Quantifying dilated perivascular spaces in children with sickle cell disease.

Sickle cell disease (SCD)-related neurological effects are particularly devastating. Dilated perivascular spaces (dPVS) are a well-described component of cerebral small vessel disease in older adults without SCD. However, the burden and association of dPVS with neurological complications in children with SCD have not been described. In this study, we used the international consensus criteria to quantify dPVS in the centrum semiovale and basal ganglia in T2-weighted magnetic resonance images (MRI) of children with SCD who were randomized as part of the Silent Cerebral Infarct Transfusion (SIT) trial. We examined the relationship between global and/or regional dPVS burden and presence or area of silent cerebral infarctions, hematological measures, demographic variables, and full-scale intelligence quotient (FSIQ) scores. The study included 156 SIT trial participants who had pre-randomization and study exit MRI. Their median age was 9.6 (5-15) years, 39% were female, and 94 (60%) participants had a high dPVS burden. Participants randomized to the blood transfusion arm and who had a high dPVS burden at baseline had a moderate decline in dPVS score over 36 months compared to no change in the observation group. On multivariable logistic regression, intelligence quotient was not associated with dPVS burden. Children with SCD included in the SIT trial have a high burden of dPVS compared to children without SCD. However, dPVS do not appear to have the same pathophysiology of silent cerebral infarcts. Further study is needed to determine both their etiology and clinical relevance.

Uncovering an undisclosed diagnosis: a glucose-6-phosphate dehydrogenase deficiency diagnosis in a critically ill adult.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects over 400 million people worldwide. The most common variant of G6PD deficiency in the United States is the A-variant, which is present amongst African-Americans. Most people with this variant, however, do not experience severe hemolysis unless under extreme circumstances. Here, we present the case of a 44-year-old African-American male who under circumstances of multiple admissions for critical illness eventually presented with a masked diagnosis of G6PD deficiency.

ß2-glycoprotein I promotes the clearance of circulating mitochondria.

ß2-glycoprotein I (ß2-Gp1) is a cardiolipin-binding plasma glycoprotein. It is evolutionarily conserved from invertebrates, and cardiolipin-bound ß2-Gp1 is a major target of antiphospholipid antibodies seen in autoimmune disorders. Cardiolipin is almost exclusively present in mitochondria, and mitochondria are present in circulating blood. We show that ß2-Gp1 binds to cell-free mitochondria (CFM) in the circulation and promotes its phagocytosis by macrophages at physiological plasma concentrations. Exogenous CFM had a short circulation time of less than 10 minutes in mice. Following infusion of CFM, ß2-Gp1-deficient mice had significantly higher levels of transfused mitochondria at 5 minutes (9.9 ± 6.4 pg/ml versus 4.0 ± 2.3 pg/ml in wildtype, p = 0.01) and at 10 minutes (3.0 ± 3.6 pg/ml versus 1.0 ± 0.06 pg/ml in wild-type, p = 0.033, n = 10). In addition, the splenic macrophages had less phagocytosed CFM in ß2-Gp1-deficient mice (24.4 ± 2.72% versus 35.6 ± 3.5 in wild-type, p = 0.001, n = 5). A patient with abnormal ß2-Gp1, unable to bind cardiolipin, has increased CFM in blood (5.09 pg/ml versus 1.26 ± 1.35 in normal) and his plasma induced less phagocytosis of CFM by macrophages (47.3 ± 1.6% versus 54.3 ± 1.3, p = 0.01) compared to normal plasma. These results show the evolutionarily conserved ß2-Gp1 is one of the mediators of the clearance of CFM in circulation.

Erythrophagocytosis in autoimmune immunoglobulin A-mediated hemolysis.

INTRODUCTION: Warm antibody-mediated autoimmune hemolysis (WAIHA) is most often due to immunoglobulin G (IgG) antibodies and is detected by direct antiglobulin test (DAT). However, about 10% cases of hemolytic anemia are DAT negative. Herein, we describe a patient with DAT-negative hemolytic anemia due to an anti-IgA antibody. We investigate if isolated IgA can promote erythrophagocytosis. METHODS: We isolated patient and control IgA on Jacalin agarose. Isolated IgA was used to sensitize healthy ABO/Rh-matched donor red blood cells (RBC). Antibody binding was examined by flowcytometry. The effect of IgA on erythrophagocytosis was evaluated using Macrophage colony stimulating factor 1 (M-CSF)-differentiated autologous macrophages by Giemsa staining and immunofluorescence microscopy. RESULTS: We show that isolated IgA from the serum can bind to red cells. In addition, RBCs were phagocytosed efficiently by autologous macrophages in the presence of patient IgA. CONCLUSION: Our results show that IgA antibodies are capable of inducing erythrophagocytosis like IgG antibodies in the absence of complement fixation.

Molecular and environmental contributors to neurological complications in sickle cell disease.

