A novel syphilis Treponema pallidum lipoprotein peptide antigen diagnostic assay using red cell kodecytes in routine blood centre column agglutination testing platforms.

BACKGROUND AND OBJECTIVES: The detection of treponemal antibodies, which are used to make a diagnosis of syphilis, is important both for diagnostic purposes and as a mandatory blood donor test in most countries. We evaluated the feasibility of using Kode Technology to make syphilis peptide red cell kodecytes for use in column agglutination serologic platforms. MATERIALS AND METHODS: Candidate Kode Technology function-spacer-lipid (FSL) constructs were made for the Treponema pallidum lipoprotein (TmpA) of T. pallidum, using the peptide and FSL selection algorithms, and then used to make kodecytes. Developmental kodecytes were evaluated against a large range of syphilis antibody reactive and non-reactive samples in column agglutination platforms and compared against established methodologies. Overall, 150 reactive and 2072 non-reactive Syphicheck assay (a modified T. pallidum particle agglutination) blood donor samples were used to evaluate the agreement rate of the developed kodecyte assay. RESULTS: From three FSL-peptide candidate constructs, one was found to be the most suitable for diagnostics. Of 150 Syphicheck assay reactive samples, 146 were TmpA-kodecyte reactive (97.3% agreement), compared with 58.0% with the rapid plasmin reagin (RPR) assay for the same samples. Against the 2072 expected syphilis non-reactive samples the agreement rate for TmpA-kodecytes was 98.8%. CONCLUSION: TmpA-kodecytes are viable for use as cost-effective serologic reagent red cells for the detection of treponemal antibodies to diagnose syphilis with a high level of specificity in blood centres. This kodecyte methodology also potentially allows for introduction of the reverse-algorithm testing into low-volume laboratories, by utilizing existing transfusion laboratory infrastructure.

Molecular basis of DEL phenotype in the Indian population: Insights from next-generation sequencing analysis of two cases.

The DEL phenotype represents an intriguing and challenging aspect of blood group serology. This condition is characterized by an extremely weak expression of the D antigen on red blood cells, to the extent that it often eludes detection through routine serological methods. Identifying the DEL phenotype necessitates more specialized techniques, such as adsorption and elution tests, to reveal the presence of the D antigen. This distinctive phenotype underscores the complexity and subtlety of blood group genetics and highlights the importance of using advanced methods to accurately classify individuals with this condition, as their ability to form anti-D antibodies can have clinical implications during transfusion and pregnancy scenarios. There is a paucity of data for the DEL phenotype in the Indian population, and the molecular basis has not been elucidated yet. Our investigation delves into the genetic underpinnings of two distinct DEL phenotype cases that pose challenges for resolution through conventional serological techniques. We employ next-generation amplicon sequencing to unravel the intricate genetic landscape underlying these cases. In the D-negative donor, the DEL phenotype was first identified serologically, which was subsequently confirmed by molecular analysis. In the second case, it was associated with an anti-D antibody in a D-positive patient. Initial data analysis unveiled a substantial reduction in coverage across the exonic segments of the RHD gene in both samples, suggesting the potential presence of RHD exon deletions. On both occasions, we identified a homozygous intronic RHD polymorphism that is well established to be linked to the RHD* 01EL.32/RHD*DEL32 variant.

Endothelial Cell Receptors in Tissue Lipid Uptake and Metabolism.

Lipid uptake and metabolism are central to the function of organs such as heart, skeletal muscle, and adipose tissue. Although most heart energy derives from fatty acids (FAs), excess lipid accumulation can cause cardiomyopathy. Similarly, high delivery of cholesterol can initiate coronary artery atherosclerosis. Hearts and arteries-unlike liver and adrenals-have nonfenestrated capillaries and lipid accumulation in both health and disease requires lipid movement from the circulation across the endothelial barrier. This review summarizes recent in vitro and in vivo findings on the importance of endothelial cell receptors and uptake pathways in regulating FAs and cholesterol uptake in normal physiology and cardiovascular disease. We highlight clinical and experimental data on the roles of ECs in lipid supply to tissues, heart, and arterial wall in particular, and how this affects organ metabolism and function. Models of FA uptake into ECs suggest that receptor-mediated uptake predominates at low FA concentrations, such as during fasting, whereas FA uptake during lipolysis of chylomicrons may involve paracellular movement. Similarly, in the setting of an intact arterial endothelial layer, recent and historic data support a role for receptor-mediated processes in the movement of lipoproteins into the subarterial space. We conclude with thoughts on the need to better understand endothelial lipid transfer for fuller comprehension of the pathophysiology of hyperlipidemia, and lipotoxic diseases such as some forms of cardiomyopathy and atherosclerosis.

