Hemoglobin-haptoglobin Complex Research Articles

Development and characterization of a portable electrochemical aptasensor for IsdA protein and Staphylococcus aureus detection.

Staphylococcus aureus (S. aureus) is recognized as one of the most common causes of gastroenteritis worldwide. This pathogen is a major foodborne pathogen that can cause many different types of various infections, from minor skin infections to lethal blood infectious diseases. Iron-regulated surface determinant protein A (IsdA) is an important protein on the S. aureus surface. It is responsible for iron scavenging via interaction with hemoglobin, haptoglobin, and hemoglobin-haptoglobin complexes. This study develops a portable aptasensor for IsdA and S. aureus detection using aptamer-modified gold nanoparticles (AuNPs) integrated into screen-printed carbon electrodes (SPCEs). The electrode system was made of three parts, including a carbon counter electrode, an AuNPs/carbon working electrode, and a silver reference electrode. The aptamer by Au-S bonding was conjugated on the electrode surface to create the aptasensor platform. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were utilized to investigate the binding interactions between the aptasensor and the IsdA protein. CV studies showed a linear correlation between varying S. aureus concentrations within the range of 101 to 106CFU/mL, resulting in a limit of detection (LOD) of 0.2CFU/mL. The results demonstrated strong reproducibility, selectivity, and sensitivity of the aptasensor for enhanced detection of IsdA, along with about 93% performance stability after 30days. The capability of the aptasensor to directly detect S. aureus via the IsdA surface protein binding was further investigated in a food matrix. Overall, the aptasensor device showed the potential for rapid detection of S. aureus, serving as a robust approach to developing real-time aptasensors to identify an extensive range of targets of foodborne pathogens and beyond.

Haptoglobin is dispensable for haemoglobin uptake by Trypanosoma brucei.

Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.

Method for quantifying free hemoglobin, distinct from the hemoglobin-haptoglobin complex, in human serum

The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1–100 μg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.

Deficiency of the hemoglobin-haptoglobin receptor, CD163, worsens insulin sensitivity in obese male mice.

Excessive iron accumulation in metabolic organs such as the adipose tissue, liver, and skeletal muscle is associated with increased diabetes risk. Tissue-resident macrophages serve multiple roles including managing inflammatory tone and regulating parachymal iron homeostasis; thus protecting against metabolic dysfunction upon iron overload. The scavenger receptor CD163 is uniquely present on tissue-resident macrophages, and plays a significant role in iron homeostasis by clearing extracellular hemoglobin-haptoglobin complexes, thereby limiting oxidative damage caused by free hemoglobin in metabolic tissues. We show that the absence of CD163 exacerbates glucose intolerance and insulin resistance in male mice with obesity. Additionally, loss of CD163 reduced the expression of iron regulatory genes (Tfr1, Cisd1, Slc40a1) in adipose tissue macrophages and anti-inflammatory (M2-like) bone marrow-derived macrophages (BMDMs). Further, CD163 deficiency mediated a pro-inflammatory shift and limited hemoglobin scavenging specifically in M2-like BMDMs. To this end, iron buffering was diminished in inguinal white adipose tissue (iWAT) macrophages in vivo, which culminated in iron spillover into adipocytes and CD45+CD11B- non-myeloid immune cells in iWAT. These findings show that CD163 on tissue-resident macrophages is critical for their anti-inflammatory and hemoglobin scavenging roles, and its absence results in impaired systemic insulin action in an obese setting.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.

Transcriptomic profile of premature ovarian insufficiency with RNA-sequencing.

