Gastrointestinal Acute Radiation Syndrome: Mechanisms, Models, Markers, and Medical Countermeasures.

There have been a number of reported human exposures to high dose radiation, resulting from accidents at nuclear power plants (e.g., Chernobyl), atomic bombings (Hiroshima and Nagasaki), and mishaps in industrial and medical settings. If absorbed radiation doses are high enough, evolution of acute radiation syndromes (ARS) will likely impact both the bone marrow as well as the gastrointestinal (GI) tract. Damage incurred in the latter can lead to nutrient malabsorption, dehydration, electrolyte imbalance, altered microbiome and metabolites, and impaired barrier function, which can lead to septicemia and death. To prepare for a medical response should such an incident arise, the National Institute of Allergy and Infectious Diseases (NIAID) funds basic and translational research to address radiation-induced GI-ARS, which remains a critical and prioritized unmet need. Areas of interest include identification of targets for damage and mitigation, animal model development, and testing of medical countermeasures (MCMs) to address GI complications resulting from radiation exposure. To appropriately model expected human responses, it is helpful to study analogous disease states in the clinic that resemble GI-ARS, to inform on best practices for diagnosis and treatment, and translate them back to inform nonclinical drug efficacy models. For these reasons, the NIAID partnered with two other U.S. government agencies (the Biomedical Advanced Research and Development Authority, and the Food and Drug Administration), to explore models, biomarkers, and diagnostics to improve understanding of the complexities of GI-ARS and investigate promising treatment approaches. A two-day workshop was convened in August 2022 that comprised presentations from academia, industry, healthcare, and government, and highlighted talks from 26 subject matter experts across five scientific sessions. This report provides an overview of information that was presented during the conference, and important discussions surrounding a broad range of topics that are critical for the research, development, licensure, and use of MCMs for GI-ARS.

White-matter correlates of anxiety: The contribution of the corpus-callosum to the study of anxiety and stress-related disorders.

OBJECTIVES: Traumatic stress has been associated with increased risk for brain alterations and development of anxiety disorders. Studies conducted in posttraumatic patients have shown white-mater volume and diffusion alterations in the corpus-callosum. Decreased cognitive performance has been demonstrated in acute stress disorder and posttraumatic patients. However, whether cognitive alterations result from stress related neuropathology or reflect a predisposition is not known. In the current study, we examined in healthy controls, whether individual differences in anxiety are associated with those cognitive and brain alterations reported in stress related pathologies. METHODS: Twenty healthy volunteers were evaluated for anxiety using the state-trait inventory (STAI), and were tested for memory performance. Brain imaging was employed to extract volumetric and diffusion characteristics of the corpus-callosum. RESULTS: Significant correlations were found between trait anxiety and all three diffusion parameters (fractional-anisotropy, mean and radial-diffusivity). Associative-memory performance and corpus-callosum volume were also significantly correlated. CONCLUSION: We suggest that cognitive and brain alterations, as tested in the current work and reported in stress related pathologies, are present early and possibly persist throughout life. Our findings support the hypothesis that individual differences in trait anxiety predispose individuals towards negative cognitive outcomes and brain alterations, and potentially to stress related disorders.

Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics.

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

The role of immune checkpoint receptors in the malignant phenotype of cutaneous T cell lymphoma.

Immune checkpoint receptors (ICR) modulate the immune response and are critical hubs for immunotherapy. However, data on their role in T lymphoid malignancies, such as cutaneous T cell lymphoma (CTCL), is sparse. We aimed to explore the role of ICR in the malignant features of transformed T lymphocytes and evaluate the effect of ICR-targeting monoclonal antibodies, often used as immunotherapy for solid tumors. We used the CTCL cell line HH and the Sézary cell line Hut78 to examine ICR expression and the effects of ICR inhibition on cell viability and proliferation. Despite their shared T cell progeny, the different CTCL cell lines exhibit markedly different ICR expression profiles. Programmed cell death-ligand 1 (PD-L1) was expressed by both cell lines, while programmed death-1 (PD-1) was expressed only by the HH cell line. Common to all malignant T cells was an autonomous hyper-proliferative state that did not require T cell receptor stimulation. A monoclonal antibody blocking PD-1 had a small but statistically significant augmenting effect on T cell proliferation. Of note, when the cells were exposed to ionizing radiation, healthy lymphocytes and those derived from the HH cell line were salvaged by anti-PD-L1. We show a regulatory role of ICR, mainly PD-1 and its ligand PD-L1, on cutaneous T cell malignancy.