Atherosclerosis Regression and Cholesterol Efflux in Hypertriglyceridemic Mice.

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Are we missing reverse TRALI? - A real world experience from a tertiary care oncology centre in India.

Transfusion related acute lung injury (TRALI) is a rare but potentially fatal pulmonary complication of transfusion that presents as acute hypoxemia and non-cardiogenic pulmonary oedema, developing during or within six hours of transfusion. Majority of the cases reported are due to transfusion of plasma rich blood components containing antibodies to human leukocyte antigen (anti-HLA) or human neutrophil antigen (anti-HNA). Rarely, anti-HLA or anti-HNA in recipients against transfused donor leukocyte antigens, cause TRALI by a reverse mechanism. Herein, we report three cases of suspected TRALI following transfusions of buffy coat derived granulocytes and peripheral blood stem cells. Three patients with hematological malignancies developed pulmonary symptoms after transfusions of leukocyte rich blood components. All cases showed findings of bilateral pulmonary infiltrates at chest radiography and patients were managed accordingly; however, all three expired within seven days of transfusion due to progressive respiratory deterioration. The patients were transfusion dependent for a long time and had received multiple non-leukoreduced blood components in the past. Clinical findings in all three cases indicate the possibility of reverse TRALI. Although, patients' anti-HLA or anti-HNA antibodies concordance with donors' cognate antigens (HLA and HNA) was not confirmed; yet these three cases suggest that reverse pathogenesis of TRALI is not as infrequent as reported in the literature. However, reverse TRALI has not been confirmed as the presence and nature of antibodies in the transfused recipient were not investigated due to the non availability of immunodiagnostic tests in India.

Regulation of lipoprotein lipase-mediated lipolysis of triglycerides.

PURPOSE OF REVIEW: To discuss the recent developments in structure, function and physiology of lipoprotein lipase (LpL) and the regulators of LpL, which are being targeted for therapy. RECENT FINDINGS: Recent studies have revealed the long elusive crystal structure of LpL and its interaction with glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1 (GPIHBP1). New light has been shed on LpL being active as a monomer, which brings into questions previous thinking that LpL inhibitors like angiopoietin-like 4 (ANGPTL4) and stabilizers like LMF1 work on disrupting or maintaining LpL in dimer form. There is increasing pharmaceutical interest in developing targets to block LpL inhibitors like ANGPTL3. Other approaches to reducing circulating triglyceride levels have been using an apoC2 mimetic and reducing apoC3. SUMMARY: Lipolysis of triglyceride-rich lipoproteins by LpL is a central event in lipid metabolism, releasing fatty acids for uptake by tissues and generating low-density lipoprotein and expanding high-density lipoprotein. Recent mechanistic insights into the structure and function of LpL have added to our understanding of triglyceride metabolism. This has also led to heightened interest in targeting its posttranslational regulators, which can be the next generation of lipid-lowering agents used to prevent hypertriglyceridemic pancreatitis and, hopefully, cardiovascular disease.

Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor.

RATIONALE: Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. OBJECTIVE: We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. METHODS AND RESULTS: We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. CONCLUSIONS: We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

Mechanism of Increased LDL (Low-Density Lipoprotein) and Decreased Triglycerides With SGLT2 (Sodium-Glucose Cotransporter 2) Inhibition.

Objective- SGLT2 (sodium-glucose cotransporter 2) inhibition in humans leads to increased levels of LDL (low-density lipoprotein) cholesterol and decreased levels of plasma triglyceride. Recent studies, however, have shown this therapy to lower cardiovascular mortality. In this study, we aimed to determine how SGLT2 inhibition alters circulating lipoproteins. Approach and Results- We used a mouse model expressing human CETP (cholesteryl ester transfer protein) and human ApoB100 (apolipoprotein B100) to determine how SGLT2 inhibition alters plasma lipoprotein metabolism. The mice were fed a high-fat diet and then were made partially insulin deficient using streptozotocin. SGLT2 was inhibited using a specific antisense oligonucleotide or canagliflozin, a clinically available oral SGLT2 inhibitor. Inhibition of SGLT2 increased circulating levels of LDL cholesterol and reduced plasma triglyceride levels. SGLT2 inhibition was associated with increased LpL (lipoprotein lipase) activity in the postheparin plasma, decreased postprandial lipemia, and faster clearance of radiolabeled VLDL (very-LDL) from circulation. Additionally, SGLT2 inhibition delayed turnover of labeled LDL from circulation. Conclusions- Our studies in diabetic CETP-ApoB100 transgenic mice recapitulate many of the changes in circulating lipids found with SGLT2 inhibition therapy in humans and suggest that the increased LDL cholesterol found with this therapy is because of reduced clearance of LDL from the circulation and greater lipolysis of triglyceride-rich lipoproteins. Most prominent effects of SGLT2 inhibition in the current mouse model were seen with antisense oligonucleotides-mediated knockdown of SGLT2.