This study aimed to explore the transcriptomic profile of premature ovarian insufficiency (POI) by investigating alterations in gene expression. A total of sixty-one women, comprising 31 individuals with POI in the POI group and 30 healthy women in the control group (HC group), aged between 24 and 40years, were recruited for this study. The transcriptomic profiles of peripheral blood samples from all study subjects were analyzed using RNA-sequencing. The results revealed 39 differentially expressed genes in individuals with POI compared to healthy controls, with 10 upregulated and 29 downregulated genes. Correlation analysis highlighted the relationship between the expression of SLC25A39, CNIH3, and PDZK1IP1 and hormone levels. Additionally, an effective classification model was developed using SLC25A39, CNIH3, PDZK1IP1, SHISA4, and LOC389834. Functional enrichment analysis demonstrated the involvement of these differentially expressed genes in the "haptoglobin-hemoglobin complex," while KEGG pathway analysis indicated their participation in the "Proteoglycans in cancer" pathway. The identified genes could play a crucial role in characterizing the genetic foundation of POI, potentially serving as valuable biomarkers for enhancing disease classification accuracy.

Serum-Soluble CD163 Levels as a Prognostic Biomarker in Patients with Diffuse Large B-Cell Lymphoma Treated with Chemoimmunotherapy.

The majority of patients with Diffuse Large B-cell Lymphoma (DLBCL) will respond to first-line treatment and be cured. However, the disease is heterogeneous, and biomarkers able to discriminate patients with suboptimal prognosis are needed. M2 CD163-positive tumor-associated macrophages (TAMs) were shown to be implicated in DLBCL disease activity regulation. Serum-soluble CD163 (sCD163) functions as a scavenger receptor for haptoglobin-hemoglobin complexes and is mostly expressed by monocytes and macrophages. Its levels are used to determine macrophage activation. We aimed to determine serum sCD163 in a sample of DLBCL patients and study eventual correlations with parameters of disease activity or survival. Serum sCD163 levels were measured in 40 frozen sera from patients diagnosed with DLBCL and 30 healthy individuals (HIs) using an enzyme-linked immunosorbent assay (ELISA). Statistical analyses were performed using SPSS version 28. The results showed that patients who achieved complete response after standard-of-care immunochemotherapy and were alive and disease-free after 12 months of follow-up but had elevated sCD163 levels (above median) at diagnosis presented a significantly worse overall survival compared to those with initial serum sCD163 levels below the median (p = 0.03). Consequently, serum sCD163 levels in patients with DLBCL may constitute a marker of long-term response to chemoimmunotherapy.

The Modulation of in Vitro Differentiation of Monocyte-derived Macrophage by Trypanosoma evansi Antigens in the Dromedary Camel

Studies on the camel immune response to Trypanosoma (T.) evansi, the causative agent of Surra, are very limited. In the present study, flow cytometry was employed to investigate the modulatory effects of different T. evansi antigens on the in vitro differentiation of camel blood monocytes into macrophages. For this, in vitro, separated camel monocytes were differentiated into monocyte-derived macrophages (MDM) in the presence or absence (control) of formalin-fixed (inactivated) T. evansi whole parasite (T. evansi group) or the purified Ro Tat 1.2 antigen (Ro Tat 1.2 group). The analysis of the antimicrobial functions of MDM (phagocytosis and reactive oxygen species (ROS) production) revealed reduced phagocytosis activity of camel MDM generated in the presence of T. evansi antigens. In addition, a lack of ROS-response was observed in camel MDM generated in the presence of T. evansi antigens after stimulation with PMA. These results indicated a compromising effect of T. evansi on the innate defense mechanisms in camels. Phenotypic analysis revealed the upregulation of major histocompatibility complex (MHC) class II molecules together with the lower abundance of the scavenger receptor for haptoglobin–hemoglobin complexes (CD163) on MDM generated in the presence of whole T. evansi parasites, indicating a polarizing effect of T. evansi on the differentiation of camel monocytes into an M1 phenotype. However, the reduced antimicrobial functions of these cells argue against their pro-inflammatory nature. Although both MDM generated in the presence of whole T. evansi antigens or their purified Ro Tat 1.2 proteins indicated similar expression levels of CD14 and MHCII molecules, the different abundance of the cell surface molecules CD172a, CD163, CD45, and CD44 indicated different phenotypes of the two MDMs. The results of the present study revealed compromising effects of T. evansi antigens on camel macrophages differentiated in vitro from blood monocytes. Whether these effects contribute to the in vivo pathogenesis of T. evansi in camels remains to be determined in future studies.

