Kravis, Leonard Abbeduto, Randi Hagerman, Christopher S. Coffey, Merit Cudkowicz, Craig A. Erickson, Andrea McDuffie, David Hessl, Lauren E. Ethridge, Flora Tassone, Walter E. Kaufmann, Katherine Friedmann, Lauren Bullard, Anne Hoffmann, Jeremy Veenstra-VanderWeele, Kevin Staley, David Klements, Michael Moshinsky, Brittney Harkey, Jeffrey D. Long, Janel Fedler, Elizabeth Klingner, Dixie J. Ecklund, Michele Costigan, Trevis Huff, Brenda Pearson
BACKGROUND. The biology of Plasmodium vivax is markedly different to that of P. falciparum; how this shapes the immune response to infection remains unclear. To address this shortfall, we inoculated human volunteers with a clonal field isolate of P. vivax and tracked their response through infection and convalescence. METHODS. Participants were injected intravenously with blood-stage parasites and infection dynamics were tracked in real-time by quantitative PCR. Whole blood samples were used for high dimensional protein analysis, RNA-sequencing and Cytometry by Time Of Flight (CyTOF), and temporal changes in the host response to P. vivax were quantified by linear regression. Comparative analyses with P. falciparum were then undertaken using analogous datasets derived from prior controlled human malaria infection studies. RESULTS.P. vivax rapidly induced a type I inflammatory response that coincided with hallmark features of clinical malaria. This acute phase response shared remarkable overlap with that induced by P. falciparum but was significantly elevated (at RNA and protein level) leading to an increased incidence of pyrexia. In contrast, T cell activation and terminal differentiation was significantly increased in volunteers infected with P. falciparum. Heterogeneous CD4+ T cells were found to dominate this adaptive response and phenotypic analysis revealed unexpected features normally associated with cytotoxicity and autoinflammatory disease. CONCLUSION.P. vivax triggers increased systemic interferon signaling (cf P. falciparum), which likely explains its reduced pyrogenic threshold. In contrast, P. falciparum drives T cell activation far in excess of P. vivax, which may partially explain why falciparum malaria more frequently causes severe disease. TRIAL REGISTRATION. ClinicalTrials.gov NCT03797989 FUNDING. Supported by the European Union's Horizon 2020 Research and Innovation programme, the Wellcome Trust and the Royal Society.
Florian A. Bach, Diana Muñoz Sandoval, Michalina Mazurczyk, Yrene Themistocleous, Thomas A. Rawlinson, Adam C. Harding, Alison Kemp, Sarah E. Silk, Jordan R. Barrett, Nick J. Edwards, Alasdair C. Ivens, Julian C. Rayner, Angela M. Minassian, Giorgio Napolitani, Simon J. Draper, Philip J. Spence
BACKGROUND. Recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) is often an incurable disease, with patients experiencing median survival of under ten months and significant morbidity. While immune checkpoint blockade (ICB) drugs are effective in ~20% of patients, the remaining experience limited clinical benefit and are exposed to potential adverse effects and financial costs. Clinically approved biomarkers, such as tumor mutational burden (TMB), have modest predictive value in HNSCC. METHODS. We analyze clinical and genomic features, generated using whole-exome sequencing, in 133 ICB-treated R/M HNSCC patients, of whom 69 had virus-associated and 64 had non-virus-associated tumors. RESULTS. Hierarchical clustering of genomic data revealed six molecular subtypes characterized by a wide range of objective response rates and survival after ICB therapy. The prognostic importance of these 6 subtypes was validated in an external cohort. A random forest-based predictive model, using several clinical and genomic features, predicted progression-free survival (PFS), overall survival (OS), and response with greater accuracy than a model based on TMB alone. Recursive partitioning analysis identified three features (systemic inflammatory response index, TMB, and smoking signature) that classified patients into risk groups with accurate discrimination of PFS and OS. CONCLUSION. These findings shed light on the immunogenomic characteristics of HNSCC tumors that drive differential response to ICB and identify a clinical-genomic classifier that outperforms the current clinically approved biomarker of TMB. This validated predictive tool may help with clinical risk stratification in patients with R/M HNSCC for whom ICB is being considered.