ABO, Rhesus, and Kell Antigens, Alleles, and Haplotypes in West Bengal, India.

BACKGROUND: Few studies have documented the blood group antigens in the population of eastern India. Frequencies of some common alleles and haplotypes were unknown. We describe phenotype, allele, and haplotype frequencies in the state of West Bengal, India. METHODS: We tested 1,528 blood donors at the Medical College Hospital, Kolkata. The common antigens of the ABO, Rhesus, and Kell blood group systems were determined by standard serologic methods in tubes. Allele and haplotype frequencies were calculated with an iterative method that yielded maximum-likelihood estimates under the assumption of a Hardy-Weinberg equilibrium. RESULTS: The prevalence of ABO antigens were B (34%), O (32%), A (25%), and AB (9%) with ABO allele frequencies for O = 0.567, A = 0.189, and B = 0.244. The D antigen (RH1) was observed in 96.6% of the blood donors with RH haplotype frequencies, such as for CDe = 0.688809, cde = 0.16983 and CdE = 0.000654. The K antigen (K1) was observed in 12 donors (0.79%) with KEL allele frequencies for K = 0.004 and k = 0.996. Conclusions: For the Bengali population living in the south of West Bengal, we established the frequencies of the major clinically relevant antigens in the ABO, Rhesus, and Kell blood group systems and derived estimates for the underlying ABO and KEL alleles and RH haplotypes. Such blood donor screening will improve the availability of compatible red cell units for transfusion. Our approach using widely available routine methods can readily be applied in other regions, where the sufficient supply of blood typed for the Rh and K antigens is lacking.

Clinical and laboratory profile of anti-M.

CONCLUSIONS: Anti-M is a frequently detected naturally occurring antibody that has been reported in various clinical settings and also in voluntary donors. We describe here the clinical and laboratory findings of 11 cases with anti-M detected at our center. This report is a retrospective study in which we reviewed our immunohematology laboratory records for cases involving anti-M. Both donor and patient data from a 28-month period (September 2014 to December 2016) were reviewed. During this period, 11 examples of anti-M were detected (8 patients, 1 voluntary whole blood donor, and 1 hematopoietic stem cell donor. Anti-M was also detected in one external quality assessment scheme sample received during this period. In conclusion, anti-M can be detected in various clinical settings. This antibody can be clinically significant; in the laboratory, it can present as a serologic problem such as an ABO group discrepancy or an incompatible crossmatch. After detection, management and course of action is determined by both the antibody characteristics and the clinical setting.

Measurement of the phospholipase activity of endothelial lipase in mouse plasma.

Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL's hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.

Angiopoietin-like 3: An important protein in regulating lipoprotein levels.

ANGPTL3 has emerged as a therapeutic target whose inhibition results in profound reductions of plasma lipids, including atherogenic triglyceride-rich lipoproteins and low-density lipoprotein cholesterol. The identification of ANGPTL3 deficiency as a cause of familial combined hypolipidemia in humans hastened the development of anti-ANGPTL3 therapeutic agents, including evinacumab (a monoclonal antibody inhibiting circulating ANGPTL3), vupanorsen (an antisense oligonucleotide [ASO] targeting hepatic ANGPTL3 mRNA for degradation), and others. Advances have also been made in ANGPTL3 vaccination and gene editing strategies, with the former still in preclinical phases and the latter in preparation for Phase 1 trials. Here, we review the discovery of ANGPTL3 as an important regulator of lipoprotein metabolism, molecular characteristics of the protein, mechanisms by which it regulates plasma lipids, and the clinical development of anti-ANGPTL3 agents. The clinical success of therapies inhibiting ANGPTL3 highlights the importance of this target as a novel approach in treating refractory hypertriglyceridemia and hypercholesterolemia.

Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity.

Angiopoietin-like protein 4 (ANGPTL4) has been associated with a variety of diseases. It is known as an endogenous inhibitor of lipoprotein lipase (LPL), and it modulates lipid deposition and energy homeostasis. ANGPTL4 is cleaved by unidentified protease(s), and the biological importance of this cleavage event is not fully understood with respect to its inhibitory effect on LPL activity. Here, we show that ANGPTL4 appears on the cell surface as the full-length form, where it can be released by heparin treatment in culture and in vivo. ANGPTL4 protein is then proteolytically cleaved into several forms by proprotein convertases (PCs). Several PCs, including furin, PC5/6, paired basic amino acid-cleaving enzyme 4, and PC7, are able to cleave human ANGPTL4 at a consensus site. PC-specific inhibitors block the processing of ANGPTL4. Blockage of ANGPTL4 cleavage reduces its inhibitory effects on LPL activity and decreases its ability to raise plasma triglyceride levels. In summary, the cleavage of ANGPTL4 by these PCs modulates its inhibitory effect on LPL activity.