Hemoglobin Receptor CD163 As a Potential Biomarker of Hemolysis Induced Hepatobiliary Injury in Sickle Cell Disease

Sickle cell disease (SCD) associated chronic hemolysis promotes oxidative stress, inflammation and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD related hepatobiliary injury has not been fully elucidated yet. Here, we show that chronic liver injury in SCD mice and patients is associated with elevated levels of hepatic CD163. Hemolysis and increase in hepatic heme, hemoglobin, iron and haptoglobin levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that HO-1 positively regulates CD163 expression and that HO-1 and NRF2 form a complex with CD163 in the SCD liver. Finally, we demonstrate that blocking CD163 activity promotes hemolysis induced liver injury in SCD. These findings indicate that CD163 is a mediator and a potential biomarker of SCD associated hepatobiliary injury. Understanding the role of HO-1 in CD163 regulation may help identify novel therapeutic targets for hemolysis induced chronic liver injury.

Abstract 11707: Differences in Erythrocyte Morphology in Thrombus From Infarct-Related Artery: Cardioembolic Thrombosis vs. Atherothrombosis, Pathological Analyses From MITO Study

Introduction: Diagnosis of cardioembolic thrombosis is important because anticoagulant therapy is recommended for its secondary prevention. However, it is difficult to make a clear distinction in the pathogenesis between cardioembolic thrombosis and atherothrombosis. It has been reported that CD163 positive macrophage which related hemoglobin-haptoglobin complex was more involved in cardioembolic thrombus than atherothrombosis. Hypothesis: Erythrocyte morphology and its hemolysis in thrombus are associated with difference in thrombus formation between cardioembolic thrombosis and atherothrombosis. Methods: The Macrophage in Thrombus (MITO) study is an observational study examining pathological and biological differences of arterial thrombus between sinus rhythm (SR) and atrial fibrillation (AF). Patients with acute thromboembolic stroke (ATS) or ST elevation myocardial infarction (STEMI) whose solid thrombus could be retrieved from the infarct-related artery were enrolled. Patients were divided into 2 groups as follows; Group C; ATS with AF (presumed cardioembolic thrombosis) and Group A; STEMI with SR: (presumed atherothrombosis), and compared erythrocyte morphology score; 1: erythrocyte morphology is maintained; 2: The erythrocyte morphology is not maintained, but part of its fragment are seen; and 3: The erythrocyte morphology is not maintained at all in the thrombus seen by х40. Circulating blood counts (CBC), haptoglobin and soluble CD163 were measured all the patients. Results: The score of Group C (n=20) was significantly higher than that of Group A (n=28) (p<0.01)Figure. Laboratory findings are shown in the Table. Conclusions: Erythrocyte morphology disruption was thought to be more involved in cardioembolic thrombus formation. Erythrocyte morphology and CBC with soluble CD163 level are likely to help distinguish cardioembolic thrombus from atherothrombus.