Sickle cell disease (SCD) is an inherited hemoglobinopathy in which affected hemoglobin polymerizes under hypoxic conditions resulting in red cell distortion and chronic hemolytic anemia. SCD affects millions of people worldwide, primarily in Sub-Saharan Africa and the Indian subcontinent. Due to vaso-occlusion of sickled red cells within the microvasculature, SCD affects virtually every organ system and causes significant morbidity and early mortality. The neurological complications of SCD are particularly devastating and diverse, ranging from overt stroke to covert cerebral injury, including silent cerebral infarctions and blood vessel tortuosity. However, even individuals without evidence of neuroanatomical changes in brain imaging have evidence of cognitive deficits compared to matched healthy controls likely due to chronic cerebral hypoxemia and neuroinflammation. In this review, we first examined the biological contributors to SCD-related neurological complications and then discussed the equally important socioenvironmental contributors. We then discuss the evidence for neuroprotection from the two primary disease-modifying therapies, chronic monthly blood transfusions and hydroxyurea, and end with several experimental therapies designed to specifically target these complications.

The Role of Inflammation in The Cellular and Molecular Mechanisms of Cardiopulmonary Complications of Sickle Cell Disease.

Cardiopulmonary complications remain the major cause of mortality despite newer therapies and improvements in the lifespan of patients with sickle cell disease (SCD). Inflammation has been identified as a major risk modifier in the pathogenesis of SCD-associated cardiopulmonary complications in recent mechanistic and observational studies. In this review, we discuss recent cellular and molecular mechanisms of cardiopulmonary complications in SCD and summarize the most recent evidence from clinical and laboratory studies. We emphasize the role of inflammation in the onset and progression of these complications to better understand the underlying pathobiological processes. We also discuss future basic and translational research in addressing questions about the complex role of inflammation in the development of SCD cardiopulmonary complications, which may lead to promising therapies and reduce morbidity and mortality in this vulnerable population.

Downregulated KLF2 in polycythemia vera and essential thrombocythemia induces prothrombotic gene expression.

Thromboses are major causes of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET) diseases associated with JAK2V617F mutation. However, the molecular mechanism(s) of increased thrombosis in PV and ET remain unknown. Kruppel-like factor 2 (KLF2) is a transcription factor that regulates expression of genes associated with inflammation and thrombosis; the absence of KLF2 in neutrophils causes thrombosis by inducing tissue factor. We studied the role of KLF2 in regulating prothrombotic gene expression in PV and ET. Neutrophils and platelets KLF2 expression in PV and ET was lower than the controls. Furthermore, in patients with thromboses, KLF2 transcripts were lower in platelets than those without thromboses. JAK2V617F allelic burden was inversely correlated with KLF2 transcript levels, suggesting JAK-STAT pathway may downregulate KLF2 expression. Whole transcriptome analyses of neutrophils and platelets showed that a lower KLF2 expression was associated with an upregulation of KLF2-regulated thrombotic genes. In addition, low KLF2 expression in platelets positively correlated with thrombotic events. In patients with PV and ET, KLF2 expression was induced by pegylated interferon alfa (PegINF-α) but not by hydroxyurea treatments. These data suggest that KLF2 may be a regulator of PV and ET thrombosis and a novel therapeutic target to prevent thrombosis.

A bone to pick-cellular and molecular mechanisms of bone pain in sickle cell disease.

The bone is one of the most commonly affected organs in sickle cell disease (SCD). Repeated ischemia, oxidative stress and inflammation within the bone is largely responsible for promoting bone pain. As more individuals with SCD survive into adulthood, they are likely to experience a synergistic impact of both aging and SCD on their bone health. As bone health deteriorates, bone pain will likely exacerbate. Recent mechanistic and observational studies emphasize an intricate relationship between bone remodeling and the peripheral nervous system. Under pathological conditions, abnormal bone remodeling plays a key role in the propagation of bone pain. In this review, we first summarize mechanisms and burden of select bone complications in SCD. We then discuss processes that contribute to pathological bone pain that have been described in both SCD as well as non-sickle cell animal models. We emphasize the role of bone-nervous system interactions and pitfalls when designing new therapies especially for the sickle cell population. Lastly, we also discuss future basic and translational research in addressing questions about the complex role of stress erythropoiesis and inflammation in the development of SCD bone complications, which may lead to promising therapies and reduce morbidity in this vulnerable population.

Thrombosis and thromboembolism: Brighton collaboration case definition and guidelines for data collection, analysis, and presentation of immunization safety data.

This is a Brighton Collaboration case definition of thrombosis and thromboembolism to be used in the evaluation of adverse events following immunization, and for epidemiologic studies for the assessment of background incidence or hypothesis testing. The case definition was developed by a group of experts convened by the Coalition for Epidemic Preparedness Innovations (CEPI) in the context of active development of SARS-CoV-2 vaccines. The case definition format of the Brighton Collaboration was followed to develop a consensus definition and defined levels of certainty, after an exhaustive review of the literature and expert consultation. The document underwent peer review by the Brighton Collaboration Network and by selected expert reviewers prior to submission.

Venous Thromboembolism in Sickle Cell Disease is Associated with Neutrophilia.

