Primaquine-5,6-Orthoquinone Is Directly Hemolytic to Older G6PD Deficient RBCs in a Humanized Mouse Model.

Primaquine and Tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinoneimine cause robust redox cycling in red blood cells (RBCs) but are so labile as to not be detected in blood or urine. Rather, the quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PDMed-) that recapitulates the human biology of RBC age-dependent enzyme decay, as well as an isogenic matched control mouse with human nondeficient G6PD hG6PDND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PDMed- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. SIGNIFICANCE STATEMENT: These findings demonstrate that 5,6-POQ, which has been considered an inert waste product of primaquine metabolism, directly induces ROS that cause clearance of older G6PDd RBCs. As 5,6-POQ is relatively stable compared with other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. The findings herein also establish a new model of G6PDd and provide the first direct evidence, to our knowledge, that young G6PDd RBCs are resistant to primaquine-induced hemolysis.

Polymerized Human Hemoglobin-Based Oxygen Carrier Preserves Lung Allograft Function During Normothermic Ex Vivo Lung Perfusion.

Normothermic ex vivo lung perfusion (EVLP) can resuscitate marginal lung allografts to increase organs available for transplantation. During normothermic perfusion, cellular metabolism is more active compared with subnormothermic perfusion, creating a need for an oxygen (O 2 ) carrier in the perfusate. As an O 2 carrier, red blood cells (RBCs) are a scarce resource and are susceptible to hemolysis in perfusion circuits, thus releasing cell-free hemoglobin (Hb), which can extravasate into the tissue space, thus promoting scavenging of nitric oxide (NO) and oxidative tissue damage. Fortunately, polymerized human Hb (PolyhHb) represents a synthetic O 2 carrier with a larger molecular diameter compared with Hb, preventing extravasation, and limiting adverse reactions. In this study, a next-generation PolyhHb-based perfusate was compared to both RBC and asanguinous perfusates in a rat EVLP model. During EVLP, the pulmonary arterial pressure and pulmonary vascular resistance were both significantly higher in lungs perfused with RBCs, which is consistent with RBC hemolysis. Lungs perfused with PolyhHb demonstrated greater oxygenation than those perfused with RBCs. Post-EVLP analysis revealed that the PolyhHb perfusate elicited less cellular damage, extravasation, iron tissue deposition, and edema than either RBCs or colloid control. These results show promise for a next-generation PolyhHb to maintain lung function throughout EVLP.

The Molecular Size of Bioengineered Oxygen Carriers Determines Tissue Oxygenation in a Hypercholesterolemia Guinea Pig Model of Hemorrhagic Shock and Resuscitation.

Polymerized human hemoglobin (PolyhHb) has shown promise in preclinical hemorrhagic shock settings. Different synthetic and purification schemes can control the size of PolyhHbs, yet research is lacking on the impact of polymerized hemoglobin size on tissue oxygenation following hemorrhage and resuscitation in specialized animal models that challenge their resuscitative capabilities. Pre-existing conditions that compromise the vasculature and end organs, such as the liver, may limit the effectiveness of resuscitation and exacerbate the toxicity of these molecules, which is an important but minimally explored therapeutic dimension. In this study, we compared the effective oxygen delivery of intermediate molecular weight PolyhHb (PolyhHb-B3; 500-750 kDa) to high molecular weight PolyhHb (PolyhHb-B4; 750 kDa-0.2 µm) for resuscitative effectiveness in guinea pig models subjected to hemorrhagic shock. We evaluated how the size of PolyhHb impacts hemodynamics and tissue oxygenation in normal guinea pigs and guinea pigs on an atherogenic diet. We observed that while PolyhHb-B3 and -B4 equivalently restore hemodynamic parameters of normal-dieted guinea pigs, high-fat-dieted guinea pigs resuscitated with PolyhHb-B4 have lower mean arterial pressures, impaired tissue oxygenation, and higher plasma lactate levels than those receiving PolyhHb-B3. We characterized the plasma of these animals following resuscitation and found that despite similar oxygen delivery kinetics, circulating PolyhHb-B3 and -B4 demonstrated a size-dependent increase in the plasma viscosity, consistent with impaired perfusion in the PolyhHb-B4 transfusion group. We conclude that intermediate-sized PolyhHbs (such as -B3) are ideal for further research given the effective resuscitation of hemorrhagic shock based on tissue oxygenation in hypercholesterolemic guinea pigs.

Extracellular Vesicle Size Reveals Cargo Specific to Coagulation and Inflammation in Pediatric and Adult Sickle Cell Disease.