Cristina Valero, Mahdi Golkaram, Joris L. Vos, Bin Xu, Conall Fitzgerald, Mark Lee, Shannon K. Kaplan, Catherine Y Han, Xin Pei, Reith Sarkar, Lillian A. Boe, Abhinav Pandey, Elizabeth S. Koh, Charlotte L. Zuur, David B. Solit, Traci Pawlowski, Li Liu, Alan L. Ho, Diego Chowell, Nadeem Riaz, Timothy A. Chan, Luc G.T. Morris
BACKGROUND. Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is neutropenia from neutrophil retention in bone marrow and is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, safety and efficacy in WHIM syndrome are undefined. METHODS. In this investigator-initiated, single-center, randomized, quadruple-masked phase 3 crossover trial, we compared the total infection severity score (TISS) as primary endpoint in an intent-to-treat manner in 19 WHIM patients for 12-months on plerixafor versus 12-months on G-CSF, the standard-of-care for severe congenital neutropenia. RESULTS. Plerixafor was non-superior to G-CSF for TISS (p=0.65). In exploratory endpoints, plerixafor was non-inferior to G-CSF for maintaining neutrophil counts >500 cells/microliter (p=0.023) and was superior to G-CSF for maintaining lymphocyte counts >1000 cells/microliter (p<0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life, or the incidence of drug failure or serious adverse events. CONCLUSIONS. Plerixafor was not superior in WHIM patients to G-CSF for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.(Funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; clinicaltrials.gov registration number, NCT02231879)
David H. McDermott, Daniel Velez, Elena Cho, Edward W. Cowen, John J. DiGiovanna, Diana V. Pastrana, Christopher B. Buck, Katherine R. Calvo, Pamela J. Gardner, Sergio D. Rosenzweig, Pamela Stratton, Melissa A. Merideth, H. Jeffrey Kim, Carmen Brewer, James D. Katz, Douglas B. Kuhns, Harry L. Malech, Dean Follmann, Michael P. Fay, Philip M. Murphy
BACKGROUND Chronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic hematopoietic cell transplantation (HCT). More accurate information regarding the risk of developing cGVHD is required. Bone marrow (BM) grafts contribute to lower cGVHD, which creates a dispute over whether risk biomarker scores should be used for peripheral blood (PB) and BM.METHODS Day 90 plasma proteomics from PB and BM recipients developing cGVHD revealed 5 risk markers that were added to 8 previous cGVHD markers to screen 982 HCT samples of 2 multicenter Blood and Marrow Transplant Clinical Trials Network (BMTCTN) cohorts. Each marker was tested for its association with cause-specific hazard ratios (HRs) of cGVHD using Cox-proportional-hazards models. We paired these clinical studies with biomarker measurements in a mouse model of cGVHD.RESULTS Spearman correlations between DKK3 and MMP3 were significant in both cohorts. In BMTCTN 0201 multivariate analyses, PB recipients with 1-log increase in CXCL9 and DKK3 were 1.3 times (95% CI: 1.1–1.4, P = 0.001) and 1.9 times (95%CI: 1.1–3.2, P = 0.019) and BM recipients with 1-log increase in CXCL10 and MMP3 were 1.3 times (95%CI: 1.0–1.6, P = 0.018 and P = 0.023) more likely to develop cGVHD. In BMTCTN 1202, PB patients with high CXCL9 and MMP3 were 1.1 times (95%CI: 1.0–1.2, P = 0.037) and 1.2 times (95%CI: 1.0–1.3, P = 0.009) more likely to develop cGVHD. PB patients with high biomarkers had increased likelihood to develop cGVHD in both cohorts (22%–32% versus 8%–12%, P = 0.002 and P < 0.001, respectively). Mice showed elevated circulating biomarkers before the signs of cGVHD.CONCLUSION Biomarker levels at 3 months after HCT identify patients at risk for cGVHD occurrence.FUNDING NIH grants R01CA168814, R21HL139934, P01CA158505, T32AI007313, and R01CA264921.
Brent R. Logan, Denggang Fu, Alan Howard, Mingwei Fei, Jianqun Kou, Morgan R. Little, Djamilatou Adom, Fathima A. Mohamed, Bruce R. Blazar, Philip R. Gafken, Sophie Paczesny
BACKGROUND. Autoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked up-regulation of HLA-DR molecules, including in post-infectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides and therefore, the reasons for these associations have frequently remained elusive. METHODS. Using immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with post-infectious LA, identified potential Borreliella burgdorferi (Bb)-mimic epitopes, and characterized T and B cell responses to these peptides or proteins. RESULTS. Of 24 post-infectious LA patients, 58% had CD4+ T cell responses to ≥1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to >1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4+ T cells in synovial fluid were T-bet-expressing Th1 cells, a small percentage were RoRyt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Treg cells. CONCLUSION. Autoreactive, proinflammatory CD4+ T cells and autoantibodies develop to ECM proteins in a subgroup of post-infectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity.
Korawit Kanjana, Klemen Strle, Robert B. Lochhead, Annalisa Pianta, Laura M. Mateyka, Qi Wang, Sheila L. Arvikar, David E. Kling, Cameron A. DeAngelo, Lucy Curham, Alan G. Barbour, Catherine E. Costello, James J. Moon, Allen C. Steere
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