Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Total-Body Irradiation Alters White Matter Volume and Microstructural Integrity in Rhesus Macaques.

PURPOSE: Long-term survivors of brain irradiation can experience irreversible injury and cognitive impairment. T1-weighted and diffusion tensor magnetic resonance imaging (MRI) are used to evaluate brain volume and white matter (WM) microstructure in neurodevelopmental and neurodegenerative conditions. The goal of this study was to evaluate the long-term effects of single-dose total-body irradiation (TBI) or TBI with 5% partial-body sparing on brain volumetrics and WM integrity in macaques. METHODS AND MATERIALS: We used MRI scans from a cohort of male rhesus macaques (age range, 3.6-22.8 years) to compare global and regional brain volumes and WM diffusion in survivors of TBI (T1-weighted, n = 137; diffusion tensor imaging, n = 121; dose range, 3.5-10 Gy) with unirradiated controls (T1-weighted, n = 48; diffusion tensor imaging, n = 38). RESULTS: In all regions of interest, radiation affected age-related changes in fractional anisotropy, which tended to increase across age in both groups but to a lesser extent in the irradiated group (interaction P < .01). Depending on the region of interest, mean diffusivity decreased or remained the same across age in unirradiated animals, whereas it increased or did not change in irradiated animals. The increases in mean diffusivity were driven by changes in radial diffusivity, which followed similar trends across age. Axial diffusivity did not differ by irradiation status. Age-related changes in relative volumes in controls reflected normal trends in humans, with increasing WM and decreasing gray matter until middle age. Cerebrospinal fluid (CSF) volume did not differ across age in controls. WM volume was lower and CSF volume was higher in young irradiated macaques. WM volume was similar between groups, and CSF volume lower in older irradiated macaques. Gray matter volume was unaffected by radiation. CONCLUSIONS: TBI results in delayed WM expansion and long-term disruption of WM integrity. Diffusion changes suggest that myelin injury in WM is a hallmark of late-delayed radiation-induced brain injury.

Assessing cerebrovascular reactivity (CVR) in rhesus macaques (Macaca mulatta) using a hypercapnic challenge and pseudo-continuous arterial spin labeling (pCASL).

Cerebrovascular reactivity (CVR) is a measure of cerebral small vessels' ability to respond to changes in metabolic demand and can be quantified using magnetic resonance imaging (MRI) coupled with a vasoactive stimulus. Reduced CVR occurs with neurodegeneration and is associated with cognitive decline. While commonly measured in humans, few studies have evaluated CVR in animal models. Herein, we describe methods to induce hypercapnia in rhesus macaques (Macaca mulatta) under gas anesthesia to measure cerebral blood flow (CBF) and CVR using pseudo-continuous arterial spin labeling (pCASL). Fifteen (13 M, 2 F) adult rhesus macaques underwent pCASL imaging that included a baseline segment (100% O2) followed by a hypercapnic challenge (isoflurane anesthesia with 5% CO2, 95% O2 mixed gas). Relative hypercapnia was defined as an end-tidal CO2 (ETCO2) ≥5 mmHg above baseline ETCO2. The mean ETCO2 during the baseline segment of the pCASL sequence was 34 mmHg (range: 23-48 mmHg). During this segment, mean whole-brain CBF was 51.48 ml/100g/min (range: 21.47-77.23 ml/100g/min). Significant increases (p<0.0001) in ETCO2 were seen upon inspiration of the mixed gas (5% CO2, 95% O2). The mean increase in ETCO2 was 8.5 mmHg and corresponded with a mean increase in CBF of 37.1% (p<0.0001). The mean CVR measured was 4.3%/mmHg. No anesthetic complications occurred as a result of the CO2 challenge. Our methods were effective at inducing a state of relative hypercapnia that corresponds with a detectable increase in whole brain CBF using pCASL MRI. Using these methods, a CO2 challenge can be performed in conjunction with pCASL imaging to evaluate CBF and CVR in rhesus macaques. The measured CVR in rhesus macaques is comparable to human CVR highlighting the translational utility of rhesus macaques in neuroscience research. These methods present a feasible means to measure CVR in comparative models of neurodegeneration and cerebrovascular dysfunction.