21 A comparison of assays measuring haptoglobin in equine plasma

Heightened inflammation is an important indicator of many diseases that affect the equine athlete. Haptoglobin, an acute phase protein present in blood, is commonly used as a biomarker for systemic inflammation. Recent literature provides a range for plasma haptoglobin in horses of 0.29–2.26mg/mL. When inflammation or infection occurs, the acute phase response of the immune system is activated, and haptoglobin concentrations can rise to 9 times that of healthy concentrations. A variety of assays have been developed to measure haptoglobin concentration, but there is disagreement in the scientific literature regarding which assay measures haptoglobin concentrations most accurately. The objective of this study was to compare 2 commonly used haptoglobin assays: The TriDelta “PHASE” Haptoglobin Assay (TD) and the ALPCO Horse Haptoglobin Assay (AP). The TD assay is based on the peroxidase activity of the haptoglobin-hemoglobin complex at lower pHs. The AP assay is a double antibody sandwich enzyme-linked immunoassay. Blood samples were collected from 8 Quarter Horse long yearlings (16–18 mo) split into a nonexercised group and exercised group. These horses and the experimental design were the focus of a different study. A total of 80 samples were collected over the 10-week study. The plasma was separated and stored in a –80°C freezer until analysis. Samples were measured in triplicate for eachassay. Results were compared using Pearson correlation analysis, linear regression, and Bland-Altman analysis, with significance set at P < 0.05. There was a good correlation (0.76) between the 2 assays. The linear regression (TD = 1.5*AP+1.9, r2 = 0.58) and the Bland-Altman (bias 2.7, 95% LOA 0.31 to 5.06) analyses indicated that the TD assay results in a higher haptoglobin measurement, with the difference between the assays increasing at higher concentrations. The TD inter-assay and intra-assay coefficient of variations were 11.4% and 4.4%, respectively. The TD measurements ranged from 1.2 to 8.8 mg/mL with a median of 3.9 mg/mL. The AP inter-assay and intra-assay coefficient of variations were 10.5% and 5.4%, respectively. The AP measurements ranged from 0.1 to 5.6 mg/mL with a median of 1.2 mg/mL. In conclusion, TD haptoglobin measurements were consistently higher than those quantified using the AP assay. The linear relationship indicates that calibration would facilitate some comparison between studies using the different assays; however, there is still significant unexplained variation. With no accepted gold standard for haptoglobin measurement, a more complete validation of one or both commonly used assays is required.

Molecular models of heme transfer in C. diphtheriae.

Corynebacterium diphtheriae is a bacteria that uses iron to assist in its survival and proliferation. It commonly obtains iron from host hemoglobin or the hemoglobin-haptoglobin complex. The protein pathway of the heme transport contains multiple proteins, including HbpA, ChtA, HtaA, HtaB, and HmuT, and the heme is shuttled between these proteins through a process that likely involves the formation of multiple transient protein/protein interfaces. However, the molecular mechanisms underlying heme acquisition and transport in these multimeric complexes remains unknown. To better understand these iron acquisition dynamics, we have performed a series of molecular dynamic (MD) simulations along the C. diphtheriae heme transfer pathway starting from unique rigid body docking structures, and used them to produce Markov State Models that reveal the kinetics and thermodynamics of the protein/protein association complexes. Results show a dynamic protein interface reveals intricacies of the heme acquisition process on the microsecond timescale.

The Potential of ANK1 to Predict Parkinson's Disease.

The main cause of Parkinson's disease (PD) remains unknown and the pathologic changes in the brain limit rapid diagnosis. Herein, differentially expressed genes (DEGs) in the Gene Expression Omnibus (GEO) database (GSE8397 and GSE22491) were assessed using linear models for microarray analysis (limma). Ankyrin 1 (ANK1) was the only common gene differentially down-regulated in lateral substantia nigra (LSN), medial substantia nigra (MSN) and blood. Additionally, DEGs between high ANK1 and low ANK1 in GSE99039 were picked out and then uploaded to the Database for Annotation, Visualization and Integrated Discovery (DAVID) for gene ontology (GO) functional annotation analysis. GO analysis displayed that these DEGs were mainly enriched in oxygen transport, myeloid cell development and gas transport (biological process (BP)); hemoglobin complex, haptoglobin-hemoglobin complex and cortical cytoskeleton (cellular component (CC)); and oxygen transporter activity, haptoglobin binding and oxygen binding (molecular function (MF)). Receiver operating characteristic (ROC) curve analysis showed ANK1 had good diagnostic accuracy and increased the area under the curve (AUC) value when combined with other biomarkers. Consistently, intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropy-ridi-ne (MPTP) in C57BL/6J mice reduced ANK1 mRNA expression in both substantia nigra and blood compared to the control group. Thus, ANK1 may serve as a candidate biomarker for PD diagnosis.

Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age.

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a ‘sub-proteome’, such as the ‘metalloproteome’, defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1–1) isoform in 15% of the population.

Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

Monocytes from Patients with Polycythemia Vera Express Molecules Related to Stress Erythropoiesis and Have Increased Erythrocyte Phagocytosis

Stress erythropoiesis (SE) is characterized by an increase in erythropoietic activity in the bone marrow and in extramedullary sites. The central macrophage present in the erythroblastic island (EBI) plays a key role in regulating SE through the expression of molecules mediating cell adhesion, iron metabolism, and those capable of identifying and engulfing damaged and senescent erythrocytes. Those receptors are also expressed in monocytes (MC), suggesting that their monocytic expression could be involved in erythropoiesis and erythrophagocytosis. CD14 +CD16 + intermediate MC (I-MC) express markers that are typically found in EBI macrophages and, together with CD14 +CD16 - classic MC (C-MC), are able to remove circulating iron. Polycythemia vera (PV) is characterized by autonomous overproduction of red blood cells (RBCs) with extramedullary hematopoiesis most often caused by an acquired JAK2 V617F mutation, resulting in a state of chronic SE. The depletion of macrophages from EBIs in animal model of PV reverses splenomegaly and erythrocytosis indicating that they are essential for the development of chronic SE. It is unknown if MC play the same role in humans or if they have different expressions of those key molecules, which could contribute to the severity of the disease.We aimed to investigate the role of MCs in SE present in PV by characterizing the expression of molecules relevant to RBC adhesion, anti-inflammation, erythrophagocytosis, and iron metabolism in MCs from PV patients and from healthy controls (HC).Peripheral blood MCs were isolated from HC (n=21) and PV patients (n=17) and phenotyped by flow cytometry (FC) for sialoadhesin (CD169), VCAM1 (CD106), the receptor for the hemoglobin-haptoglobin complex (CD163), mannose receptor (CD206), SIRPα (CD172), ferroportin (Fpn), and separated into subtypes according to expression of CD14 and CD16. We also evaluated MC erythrophagocytosis by determining positivity for intracellular glycophorin (CD235a) (nHC=13 and nPV=17).In PV, we observed significantly higher expression of CD169 and CD106, and lower expression of CD172 in C-MC (1,167±216.6 vs 1,834±241.9, p=0.009; 1,427±217.1 vs 2,849±182.3, p=0.0004; 104,707±9,546 vs 77,070±8,756, p=0.0428, respectively); higher CD169 and CD106 in I-MC (2,221±322.4 vs 3,150±321.8, p=0.0371; 2,186±201.7 vs 2,721±153.5, p=0.0238, respectively); and higher CD206, CD163, CD172, and CD106 in CD14 lowCD16 + non-classical MCs (NC-MC) (181.2±8.5 vs 268.6±13.4, p<0.0001; 312.3±15.1 vs 368.8±12.1, p=0.0174; 13,923±2256 vs 22,792±3211, p=0.0161; 1,234±96 vs 1,498±58.9 p=0.004, respectively). Fpn expression was not significantly different. A lower expression of CD172 in the C-MC suggests less inhibitory signaling for erythrophagocytosis in those cells. Although C-MC and I-MC have been previously linked to erythropoiesis, we saw a larger number of the investigated molecules being more expressed in NC-MC, supporting their possible involvement in regulating erythropoiesis. Our results suggest that MCs in PV could be more likely to attach erythroid cells and could therefore contribute to form EBIs if differentiated to macrophages. Higher molecule expression was associated to a higher percentage of MCs containing intracellular CD235a (0.35±0.07 vs 4.48±1.08, p<0.0001). This is evidence of circulating PV MCs performing erythrophagocytosis and supports a role for them in RBC clearance.Our findings reveal an increase in the expression of markers relevant to the adhesion of erythrocytes to MCs in all MC subsets from PV patients along with more frequent erythrocyte phagocytosis in circulating cells. Further studies should yield better understanding of the role of MCs in SE and in the formation of EBIs, providing future targets for the treatment of chronic SE in PV patients. DisclosuresFertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Articles of Significant Interest in This Issue.