Optimization of Detection of Gadodiamide Brain Retention in Rats Using Quantitative T2 Mapping and Intraperitoneal Administration.

BACKGROUND: Currently, the gadolinium retention in the brain after the use of contrast agents is studied by T1 -weighted magnetic resonance imaging (MRI) (T1 w) and T1 mapping. The former does not provide easily quantifiable data and the latter requires prolonged scanning and is sensitive to motion. T2 mapping may provide an alternative approach. Animal studies of gadolinium retention are complicated by repeated intravenous (IV) dosing, whereas intraperitoneal (IP) injections might be sufficient. HYPOTHESIS: T2 mapping will detect the changes in the rat brain due to gadolinium retention, and IP administration is equivalent to IV for long-term studies. STUDY TYPE: Prospective longitudinal. ANIMAL MODEL: A total of 31 Sprague-Dawley rats administered gadodiamide IV (N = 8) or IP (N = 8), or saline IV (N = 6) or IP (N = 9) 4 days per week for 5 weeks. FIELD STRENGTH/SEQUENCES: A 7 T, T1 w, and T2 mapping. ASSESSMENT: T2 relaxation and image intensities in the deep cerebellar nuclei were measured pre-treatment and weekly for 5 weeks. Then brains were assessed for neuropathology (N = 4) or gadolinium content using inductively coupled plasma mass spectrometry (ICP-MS, N = 12). STATISTICAL TESTS: Repeated measures analysis of variance with post hoc Student-Newman-Keuls tests and Hedges' effect size. RESULTS: Gadolinium was detected by both approaches; however, T2 mapping was more sensitive (effect size 2.32 for T2 vs. 0.95 for T1 w), and earlier detection (week 3 for T2 vs. week 4 for T1 w). ICP-MS confirmed the presence of gadolinium (3.076 ± 0.909 nmol/g in the IV group and 3.948 ± 0.806 nmol/g in the IP group). There was no significant difference between IP and IV groups (ICP-MS, P = 0.109; MRI, P = 0.696). No histopathological abnormalities were detected in any studied animal. CONCLUSION: T2 relaxometry detects gadolinium retention in the rat brain after multiple doses of gadodiamide irrespective of the route of administration. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

Liquid biopsy reveals collateral tissue damage in cancer.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

A phase 1a/1b trial of CSF-1R inhibitor LY3022855 in combination with durvalumab or tremelimumab in patients with advanced solid tumors.

Background LY3022855 is a recombinant, immunoglobulin, human monoclonal antibody targeting the colony-stimulating factor-1 receptor. This phase 1 trial determined the safety, pharmacokinetics, and antitumor activity of LY3022855 in combination with durvalumab or tremelimumab in patients with advanced solid cancers who had received standard anti-cancer treatments. Methods In Part A (dose-escalation), patients received intravenous (IV) LY3022855 25/50/75/100 mg once weekly (QW) combined with durvalumab 750 mg once every two weeks (Q2W) IV or LY3022855 50 or 100 mg QW IV with tremelimumab 75/225/750 mg once every four weeks. In Part B (dose-expansion), patients with non-small cell lung cancer (NSCLC) or ovarian cancer (OC) received recommended phase 2 dose (RP2D) of LY3022855 from Part A and durvalumab 750 mg Q2W. Results Seventy-two patients were enrolled (median age 61 years): Part A = 33, Part B = 39. In Part A, maximum tolerated dose was not reached, and LY3022855 100 mg QW and durvalumab 750 mg Q2W was the RP2D. Four dose-limiting equivalent toxicities occurred in two patients from OC cohort. In Part A, maximum concentration, area under the concentration-time curve, and serum concentration showed dose-dependent increase over two cycles of therapy. Overall rates of complete response, partial response, and disease control were 1.4%, 2.8%, and 33.3%. Treatment-emergent anti-drug antibodies were observed in 21.2% of patients. Conclusions LY3022855 combined with durvalumab or tremelimumab in patients with advanced NSCLC or OC had limited clinical activity, was well tolerated. The RP2D was LY3022855 100 mg QW with durvalumab 750 mg Q2W. ClinicalTrials.gov ID: NCT02718911 (Registration Date: May 3, 2011).

ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

Checkpoint inhibitors: Better outcomes among advanced cutaneous head and neck melanoma patients.

OBJECTIVE: The aim of this study was to investigate if the treatment outcomes of checkpoint inhibitors (CPI) in patients with advanced-stage skin head and neck melanoma (HNM) differs from outcomes in patients with non-HNM. DESIGN: A retrospective cohort study of patients with unresectable AJCC stage III and stage IV, who received CPI between 2010 and 2017. PARTICIPANTS: Overall, 122 unresectable AJCC stage III and metastatic stage IV melanoma adult patients were treated with CPI during the study period (consecutive patients). The HNM group of patients was comparable with limbs and trunk melanoma group except different distant metastatic (M1a/b/c/d) pattern (p = 0.025). MAIN OUTCOMES: Comparison of overall survival and clinical response to CPI in patients with advanced-stage skin melanoma of the head and neck with non-HNM. RESULTS: We analyzed 38 patients with melanoma arising in the head and neck skin regions, 33 with melanoma of limbs and 51 with trunk melanoma. Most of the head and neck patients were men (89.5%), the average age of melanoma diagnosis was 61.4±16.7 years (range 16.4-85.6). More than a third of HNM group of patients (36.8%) were 70 years and older. Overall response rate (ORR) to CPI was 50% (CR 31.6% and PR 18.4%) in the head and neck study group of patients, compared to an ORR of 36.3% and 23.5% in melanoma of the limbs and of the trunk, respectively (p = 0.03). The median overall survival of HNM group of patients was 60.2±6.3 months, CI 95% [47.7-72.7], 63% were alive at 30 months, reaching a plateau. Whereas, the median survival time of limbs and trunk melanoma were 51.2 and 53.4 months, which did not reach significance. CONCLUSIONS AND RELEVANCE: Response rate to CPI is significantly improved in patients with melanoma of the head and neck and they have a trend towards improved, long standing, overall survival.

SLAMF6​ deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint.

SLAMF6 is a homotypic receptor of the Ig-superfamily whose exact role in immune modulation has remained elusive. Its constitutive expression on resting and activated T cells precludes it from being a bona fide exhaustion marker. By breeding Pmel-1 mice with SLAMF6 -/- mice, we generated donors for T cells lacking SLAMF6 and expressing a transgenic TCR for gp100-melanoma antigen. Activated Pmel-1xSLAMF6 -/- CD8+ T cells displayed improved polyfunctionality and strong tumor cytolysis. T-bet was the dominant transcription factor in Pmel-1 x SLAMF6 -/- cells, and upon activation, they acquired an effector-memory phenotype. Adoptive transfer of Pmel-1 x SLAMF6 -/- T cells to melanoma-bearing mice resulted in lasting tumor regression in contrast to temporary responses achieved with Pmel-1 T cells. LAG-3 expression was elevated in the SLAMF6 -/- cells, and the addition of the LAG-3-blocking antibody to the adoptive transfer protocol improved the SLAMF6 -/- T cells and expedited the antitumor response even further. The results from this study support the notion that SLAMF6 is an inhibitory immune receptor whose absence enables powerful CD8+ T cells to eradicate tumors.