The TLR2/TLR6 ligand FSL-1 mitigates radiation-induced hematopoietic injury in mice and nonhuman primates.

Thrombocytopenia, hemorrhage, anemia, and infection are life-threatening issues following accidental or intentional radiation exposure. Since few therapeutics are available, safe and efficacious small molecules to mitigate radiation-induced injury need to be developed. Our previous study showed the synthetic TLR2/TLR6 ligand fibroblast stimulating lipopeptide (FSL-1) prolonged survival and provided MyD88-dependent mitigation of hematopoietic acute radiation syndrome (H-ARS) in mice. Although mice and humans differ in TLR number, expression, and function, nonhuman primate (NHP) TLRs are like those of humans; therefore, studying both animal models is critical for drug development. The objectives of this study were to determine the efficacy of FSL-1 on hematopoietic recovery in small and large animal models subjected to sublethal total body irradiation and investigate its mechanism of action. In mice, we demonstrate a lack of adverse effects, an easy route of delivery (subcutaneous) and efficacy in promoting hematopoietic progenitor cell proliferation by FSL-1. NHP given radiation, followed a day later with a single subcutaneous administration of FSL-1, displayed no adversity but showed elevated hematopoietic cells. Our analyses revealed that FSL-1 promoted red blood cell development and induced soluble effectors following radiation exposure. Cytologic analysis of bone marrow aspirates revealed a striking enhancement of mononuclear progenitor cells in FSL-1-treated NHP. Combining the efficacy of FSL-1 in promoting hematopoietic cell recovery with the lack of adverse effects induced by a single administration supports the application of FSL-1 as a viable countermeasure against H-ARS.

A New Method for Estimating Glomerular Filtration Rate in Rhesus Macaques (Macaca mulatta).

Late effects of total- or partial-body irradiation include chronic kidney injury (CKI), which increases morbidity and mortality. Glomerular filtration rate (GFR) is the gold standard measure of kidney function. Renal function markers, such as blood urea nitrogen (BUN) and serum creatinine (Cr), may not be higher than reference ranges until 50% or more of nephrons are affected. Currently available methods to measure GFR are difficult and expensive, requiring multiple blood draws or timed urine collections, but their use can provide a framework for the development of simpler GFR estimates. The measurement of iohexol clearance is a validated tool used to determine GFR in veterinary patients. In this study, we aimed to determine if the Schwartz formula as used in human pediatric medicine can estimate GFR in rhesus macaques. We hypothesized that iohexol-GFR would correlate with the Schwartz formula-estimated GFR (eGFR) in irradiated and non-irradiated rhesus macaques. Twelve rhesus macaques [age 5-14 years (mean 7 years); 5 females, 7 males] with a range of BUN levels were selected for comparison to 4 non-irradiated controls (2 females, 2 males). Irradiated animals were divided by BUN into 3 groups: BUN ≤20 mg/dL (n = 4), BUN >20-24 mg/dL (n = 4), and BUN ≥25 mg/dL (n = 4). Baseline serum chemistry and urinalysis were used to assess renal function. For measurement of GFR, macaques were maintained under general anesthesia and received an intravenous injection of iohexol (2 mL/kg, 300 mg I/mL). Whole blood was collected at 10, 30, 60 and 90 min post-iohexol injection. Plasma iohexol concentrations were determined by mass spectrometry. GFR was calculated from the peak iohexol concentration and trapezoidal area under the curve (tAUC). The iohexol-GFR significantly correlated with the Schwartz formula-eGFR. In macaques with renal irradiation doses below 6 Gy, GFR was higher for males than females. GFR was lower in macaques with renal irradiation doses greater than 6 Gy compared to macaques with renal doses less than 6 Gy. We conclude that use of the Schwartz formula can provide a rapid, non-invasive, cost-effective, and accurate estimation of GFR to aid in the clinical assessment of renal function in irradiated rhesus macaques.

Dose-Dependent Testicular Injury and Recovery after Total-Body Irradiation in Rhesus Monkeys.