Venous thromboembolism (VTE) in individuals with sickle cell disease is common and portends a poor prognosis. The role of leukocyte count and its subsets on risk of VTE in sickle cell disease are not known. We conducted a retrospective case-control study and analyzed for leukocyte count at the time of VTE and 3 months prior. Leukocyte and neutrophil counts were elevated at the time of VTE (p = 0.003 and p = 0.0006, respectively) and 3 months prior (p = 0.001 and p = 0.0096, respectively) when compared to controls. Baseline leukocytosis and neutrophilia may be associated with subsequent risk for thrombosis in sickle cell disease.

Microvascular thrombosis: experimental and clinical implications.

A significant amount of clinical and research interest in thrombosis is focused on large vessels (eg, stroke, myocardial infarction, deep venous thrombosis, etc.); however, thrombosis is often present in the microcirculation in a variety of significant human diseases, such as disseminated intravascular coagulation, thrombotic microangiopathy, sickle cell disease, and others. Further, microvascular thrombosis has recently been demonstrated in patients with COVID-19, and has been proposed to mediate the pathogenesis of organ injury in this disease. In many of these conditions, microvascular thrombosis is accompanied by inflammation, an association referred to as thromboinflammation. In this review, we discuss endogenous regulatory mechanisms that prevent thrombosis in the microcirculation, experimental approaches to induce microvascular thrombi, and clinical conditions associated with microvascular thrombosis. A greater understanding of the links between inflammation and thrombosis in the microcirculation is anticipated to provide optimal therapeutic targets for patients with diseases accompanied by microvascular thrombosis.

High UDG and BRCA1 expression is associated with adverse outcome in patients with pemetrexed treated non-small cell lung Cancer.

OBJECTIVE: The antifolate chemotherapy agent pemetrexed has been widely used to treat non-small-cell-lung-cancer (NSCLC), but there is no clinically validated biomarker to select patients likely to respond. The aim of this study was to assess two proteins involved in DNA repair mechanisms, uracil DNA glycosylase (UDG) and BRCA1 as potential prognostic biomarkers in NSCLC patients treated with pemetrexed-based chemotherapy. MATERIAL AND METHODS: Formalin-fixed-paraffin-embedded tumor specimens from 119 patients with advanced NSCLC treated with pemetrexed between 2004 and 2011 were retrospectively analyzed. Expression of UDG, BRCA1, and known prognostic factors ALK, TTF-1, thymidylate synthase and folylpolyglutamate synthase was assessed by immunohistochemistry using H-SCORE (product of percent stained cells and intensity of expression). Progression-free (PFS) and overall survival (OS) served as reference endpoint. RESULTS: Most NSCLC tumor samples had UDG positivity in at least 5% of tumor cells and 34% samples had more than 50% positive tumor cells. Using the median expression value as threshold, high UDG expression (H-SCORE≥75) was significantly associated with shorter median PFS (3-year PFS 7% vs. 37%, p = 0.045) and a trend for shorter OS (3-year OS 15% vs 42%, p = 0.066) compared to patients with low UDG. In multivariable Cox analysis, the association between high UDG and shorter PFS was close to statistically significant (p = 0.08) at a significance level of 0.05 after controlling for age, gender, ALK- and TTF1-status with hazard ratio of 2.1. Grouping patients according to combined UDG and BRCA1 expression, patients with a profile of UDGhigh/BRCA1high had the shortest PFS and OS compared to all other patient groups (p = 0.007 and 0.02, respectively). CONCLUSION: Our results demonstrate an important prognostic role for high UDG expression in pemetrexed-treated NSCLC patients, in addition to its previously reported role in pemetrexed cytotoxicity. High UDG expression was predictive of shorter PFS and OS, and patients with a combined profile of UDGhigh/BRCA1high had the poorest outcome following pemetrexed treatment.

Neoadjuvant therapy for early-stage breast cancer: the clinical utility of pertuzumab.

Approximately 20% of breast cancer patients harbor tumors that overexpress human epidermal growth factor receptor 2 (HER2; also known as ErbB2), a receptor tyrosine kinase that belongs to the epidermal growth factor receptor family of receptor tyrosine kinases. HER2 amplification and hyperactivation drive the growth and survival of breast cancers through the aberrant activation of proto-oncogenic signaling systems, particularly the Ras/MAP kinase and PI3K/AKT pathways. Although HER2-positive (HER2(+)) breast cancer was originally considered to be a highly aggressive form of the disease, the clinical landscape of HER2(+) breast cancers has literally been transformed by the approval of anti-HER2 agents for adjuvant and neoadjuvant settings. Indeed, pertuzumab is a novel monoclonal antibody that functions as an anti-HER2 agent by targeting the extracellular dimerization domain of the HER2 receptor; it is also the first drug to receive an accelerated approval by the US Food and Drug Administration for use in neoadjuvant settings in early-stage HER2(+) breast cancer. Here, we review the molecular and cellular factors that contribute to the pathophysiology of HER2 in breast cancer, as well as summarize the landmark preclinical and clinical findings underlying the approval and use of pertuzumab in the neoadjuvant setting. Finally, the molecular mechanisms operant in mediating resistance to anti-HER2 agents, and perhaps to pertuzumab as well, will be discussed, as will the anticipated clinical impact and future directions of pertuzumab in breast cancer patients.