The immune system helps to protect our bodies from illnesses and infections. Immunotherapies are medicines designed to treat diseases, such as cancer, by boosting the immune system against the condition. This is a powerful approach but so far immunotherapies have only had partial success and there is a need for further improvements. One protein called SLAMF6 is found on cells from the immune system that attack and kill cancer cells. Immunotherapies that suppress SLAMF6 on immune cells called killer T cells could increase immune system activity helping to treat cancers, particularly melanoma skin cancers. So far the potential for SLAMF6 as a target for immunotherapy has not been fully explored. Hajaj et al. created mice with killer T cells that recognized skin cancer cells and lacked SLAMF6. These modified cells were better at fighting cancer, producing more anti-cancer chemicals called cytokines and killing more cancer cells. The modified cells had a lasting effect on tumors and helped the mice to live longer. The effects could be further boosted by treating the mice in combination with other immunotherapies. SLAMF6 is a possible new target for skin cancer immunotherapy that could help more people to live longer following cancer diagnosis. The next step is to create a drug to target SLAMF6 in humans and to test it in clinical trials.

Coordinated Activity of Transcriptional Networks Responding to the Pattern of Action Potential Firing in Neurons.

Transcriptional responses to the appropriate temporal pattern of action potential firing are essential for long-term adaption of neuronal properties to the functional activity of neural circuits and environmental experience. However, standard transcriptome analysis methods can be too limited in identifying critical aspects that coordinate temporal coding of action potential firing with transcriptome response. A Pearson correlation analysis was applied to determine how pairs of genes in the mouse dorsal root ganglion (DRG) neurons are coordinately expressed in response to stimulation producing the same number of action potentials by two different temporal patterns. Analysis of 4728 distinct gene-pairs related to calcium signaling, 435,711 pairs of transcription factors, 820 pairs of voltage-gated ion channels, and 86,862 pairs of calcium signaling genes with transcription factors indicated that genes become coordinately activated by distinct action potential firing patterns and this depends on the duration of stimulation. Moreover, a measure of expression variance revealed that the control of transcripts abundances is sensitive to the pattern of stimulation. Thus, action potentials impact intracellular signaling and the transcriptome in dynamic manner that not only alter gene expression levels significantly (as previously reported) but also affects the control of their expression fluctuations and profoundly remodel the transcriptional networks.

Intratumoral immune-biomarkers and mismatch repair status in leiyomyosarcoma -potential predictive markers for adjuvant treatment: a pilot study.

Leiomyosarcoma is the second most frequent soft-tissue sarcoma. Tumor lymphocytic infiltration (TIL) and programed cell death ligand-1 (PD-L1) have been associated with prognosis in different malignancies while DNA mismatch-repair deficiency (MMR-D) has been associated with response to check-point inhibitors. In this pilot study, we sought to examine TIL, PD-L1 and mismatch-repair (MMR) protein expression in 11 leiomyosarcoma and its association with outcome as potential biomarkers for adjuvant treatment. Eleven primary leiomyosarcoma archived-tissues were analyzed for expression of MMR proteins (MSH2, MLH1, MSH6 and PSM2), PD-L1 expression and PD-1, CD3 or CD8. MMR-D was detected in tumor tissue from 2/11 leiomyosarcoma patients. CD3 T-cells were present in all samples, whereas CD8 staining was positive in all but one. PDL-1 was positive in 4/11 and PD-L1 in 6/11. Interestingly, the three patients with the poorest outcome had strongly positive staining for PD-L1 and CD8 while in the two patients who are alive and recurrence-free, both PD-L1 and CD8 infiltration were lacking. We found an association between tumor infiltrating CD8 cytotoxic lymphocytes, strong PD-L1 staining and survival; suggesting a role as biomarkers for treatment decisions regarding peri-operative chemotherapy. We also identified MMR-D in two patients with leiomyosarcoma comprising 18% of our sample.

Comparative pathogenomics of Clostridium tetani.

Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP) and four distinct constituents of the mobile genome (mobilome): a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a common ancestor.

Gene networks activated by specific patterns of action potentials in dorsal root ganglia neurons.