Testicular injury is a well-documented acute effect of radiation exposure, though little is known about recovery years after irradiation, especially at higher doses. We examined the testes from 143 irradiated and control male rhesus monkeys, who were part of the Radiation Late Effects Cohort over a four-year period. Irradiated animals were exposed to doses ranging from 3.5 to 8.5 Gy of total-body irradiation. The testes were assessed using computed tomography (CT) volumetry, serum testosterone, and histology for deceased members of the cohort. Irradiated animals exhibited dose-dependent testicular atrophy as well as decreased serum testosterone during the winter breeding season when compared to age-matched unirradiated controls. No significant difference in summer testosterone levels was observed. Volumetric and histologic evidence of testicular recovery was present approximately three years postirradiation for animals who received ≤8 Gy. The study demonstrates dose-dependent testicular injury after total-body irradiation and provides evidence for volumetric and spermatogonial recovery even at lethal doses of total-body irradiation in rhesus monkeys.

Longitudinal Analysis of Leukocyte Total and Differential Count of Rhesus Macaques (Macaca mulatta) after Total-Body Irradiation.

Archival data of leukocyte count and the differentials obtained from control and irradiated Rhesus Macaques (Macaca mulatta) were statistically analyzed to understand the long-term effect of ionizing radiation exposure. Nine animals received total-body irradiation (TBI) of 7.2-8.4 Gy at 3-4 years old. Twelve animals served as age-matched controls with no radiation exposure. The complete blood cell count dataset was obtained during regular health exams every 2-6 months for 8 years from their age of 8 to 17 years old. Linear mixed models for leukocyte, neutrophil, lymphocyte, and monocyte counts and their percentages were successfully developed. Estimated marginal means calculated based on the models revealed statistically significant elevations in leukocyte and neutrophil counts and neutrophil percentages in irradiated animals compared to the controls. Lymphocyte percentage was significantly lower in irradiated animals. Longitudinal trends for both control and irradiated animals were consistent with expected trends of aging in hematopoiesis, which is skewed towards production of myeloid lineage cells such as neutrophils and monocytes rather than lymphoid cells. Longitudinal trends from irradiated animals suggested the age-related increase in neutrophils and decrease in lymphocytes were stronger than in the controls, although the difference did not reach statistical significance. The mechanism of the long-term effects in the hematopoietic system were not investigated. However, the results suggest ionizing radiation causes long-term effects on some of the factors implicated in hematopoietic aging, possibly inducing early-onset or accelerated aging in the hematopoietic system. Extended analysis with observations including before and after the follow-up period in this study will be beneficial to understand the timeline and features of the long-term response.

Measurement of renal cortical fibrosis by CT scan.

Rationale and objectives: The accurate, non-invasive, and rapid measurement of renal cortical fibrosis is needed for well-defined benchmarks of permanent injury and for use of anti-fibrotic agents. It is also needed for non-invasive and rapid assessment of the chronicity of human renal diseases. Materials and methods: We have used a non-human primate model of radiation nephropathy to develop a novel method of size-corrected CT imaging to quantify renal cortical fibrosis. Results: Our method has an area under the receiver operating curve of 0.96, which is superior to any other non-invasive method of measuring renal fibrosis. Conclusion: Our method is suitable for immediate translation to human clinical renal diseases.

Assessment of Blood Pressure in Irradiated Rhesus Macaques (Macaca mulatta).

There is increasing evidence that circulatory disease incidence and mortality is associated with radiation exposure. Wake Forest School of Medicine is home to a unique cohort of total-body irradiated macaques, some with evidence of vascular end-organ disease in the brain, kidney and heart. Because there is a link between high blood pressure and vascular disease in all these sites, we undertook a retrospective study to evaluate blood pressure and radiation in this cohort of animals. In this work, we utilized a cohort of nonhuman primates (rhesus macaques, Macaca mulatta) long-term survivors of high-dose total-body irradiation (1.1-8.5 Gy, N = 129) and controls (N = 37) to evaluate the effects of radiation on blood pressure and obesity. Subjects were between 3 and 22 years of age (median 9 years). Blood pressure (BP) was measured 1-14 years postirradiation (median 4 years). Subjects were sedated with a combination of ketamine HCl (15 mg/kg body weight, IM) and midazolam (0.1 mg/kg body weight, IM) and systolic, diastolic, and mean arterial pressures were measured using a high definition oscillometer. Obesity was defined by dual energy X-ray absorptiometry as a body fat percentage >35%. Statistical analysis of the collected data indicated significant increases in blood pressure with increasing age and obesity. However, radiation did not significantly alter blood pressure in irradiated animals relative to controls, radiation dose, or age of irradiation.