The HbpA protein in Corynebacterium diphtheriae is crucial for iron acquisition from the hemoglobin-haptoglobin (Hb-Hp) complex. Lyman et al. (e00196-21) show that the C-terminal domain of HbpA is essential for cellular localization, hemin iron acquisition, and optimal hemoprotein binding. The C-terminal domain is also required for the formation of large, secreted complexes of HbpA that are involved in iron uptake from Hb-Hp. HbpA lacks homology to known hemin transport proteins and has exceptional affinity for hemoglobin. The multiple functions associated with this unusual C-terminal domain expand our understanding of the mechanism of iron acquisition by bacterial pathogens.

First-in-Human Autologous Implantation of Genetically Modified Adipocytes Expressing LCAT for the Treatment of Familial LCAT Deficiency

Familial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a severe inherited disease without a cure. Genetically modified adipocyte (GMAC) secreting LCAT (LCAT-GMAC) was developed to treat FLD. First-in-human autologous implantation of LCAT-GMACs was performed, showing to be safe and effective. The LCAT-GMAC implantation increased serum LCAT activity by approximately 50% of the baseline and sustained over three years. The patient exhibited apparent hemolysis by visual inspection. We found the presence of hemoglobin/haptoglobin complex in the patient's serum. The hemoglobin-haptoglobin complex was almost disappeared seven days after the implantation. Upon implantation of LCAT-GMAC, the patient's proteinuria decreased temporarily to mild levels and then gradually increased to the baseline. At 48 weeks after implantation, proteinuria deteriorated with the development of mild hypertension. To treat hypertension, the patient received antihypertensives, leading to the normalization of blood pressure. With the reduction of blood pressure, the proteinuria rapidly decreased to mild proteinuria levels. Trial registration: registered in Japan Registry of Clinical Trials: jRCTa030190230 Ethics: Approved by the Ministry of Health, Labour, and Welfare of Japan Funding: This research was supported by Japan Agency for Medical Research and Development (AMED) to M. A. under Grant Number JP 17im0110606h0004 Conflict of Interests: None.

Impaired macrophages and failure of steroidogenesis and spermatogenesis in rat testes with cytokines deficiency induced by diacerein.

The role of cytokines in testicular function under normal conditions has not been completely understood. Here, we evaluated testicular macrophages (TM), steroidogenesis by Leydig cells (LC) and seminiferous tubules integrity in cytokines-deficient rat testes induced by diacerein, an anti-inflammatory drug that inhibits interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). Male rats received daily 100 mg/kg of diacerein (DIAG; n = 8) or saline (CG; n = 8) for 30 days. Serum testosterone (T) levels were measured and the seminiferous tubule (ST) area, epithelial area (EA), frequency of damaged ST and number of Sertoli cells (SC) were evaluated. TUNEL method and immunoreactions for detection of pro-IL-1β, TNF-α, steroidogenic acute regulatory protein (StAR), 17β-hydroxysteroid dehydrogenase (17β-HSD), androgen receptor (AR) and scavenger receptor for hemoglobin-haptoglobin complexes (CD163), a TM marker, were performed. Testicular AR, 17β-HSD and IL-1β levels were detected by Western blot. Data were submitted to Student t test (p ≤ 0.05). In DIAG, T and testicular AR, 17β-HSD and IL-1β levels decreased significantly (p < 0.05). The number of TUNEL-positive interstitial cells increased and LC showed weak StAR, 17β-HSD and AR immunoexpression in association with reduced IL-1β immunoexpression and number of CD163-positive TM in the interstitial tissue from diacerein-treated rats. Numerous damaged ST were found in DIAG, and reduction in the EA were associated with germ cells death. Moreover, the number of SC reduced and weak AR and TNF-α immunoexpression was observed in SC and germ cells, respectively. The cytokines deficiency induced by diacerein impairs TM, LC and spermatogenesis, and points to a role of IL-1β in steroidogenesis under normal conditions. In the ST, the weak AR and TNF-α immunoexpression in SC and germ cells, respectively, reinforces the idea that TNF-α plays a role in the SC androgenic control.