Gene regulatory networks underlie the long-term changes in cell specification, growth of synaptic connections, and adaptation that occur throughout neonatal and postnatal life. Here we show that the transcriptional response in neurons is exquisitely sensitive to the temporal nature of action potential firing patterns. Neurons were electrically stimulated with the same number of action potentials, but with different inter-burst intervals. We found that these subtle alterations in the timing of action potential firing differentially regulates hundreds of genes, across many functional categories, through the activation or repression of distinct transcriptional networks. Our results demonstrate that the transcriptional response in neurons to environmental stimuli, coded in the pattern of action potential firing, can be very sensitive to the temporal nature of action potential delivery rather than the intensity of stimulation or the total number of action potentials delivered. These data identify temporal kinetics of action potential firing as critical components regulating intracellular signalling pathways and gene expression in neurons to extracellular cues during early development and throughout life.

Adoptive cell therapy: past, present and future.

The immune system is a potent inhibitor of tumor growth with curative potential, constituting in many eyes the future of antineoplastic therapy. Adoptive cell therapy (ACT) is a form of immunotherapy in which autologous cancer-cognate lymphocytes are expanded and modified ex vivo and re-infused to combat the tumor. This review follows the evolvement of ACT and treatment protocols, focusing on unresolved dilemmas regarding this treatment while providing evidence for its effectiveness in refractory patients. Future directions of ACT are discussed, in particular with regard to genetic engineering of autologous cells, and the role of ACT in the era of checkpoint inhibitors is addressed.

Breakdown of Inter-Hemispheric Connectivity Is Associated with Posttraumatic Symptomatology and Memory Impairment.

Altered brain anatomy in specific gray-matter regions has been shown in patients with posttraumatic stress disorder (PTSD). Recently, white-matter tracts have become a focus of research in PTSD. The corpus callosum (CC) is the principal white-matter fiber bundle, crucial in relaying sensory, motor and cognitive information between hemispheres. Alterations in CC fibers have been reported in PTSD and might be assumed to underlie substantial behavioral and cognitive sequelae; however most diffusion tensor imaging (DTI) studies in adult-onset PTSD failed to address the clinical correlates between imaging and PTSD symptoms severity, behavioral manifestation and cognitive functions. In the current study we examined (a) to what extent microstructural integrity of the CC is associated with memory performance and (b) whether imaging and cognitive parameters are associated with PTSD symptom severity. DTI data were obtained and fractional anisotropy (FA) values were computed for 16 patients and 14 controls. PTSD symptom severity was assessed by employing the clinician administered PTSD scale (CAPS) and memory was tested using a task probing item and associative memory for words and pictures. Significant correlations were found between PTSD symptoms severity, memory accuracy and reaction-time to CC FA values in the PTSD group. This study demonstrates meaningful clinical and cognitive correlates of microstructural connectivity. These results have implications for diagnostic tools and future studies aimed at identifying individuals at risk for PTSD.

Retrospective Study of the Predictive Value of Target Now in Systemic Therapy for Metastatic Colorectal and Gastric Carcinomas.

BACKGROUND: Predictive biomarkers for personalized treatment of neoplasms are suggested to be a major advancement in oncology and are increasingly used in clinical practice, albeit based on level II evidence. Target Now (TN) employs immunostaining and RNA expression on tumor samples to identify potentially beneficial or ineffective drugs. OBJECTIVES: To explore retrospectively the predictive value of TN for patients with colorectal and gastric carcinomas. METHODS: The study group comprised colorectal and gastric carcinoma patients with TN test reports. We identified chemotherapy regimens given for stage IV disease for which TN reports indicated prediction. Protocols were classified as having clinical benefit (CB; i.e., stable disease or any objective response) or progressive disease, and this was compared with the TN prediction. RESULTS: Nineteen patients--12 colorectal and 7 gastric carcinomas--met the inclusion criteria. There were 26 evaluable treatment protocols; of 18 with a CB15 were predicted to have a CB while 3 were predicted to have a lack of CB. Of eight protocols that had no CB, seven were predicted to have a CB and one a lack of CB. A chi-square test was non-significant (P = 0.78). An exploratory analysis yielded a positive predictive value of 68% and a sensitivity of 83% for the TN test. CONCLUSIONS: This study emphasizes the need for larger multi-center studies to validate the TN test before it is adopted into clinical practice.