Quantitative Assessment and Comparative Analysis of Longitudinal Lung CT Scans of Chest-Irradiated Nonhuman Primates.

Computed tomography (CT) imaging has been used to diagnose radiation-induced lung injury for decades. However, histogram-based quantitative tools have rarely been applied to assess lung abnormality due to radiation-induced lung injury (RILI). Here, we used first-order summary statistics to derive and assess threshold measures extracted from whole lung histograms of CT radiodensity in rhesus macaques. For the present study, CT scans of animals exposed to 10 Gy of whole thorax irradiation were utilized from a previous study spanning 2-9 months postirradiation. These animals were grouped into survivors and non-survivors based on their clinical and experimental endpoints. We quantified the change in lung attenuation after irradiation relative to baseline using three density parameters; average lung density (ALD), percent change in hyper-dense lung volume (PCHV), hyperdense volume as a percent of total volume (PCHV/TV) at 2-month intervals and compared each parameter between the two irradiated groups (non-survivors and survivors). We also correlated our results with histological findings. All the three indices (ALD, PCHV, PCHV/TV) obtained from density histograms showed a significant increase in lung injury in non-survivors relative to survivors, with PCHV relatively more sensitive to detect early RILI changes. We observed a significant positive correlation between histologic pneumonitis scores and each of the three CT measurements, indicating that CT density is useful as a surrogate for histologic disease severity in RILI. CT-based three density parameters, ALD, PCHV, PCHV/TV, may serve as surrogates for likely histopathology patterns in future studies of RILI disease progression.

Long-Term Immunological Consequences of Radiation Exposure in a Diverse Cohort of Rhesus Macaques.

PURPOSE: The aim of this study was to develop an improved understanding of the delayed immunologic effects of acute total body irradiation (TBI) using a diverse cohort of nonhuman primates as a model for an irradiated human population. METHODS AND MATERIALS: Immune recovery was evaluated in 221 rhesus macaques either left unirradiated (n = 36) or previously irradiated (n = 185) at 1.1 to 8.5 Gy TBI (median, 6.5 Gy) when aged 2.1 to 15.5 years (median, 4.2 years). Blood was drawn annually for up to 5 years total between 0.5 and 14.3 years after exposure. Blood was analyzed by complete blood count, immunophenotyping of monocytes, dendritic cells (DC) and lymphocytes by flow cytometry, and signal joint T-cell receptor exclusion circle quantification in isolated peripheral blood CD4 and CD8 T cells. Animals were categorized by age, irradiation status, and time since irradiation. Sex-adjusted means of immune metrics were evaluated by generalized estimating equation models to identify cell populations altered by TBI. RESULTS: Overall, the differences between irradiated and nonirradiated animals were subtle and largely restricted to younger animals and select cell populations. Subsets of monocytes, DC, T cells, and B cells showed significant interaction effects between radiation dose and age after adjustment for sex. Irradiation at a young age caused transient increases in the percentage of peripheral blood myeloid DC and dose-dependent changes in monocyte balance for at least 5 years after TBI. TBI also led to a sustained decrease in the percentage of circulating memory B cells. Young irradiated animals exhibited statistically significant and prolonged disruption of the naïve/effector memory/central memory CD4 and CD8 T-cell equilibrium and exhibited a dose-dependent increase in thymopoiesis for 2 to 3 years after exposure. CONCLUSIONS: This study indicates TBI subtly but significantly alters the circulating proportions of cellular mediators of adaptive immune memory for several years after irradiation, especially in macaques under 5 years of age and those receiving a high dose of radiation.

Analysis of College of American Pathologists von Willebrand Factor Proficiency Testing Program.

Von Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

Analysis of College of American Pathologists von Willebrand Factor Proficiency Testing Program.

Von Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

Total-Body Irradiation Is Associated With Increased Incidence of Mesenchymal Neoplasia in a Radiation Late Effects Cohort of Rhesus Macaques (Macaca mulatta).