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay.

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.

Comparison of Quality and Efficacy of Apheresis Platelets Stored in Platelet Additive Solution Vis a Vis Plasma.

PAS, by replacing part of the plasma in the platelet storage bag, reduces post transfusion allergic reactions and DHTR in the recipient. In this study we compared quality and efficacy of PAS and usual plasma stored platelets. Platelet concentration, content, MPV, pH, swirling, LDH and glucose concentration were tested in SDPs after preparation and on the day of transfusion; and compared between control (plasma-stored SDP) and study (PAS-stored SDP) groups. CCI was compared between the two groups. Transfusion reactions were also noted. In both groups quality parameters were similar except glucose [significantly decreased (p < 0.001) in plasma] and LDH [increased significantly (p: -0.005) in PAS]. CCI was similar in both groups. Transfusion reaction rate were 0.012% and 0.049% in both groups respectively. Quality and post-transfusion efficacy in both groups were similar. PAS stored platelets may be transfused in multi-transfused patients with allergic manifestations and in minor ABO incompatible transfusions.

Lipolytic enzymes and free fatty acids at the endothelial interface.

Lipids released from circulating lipoproteins by intravascular action of lipoprotein lipase (LpL) reach parenchymal cells in tissues with a non-fenestrated endothelium by transfer through or around endothelial cells. The actions of LpL are controlled at multiple sites, its synthesis and release by myocytes and adipocytes, its transit and association with the endothelial cell luminal surface, and finally its activation and inhibition by a number of proteins and by its product non-esterified fatty acids. Multiple pathways mediate endothelial transit of lipids into muscle and adipose tissues. These include movement of fatty acids via the endothelial cell fatty acid transporter CD36 and movement of whole or partially LpL-hydrolyzed lipoproteins via other apical endothelial cell receptors such as SR-B1and Alk1. Lipids also likely change the barrier function of the endothelium and operation of the paracellular pathway around endothelial cells. This review summarizes in vitro and in vivo support for the key role of endothelial cells in delivery of lipids and highlights incompletely understood processes that are the focus of active investigation.

CREBH normalizes dyslipidemia and halts atherosclerosis in diabetes by decreasing circulating remnant lipoproteins.

Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the activity of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol. Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL, as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe-/- mice. Importantly, individuals with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOE-dependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.

Hypertriglyceridemia and Atherosclerosis: Using Human Research to Guide Mechanistic Studies in Animal Models.

Human studies support a strong association between hypertriglyceridemia and atherosclerotic cardiovascular disease (CVD). However, whether a causal relationship exists between hypertriglyceridemia and increased CVD risk is still unclear. One plausible explanation for the difficulty establishing a clear causal role for hypertriglyceridemia in CVD risk is that lipolysis products of triglyceride-rich lipoproteins (TRLs), rather than the TRLs themselves, are the likely mediators of increased CVD risk. This hypothesis is supported by studies of rare mutations in humans resulting in impaired clearance of such lipolysis products (remnant lipoprotein particles; RLPs). Several animal models of hypertriglyceridemia support this hypothesis and have provided additional mechanistic understanding. Mice deficient in lipoprotein lipase (LPL), the major vascular enzyme responsible for TRL lipolysis and generation of RLPs, or its endothelial anchor GPIHBP1, are severely hypertriglyceridemic but develop only minimal atherosclerosis as compared with animal models deficient in apolipoprotein (APO) E, which is required to clear TRLs and RLPs. Likewise, animal models convincingly show that increased clearance of TRLs and RLPs by LPL activation (achieved by inhibition of APOC3, ANGPTL3, or ANGPTL4 action, or increased APOA5) results in protection from atherosclerosis. Mechanistic studies suggest that RLPs are more atherogenic than large TRLs because they more readily enter the artery wall, and because they are enriched in cholesterol relative to triglycerides, which promotes pro-atherogenic effects in lesional cells. Other mechanistic studies show that hepatic receptors (LDLR and LRP1) and APOE are critical for RLP clearance. Thus, studies in animal models have provided additional mechanistic insight and generally agree with the hypothesis that RLPs derived from TRLs are highly atherogenic whereas hypertriglyceridemia due to accumulation of very large TRLs in plasma is not markedly atherogenic in the absence of TRL lipolysis products.