Aberrant coagulation in sickle cell disease (SCD) is linked to extracellular vesicle (EV) exposure. However, there is no consensus on the contributions of small EVs (SEVs) and large EVs (LEVs) toward underlying coagulopathy or on their molecular cargo. The present observational study compared the thrombin potential of SEVs and LEVs isolated from the plasma of stable pediatric and adult SCD patients. Further, EV lipid and protein contents were analyzed to define markers consistent with activation of thrombin and markers of underlying coagulopathy. Results suggested that LEVs-but not SEVs-from pediatrics and adults similarly enhanced phosphatidylserine (PS)-dependent thrombin generation, and cell membrane procoagulant PS (18:0;20:4 and 18:0;18:1) were the most abundant lipids found in LEVs. Further, LEVs showed activated coagulation in protein pathway analyses, while SEVs demonstrated high levels of cholesterol esters and a protein pathway analysis that identified complement factors and inflammation. We suggest that thrombin potential of EVs from both stable pediatric and adult SCD patients is similarly dependent on size and show lipid and protein contents that identify underlying markers of coagulation and inflammation.

Communication of Positive Lung Cancer Screening Findings and Receipt of Recommended Follow-up Care.

This cohort study evaluates associations of communication methods and content of positive lung cancer screening findings with receipt of recommended follow-up care.

Biophysical Analysis and Preclinical Pharmacokinetics-Pharmacodynamics of Tangential Flow Filtration Fractionated Polymerized Human Hemoglobin as a Red Blood Cell Substitute.

Red blood cell (RBC) substitutes tested in late-phase clinical trials contained low-molecular-weight hemoglobin species (<500 kDa), resulting in vasoconstriction, hypertension, and oxidative tissue injury; therefore, contributing to poor clinical outcomes. This work aims to improve the safety profile of the RBC substitute, polymerized human hemoglobin (PolyhHb), via in vitro and in vivo screening of PolyhHb fractionated into four molecular weight brackets (50-300 kDa [PolyhHb-B1]; 100-500 kDa [PolyhHb-B2]; 500-750 kDa [PolyhHb-B3]; and 750 kDa to 0.2 µm [PolyhHb-B4]) using a two-stage tangential flow filtration purification process. Analysis showed that PolyhHb's oxygen affinity, and haptoglobin binding kinetics decreased with increasing bracket size. A 25% blood-for-PolyhHb exchange transfusion guinea pig model suggests that hypertension and tissue extravasation decreased with increasing bracket size. PolyhHb-B3 demonstrated extended circulatory pharmacokinetics, no renal tissue distribution, no aberrant blood pressure, or cardiac conduction effects, and may therefore be appropriate material for further evaluation.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Receipt of Recommended Follow-up Care After a Positive Lung Cancer Screening Examination.

Importance: Maximizing benefits of lung cancer screening requires timely follow-up after a positive screening test. The American College of Radiology (ACR) Lung CT Screening Reporting and Data System (Lung-RADS) recommends testing and follow-up timing based on the screening result. Objective: To determine rates of and factors associated with recommended follow-up after a positive lung cancer screening examination by Lung-RADS category. Design, Setting, and Participants: This prospective cohort study of lung cancer screening examinations performed from January 1, 2015, through July 31, 2020, with follow-up through July 31, 2021, was conducted at 5 academic and community lung cancer screening sites in North Carolina. Participants included 685 adults with a positive screening examination, Lung-RADS categories 3, 4A, 4B, or 4X. Statistical analysis was performed from December 2020 to March 2022. Exposures: Individual age, race, sex, smoking exposure, year of lung cancer screening examination, chronic obstructive pulmonary disease, body mass index, referring clinician specialty, rural or urban residence. Main Outcomes and Measures: Adherence, defined as receipt of recommended follow-up test or procedure after the positive screen per ACR Lung-RADS timeframes: 6 months for Lung-RADS 3 and 3 months for Lung-RADS 4A. For Lung-RADS 4B or 4X, adherence was defined as follow-up care within 4 weeks, as ACR Lung-RADS does not specify a timeframe. Results: Among the 685 individuals included in this study who underwent lung cancer screening with low-dose computed tomography, 416 (60.7%) were aged at least 65 years, 123 (18.0%) were Black, 562 (82.0%) were White, and 352 (51.4%) were male. Overall adherence to recommended follow-up was 42.6% (292 of 685) and varied by Lung-RADS category: Lung-RADS 3 = 30.0% (109 of 363), Lung-RADS 4A = 49.5% (96 of 194), Lung-RADS 4B or 4X = 68.0% (87 of 128). Extending the follow-up time increased adherence: Lung-RADS 3 = 68.6% (249 of 363) within 9 months, Lung-RADS 4A = 77.3% (150 of 194) within 5 months, and Lung-RADS 4B or 4X = 80.5% (103 of 128) within 62 days. For Lung-RADS 3, recommended follow-up was less likely among those currently smoking vs those who quit (adjusted odds ratio [aOR], 0.48; 95% CI, 0.29-0.78). In Lung-RADS 4A, recommended follow-up was less likely in Black individuals vs White individuals (aOR, 0.35; 95% CI, 0.15-0.86). For Lung-RADS 4B or 4X, recommended follow-up was more likely in female individuals vs male individuals (aOR, 2.82; 95% CI, 1.09-7.28) and less likely in those currently smoking vs those who quit (aOR, 0.31; 95% CI, 0.12-0.80). Conclusions and Relevance: In this cohort study, adherence to recommended follow-up after a positive screening examination was low but improved among nodules with a higher suspicion of cancer and after extending the follow-up timeline. However, the association of extending the follow-up time of screen-detected nodules with outcomes at the population level, outside of a clinical trial, is unknown. These findings suggest that studies to understand why recommended follow-up is lower in Black individuals, male individuals, and individuals currently smoking are needed to develop strategies to improve adherence.