PURPOSE: Cancer is a severe delayed effect of acute radiation exposure. Total-body irradiation has been associated with an increased risk of solid cancer and leukemia in Japanese atomic bomb survivors, and secondary malignancies, such as sarcoma, are a serious consequence of cancer radiation therapy. The radiation late effects cohort (RLEC) of rhesus macaques (Macaca mulatta) is a unique resource of more than 200 animals for studying the long-term consequences of total-body irradiation in an animal model that closely resembles humans at the genetic and physiologic levels. METHODS AND MATERIALS: Using clinical records, clinical imaging, histopathology, and immunohistochemistry, this retrospective study characterized the incidence of neoplasia in the RLEC. RESULTS: Since 2007, 61 neoplasms in 44 of 239 irradiated animals were documented (18.4% of the irradiated population). Only 1 neoplasm was diagnosed among the 51 nonirradiated controls of the RLEC (2.0%). The most common malignancies in the RLEC were sarcomas (38.3% of diagnoses), which are rare neoplasms in nonirradiated macaques. The most common sarcomas included malignant nerve sheath tumors and malignant glomus tumors. Carcinomas were less common (19.7% of diagnoses), and consisted primarily of renal cell and hepatocellular carcinomas. Neoplasia occurred in most major body systems, with the skin and subcutis being the most common site (40%). RNA analysis showed similarities in transcriptional profiles between RLEC and human malignant nerve sheath tumors. CONCLUSIONS: This study indicates that total-body irradiation is associated with an increased incidence of neoplasia years following irradiation, at more than double the incidence described in aging, nonirradiated animals, and promotes tumor histotypes that are rarely observed in nonirradiated, aging rhesus macaques.

Age effects on radiation response: summary of a recent symposium and future perspectives.

One of the principal uncertainties when estimating population risk of late effects from epidemiological data is that few radiation-exposed cohorts have been followed up to extinction. Therefore, the relative risk model has often been used to estimate radiation-associated risk and to extrapolate risk to the end of life. Epidemiological studies provide evidence that children are generally at higher risk of cancer induction than adults for a given radiation dose. However, the strength of evidence varies by cancer site and questions remain about site-specific age at exposure patterns. For solid cancers, there is a large body of evidence that excess relative risk (ERR) diminishes with increasing age at exposure. This pattern of risk is observed in the Life Span Study (LSS) as well as in other radiation-exposed populations for overall solid cancer incidence and mortality and for most site-specific solid cancers. However, there are some disparities by endpoint in the degree of variation of ERR with exposure age, with some sites (e.g., colon, lung) in the LSS incidence data showing no variation, or even increasing ERR with increasing age at exposure. The pattern of variation of excess absolute risk (EAR) with age at exposure is often similar, with EAR for solid cancers or solid cancer mortality decreasing with increasing age at exposure in the LSS. We shall review the human data from the Japanese LSS cohort, and a variety of other epidemiological data sets, including a review of types of medical diagnostic exposures, also some radiobiological animal data, all bearing on the issue of variations of radiation late-effects risk with age at exposure and with attained age. The paper includes a summary of several oral presentations given in a Symposium on "Age effects on radiation response" as part of the 67th Annual Meeting of the Radiation Research Society, held virtually on 3-6 October 2021.

Interlaboratory Performance in Measurement of Dabigatran and Rivaroxaban.

