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This session, held during the 41st Annual STP Symposium, focused on mechanisms of decreased erythropoiesis and erythroid cell injury. The speakers provided comprehensive overviews of physiologic and pathologic erythropoiesis, reviewed various mechanisms of erythroid cell injury, and shared innovative investigative research with the audience.

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible. In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice. Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals. These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary. Disclosures No relevant conflicts of interest to declare.

The continuing education course on integrating clinical and anatomical pathology data was designed to communicate the importance of using a weight of evidence approach to interpret safety findings in toxicology studies. This approach is necessary, as neither clinical nor anatomic pathology data can be relied upon in isolation to fully understand the relationship between study findings and the test article. Basic principles for correlating anatomic pathology and clinical pathology findings and for integrating these with other study end points were reviewed. To highlight these relationships, a series of case examples, presented jointly by a clinical pathologist and an anatomic pathologist, were used to illustrate the collaborative effort required between clinical and anatomical pathologists. In addition, the diagnostic utility of traditional liver biomarkers was discussed using results from a meta-analysis of rat hepatobiliary marker and histopathology data. This discussion also included examples of traditional and novel liver and renal biomarker data implementation in nonclinical toxicology studies to illustrate the relationship between discrete changes in biochemistry and tissue morphology.

The Society of Toxicologic Pathology formed a working group in collaboration with the American Society for Veterinary Clinical Pathology to provide recommendations for the appropriate inclusion of clinical pathology evaluation in recovery arms of nonclinical toxicity studies but not on when to perform recovery studies. Evaluation of the recovery of clinical pathology findings is not required routinely but provides useful information on risk assessment in nonclinical toxicity studies and is recommended when the ability of the organ to recover is uncertain. The study design generally requires inclusion of concurrent controls to separate procedure-related changes from test article–related changes, but return of clinical pathology values toward baseline may be sufficient in some cases. Evaluation of either a select or full panel of standard hematology, coagulation, and serum and urine chemistry biomarkers can be scientifically justified. It is also acceptable to redesignate dosing phase animals to the recovery phase or vice versa to optimize data interpretation. Assessment of delayed toxicity during the recovery phase is not required but may be appropriate in development programs with unique concerns. Evaluation of the recovery of clinical pathology data for vaccine development is required and, for efficacy markers, is recommended if it furthers pharmacologic understanding.

The toxicologic pathologist plays a vital role in the scientific community, using their unique blend of diagnostic and investigative skills to advance biomedical research, public health, drug discovery, or regulatory practices. But what exactly do toxicologic pathologists contribute? Where do these specialized professionals work? How can toxicologic pathologists maximize their efficiency and potential? To enlighten students and trainees, as well as early- or mid-career toxicologic pathologists, or even those approaching retirement, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled “Practical Strategies for Navigating Toxicologic Pathology in One’s Early Career…and Beyond!” in conjunction with the STP 37th annual symposium. The workshop featured toxicologic pathologists from contract research organizations and the pharmaceutical industry, who provided their perspectives on career preparation, evolving veterinary pathologist roles within various sectors of toxicologic pathology, the fundamentals of safety assessment, logistics of projects involving good laboratory practices, tools for effective interpretation and communication of anatomic and clinical pathology results, and a recap of scientific resources available to support the toxicologic pathologist in his or her journey. This article provides brief summaries of the talks presented during this career development workshop.

There is limited direction in the literature or regulatory guidance on determination of adversity for clinical pathology (CP) biomarkers in preclinical safety studies. Toxicologic clinical pathologists representing the American Society for Veterinary Clinical Pathology—Regulatory Affairs Committee and Society of Toxicologic Pathology—Clinical Pathology Interest Group identified principles, overall approach, and unique considerations for assessing adversity in CP data interpretation to provide a consensus opinion. Emphasized is the need for pathophysiologic context and a weight-of-evidence approach. Most CP biomarkers do not have the potential to be adverse in isolation, regardless of magnitude of change. Rather, they quantify or describe the impact of effects, provide adjunct or supportive information regarding a process or pathogenesis, and provide translational biomarkers of effect. Most often, CP changes are part of a constellation of findings that collectively are adverse. Thus, most CP changes must be interpreted in conjunction with other study findings and require contextual and integrative interpretation. Exceptions include critical CP changes without correlates that indicate a health risk in the tested species. Overall, CP changes should not be interpreted in isolation and their adversity is best addressed with an integrated approach.

The phenomenon by which irradiated cells influence non-irradiated neighboring cells, referred to as the bystander effect (BSE), is not well understood in terms of the underlying pathways involved. We sought to enlighten connections between DNA damage repair and the BSE. Utilizing sister chromatid exchange (SCE) frequencies as a marker of the BSE, we performed cell transfer strategies that enabled us to distinguish between generation versus reception of a bystander signal. We find that DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) and Ataxia Telangectasia Mutated (ATM) are necessary for the generation of such a bystander signal in normal human cells following gamma (gamma)-ray exposure, but are not required for its reception. Importantly, we also show that directly irradiated human cells do not respond to receipt of a bystander signal, helping to explain why the BSE is a low-dose phenomenon. These studies provide the first evidence for a role of the DNA damage response proteins DNA-PKcs and ATM specifically in the generation of a bystander signal and intercellular signaling.