Lung Cancer Screening in Individuals With and Without Lung-Related Comorbidities.

Importance: Comorbidities characterize the underlying health status of individuals. In the context of lung cancer screening (LCS), lung-related comorbidities may influence the observed benefits and harms. Objective: To compare the characteristics of individuals undergoing LCS, the LCS examination result, the cancer detection rate (CDR), and the false-positive rate (FPR) in those with and without lung-related comorbidities. Design, Setting, and Participants: A prospective cohort study was conducted in 5 academic and community screening sites across North Carolina from January 1, 2014, to November 7, 2020. Participants included 611 individuals screened for lung cancer who completed a 1-page health history questionnaire. Exposures: Presence of at least 1 self-reported lung-related comorbidity, including chronic obstructive pulmonary disease, chronic bronchitis, emphysema, asthma, bronchiectasis, pulmonary fibrosis, silicosis, asbestosis, sarcoidosis, and tuberculosis. Main Outcomes and Measures: The LCS examination result was determined from the radiologist's Lung Imaging Reporting and Data System assessment (negative, 1 or 2; positive, 3 or 4). The age-adjusted CDR and FPR were calculated per 100 LCS examinations, using binary logistic regression. Results: Among the 611 individuals screened for lung cancer (308 men [50.4%]; mean [SD] age, 64 [6.2] years), 335 (54.8%) had at least 1 lung-related comorbidity. Individuals with vs without lung-related comorbidities were more likely to be female than male (180 of 335 [53.7%] vs 123 of 276 [44.6%]; P = .02), White vs non-White race (275 of 326 [84.4%] vs 193 of 272 [71.0%]; P < .001), and have high school or less education vs greater than a high school education (108 of 231 [46.7%] vs 64 of 208 [30.8%]; P = .001). There were no significant differences in the proportion of positive LCS examinations in those with vs without a lung-related comorbidity at baseline (37 [16.0%] vs 22 [11.1%]; P = .14) or subsequent (40 [12.3%] vs 23 [10.6%]; P = .54) LCS examination. Comparing individuals with vs without lung-related comorbidities, there was no statistically significant difference in the CDR (1.6 vs 1.9 per 100; P = .73) or FPR (13.0 vs 9.3 per 100; P = .16). Of the 17 individuals with lung cancer, 13 patients (76.5%) were diagnosed with stage I lung cancer. Conclusions and Relevance: The findings of this study suggest that individuals with self-reported lung-related comorbidities undergoing LCS were more likely to be female, of White race, and have less education than those without lung-related comorbidity. Although no statistically significant differences in the proportion of positive examinations, CDR, or FPR by self-reported lung comorbidities were noted, additional studies with larger numbers of individuals undergoing screening are needed to understand LCS outcomes in those with lung-related comorbidities.

Angiogenesis and CD34 expression as a predictor of recurrence in oral squamous cell carcinoma.

PURPOSE: To assess immunohistologic features of angiogenesis of T1N0M0 oral squamous cell carcinoma (OSCC), and to identify predictors of regional recurrence. The identification of prognostic markers of early lymph node involvement in OSCC could allow for the use of more targeted biologic therapies for patients with early-stage tumors. PATIENTS AND METHODS: The study included patients treated for T1N0M0 OSCC at the Mayo Clinic from 1986 to 2001. All patients had initial surgical resection without neck dissection, and all had adequate follow-up with histologic specimens for review. Patients with lip, pharyngeal, or salivary gland tumors were excluded. Patient specimens were regraded and assessed for the histologic markers p53 and CD34 (penetrating and circumscribing patterns). The Kaplan-Meier method was used to estimate patient survival and survival free of regional recurrence. RESULTS: The study included 175 patients. The overall 5-year survival was 75%, and 5-year survival free of regional recurrence was 80.3%. Twenty-eight patients had regional recurrence. High-grade tumors (P = .03) and the penetrating pattern of CD34 (P = .02) were significantly associated with early regional metastasis from early-stage OSCC. The presence of p53 was not independently associated as a marker for regional metastasis. CONCLUSION: Early-stage T1 OSCC with high-grade lesions and a penetrating pattern of CD34 was associated with a statistically significant risk of cervical lymph node metastasis, compared with a circumscribing pattern of CD34.