CONTEXT.­: Assessing direct oral anticoagulant (DOAC) drug levels by reliable laboratory assays is necessary in a number of clinical scenarios. OBJECTIVE.­: To evaluate the performance of DOAC-specific assays for various concentrations of dabigatran and rivaroxaban, assess the interlaboratory variability in measurement of these DOACs, and investigate the responsiveness of the routine clotting assays to various concentrations of these oral anticoagulants. DESIGN.­: College of American Pathologists proficiency testing survey data from 2013 to 2016 were summarized and analyzed. RESULTS.­: For dabigatran, the interlaboratory coefficient of variation (CV) of ecarin chromogenic assay was broad (ranging from 7.5% to 29.1%, 6.3% to 15.5%, and 6.8% to 9.0% for 100-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The CV for diluted thrombin time for dabigatran was better overall (ranging from 11.6% to 17.2%, 9.3% to 12.3, and 7.1% to 11.2% for 100 ng/mL, 200 ng/mL, and 400 ng/mL, respectively). The rivaroxaban-calibrated anti-Xa assay CVs also showed variability (ranging from 11.5% to 22.2%, 7.2% to 10.9%, and 6.4% to 8.1% for 50-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The prothrombin time (PT) and activated partial thromboplastin time (aPTT) showed variable dose- and reagent-dependent responsiveness to DOACs: PT was more responsive to rivaroxaban and aPTT to dabigatran. The undiluted thrombin time showed maximum prolongation across all 3 dabigatran concentrations, making it too sensitive for drug-level monitoring, but supporting its use as a qualitative screening assay. CONCLUSIONS.­: DOAC-specific assays performed reasonably well. While PT and aPTT cannot be used safely to determine DOAC degree of anticoagulation, a normal thrombin time excludes the presence of dabigatran.

Pseudo Pelger-Huët anomalies as potential biomarkers for acute exposure radiation dose in rhesus macaques (Macaca mulatta).

PURPOSE: The potential for malicious use of radiation, or radiation accidents could potentially lead to acute, high radiation doses to the public. Following acute accidental exposure to high doses of radiation, medical intervention is pivotal to the survivability of the patient, and the sooner the appropriate measures are taken the better the odds for survival. Early estimates of acute accidental radiation doses can be determined via biomarkers such as dicentric chromosome analysis or scenario reconstruction using computer software. However, both take valuable time and can be expensive. Increased frequencies of abnormal neutrophils in peripheral blood, referred to as pseudo Pelger-Huët anomalies (PPHAs), have been shown to be potential biomarkers of radiation exposure in several scenarios, including the 1958 Y-12 criticality accident and the radium dial painters. PPHAs are potentially a faster and cheaper quantitative biomarker for radiation exposure, and here they were evaluated in acutely exposed rhesus macaques. METHODS AND MATERIALS: Peripheral blood smears from acutely exposed rhesus macaques were evaluated for the percentage of neutrophils that displayed the PPHA morphology using light microscopy. Irradiated animals received 0 to 8.5 Gy total body radiation using one of two strategies: (1) linear accelerator-produced 6 MV photons delivered at 80 cGy/minute; or (2) Cobalt 60-produced gamma irradiation delivered at 60 cGy/min. Zero dose animals were used to determine a baseline percentage of PPHAs, and blood smears taken periodically throughout the lifetime of exposed animals post-irradiation were used to determine the persistence and biokinetics of PPHAs. RESULTS: The baseline prevalence of the PPHA in rhesus macaques was determined to be 0.58 ± 0.46%. The dose-response curve with doses ranging from 0 Gy to 8.5 Gy (LD90/30) displayed a strong positive correlation between PPHA percentage and acute radiation dose (R2 of 0.88 p = 3.62 × 10-22). Statistically significant differences were found when animals were separated into dose cohorts of 0, 4, 6.4-6.5, and 8-8.5 Gy. The biokinetics model utilized only 4 Gy exposures and blood smears taken periodically over 3.1 years post-irradiation. PPHA morphology increases quickly following irradiation and appears stable over 3.1 years post-irradiation. CONCLUSION: PPHA morphology was confirmed to be present in rhesus macaques, a dose-response relationship was constructed, and it is stable over 3 years post-irradiation. This study demonstrates that PPHA analysis can be a fast and cheap method of biodosimetry. Future studies will work to determine the accuracy of dose determination and lower limits of detection.

Apolipoprotein E levels in the amygdala and prefrontal cortex predict relative regional brain volumes in irradiated Rhesus macaques.

In the brain, apolipoprotein E (apoE) plays an important role in lipid transport and response to environmental and age-related challenges, including neuronal repair following injury. While much has been learned from radiation studies in rodents, a gap in our knowledge is how radiation might affect the brain in primates. This is important for assessing risk to the brain following radiotherapy as part of cancer treatment or environmental radiation exposure as part of a nuclear accident, bioterrorism, or a nuclear attack. In this study, we investigated the effects of ionizing radiation on brain volumes and apoE levels in the prefrontal cortex, amygdala, and hippocampus of Rhesus macaques that were part of the Nonhuman Primate Radiation Survivor Cohort at the Wake Forest University. This unique cohort is composed of Rhesus macaques that had previously received single total body doses of 6.5-8.05 Gy of ionizing radiation. Regional apoE levels predicted regional volume in the amygdala and the prefrontal cortex. In addition, apoE levels in the amygdala, but not the hippocampus, strongly predicted relative hippocampal volume. Finally, radiation dose negatively affected relative hippocampal volume when apoE levels in the amygdala were controlled for, suggesting a protective compensatory role of regional apoE levels following radiation exposure. In a supplementary analysis, there also was a robust positive relationship between the neuroprotective protein α-klotho and apoE levels in the amygdala, further supporting the potentially protective role of apoE. Increased understanding of the effects of IR in the primate brain and the role of apoE in the irradiated brain could inform future therapies to mitigate the adverse effects of IR on the CNS.

Clinicopathologic and Transcriptomic Analysis of Radiation-Induced Lung Injury in Nonhuman Primates.

PURPOSE: Radiation-induced lung injury (RILI) is a progressive condition with an early phase (radiation pneumonitis) and a late phase (lung fibrosis). RILI may occur after partial-body ionizing radiation exposures or internal radioisotope exposure, with wide individual variability in timing and extent of lung injury. This study aimed to provide new insights into the pathogenesis and progression of RILI in the nonhuman primate (NHP) rhesus macaque model. METHODS AND MATERIALS: We used an integrative approach to understand RILI and its evolution at clinical and molecular levels in 17 NHPs exposed to 10 Gy of whole-thorax irradiation in comparison with 3 sham-irradiated control NHPs. Clinically, we monitored respiratory rates, computed tomography (CT) scans, plasma cytokine levels, and bronchoalveolar lavage (BAL) over 8 months and lung samples collected at necropsy for molecular and histopathologic analyses using RNA sequencing and immunohistochemistry. RESULTS: Elevated respiratory rates, greater CT density, and more severe pneumonitis with increased macrophage content were associated with early mortality. Radiation-induced lung fibrosis included polarization of macrophages toward the M2-like phenotype, TGF-ß signaling, expression of CDKN1A/p21 in epithelial cells, and expression of α-SMA in lung stroma. RNA sequencing analysis of lung tissue revealed SERPINA3, ATP12A, GJB2, CLDN10, TOX3, and LPA as top dysregulated transcripts in irradiated animals. In addition to transcriptomic data, we observed increased protein expression of SERPINA3, TGF-ß1, CCL2, and CCL11 in BAL and plasma samples. CONCLUSIONS: Our combined clinical, imaging, histologic, and transcriptomic analysis provides new insights into the early and late phases of RILI and highlights possible biomarkers and potential therapeutic targets of RILI. Activation of TGF-ß and macrophage polarization appear to be key mechanisms involved in RILI.

A Flow Cytometric Study of Reagent Cells to Resolve ABO Typing Discrepancy.

OBJECTIVES: RBC alloantibodies can lead to ABO grouping discrepancies unrelated to A or B antigens or antibodies posing challenges in the blood bank testing. Routine blood bank testing and flow cytometry were used to immunophenotype reagent cells and elucidate the cause of ABO discrepancies in two patients. METHODS: ABO discrepancy was identified in two patients after transfusion with several units of RBCs. For both patients, the pretransfusion type and screen demonstrated blood group A. Eight and 16 days later, both patients showed an apparent antibody to reagent group A cells, which prompted additional study with patients' samples and flow cytometric testing of commercial reagent cells. RESULTS: In both patients' specimens, posttransfusion evaluation demonstrated an emerging antibody to the Kell antigen (K). The RBCs of both patients typed negative for K, and both were transfused with K-positive RBCs. Flow cytometric analysis of reagent RBCs demonstrated that five of seven lot numbers were positive for K. CONCLUSIONS: Emerging anti-K antibody led to agglutination of the K-positive reagent A1 cells, highlighting the importance of considering RBC alloantibodies and the composition of reagent cells when interpreting cases with an apparent ABO grouping discrepancy.