Assessing and mitigating the interference of ALX148, a novel CD47 blocking agent, in pretransfusion compatibility testing.

ALX148, a novel CD47 blocking agent, is in clinical development for the treatment of advanced solid tumors and lymphoma. Because CD47 is highly expressed on red blood cells (RBCs), its therapeutic blockade can potentially interfere with pretransfusion compatibility testing. This study describes the interference of ALX148 in pretransfusion compatibility testing and evaluates the methods used for mitigating such interference. STUDY DESIGN AND METHODS: Routine serologic tests were performed on six samples from four patients treated with ALX148. Antibody screening tests were performed on ALX148-spiked plasma, and RBC testing including antigen typing was performed on ALX148-coated RBCs. Soluble CD47 or high-affinity signal regulatory protein α (SIRPα) monomers were used to remove the false-positive reactivity of ALX148-spiked plasma with or without anti-E. RESULTS: ALX148 caused false-positive reactivity in antibody screening using indirect antiglobulin testing (IAT) and two-stage papain testing. However, false-positive reactivity was not observed at the immediate spin (IS), room temperature (RT), and 37°C phases. Direct antiglobulin testing, autologous controls, and eluates showed positive results. ALX148 did not affect blood group antigen typing performed at the IS or RT phases. The use of 50- to 100-fold molar excess of soluble CD47 or 300-fold molar excess of high-affinity SIRPα monomers removed false-positive reactivity in IAT without affecting anti-E detection. CONCLUSION: ALX148 generates false-positive reactivity in IAT, interfering with pretransfusion compatibility testing. The use of soluble CD47 or high-affinity SIRPα monomers can resolve the interference without possibly missing clinically significant alloantibodies.

Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR.

Obesity is associated with increased risk of Alzheimer's disease. Previous studies have demonstrated that amyloid-beta precursor protein (APP) is expressed in subcutaneous adipose tissue (SAT), upregulated with obesity, and correlates with insulin resistance and adipose tissue inflammation. APP is alternatively spliced into several isoforms, which may be indicative of the pathogenesis of APP-related diseases, but the accurate quantification has been difficult to standardize and reproduce. In light of this, we developed isoform-specific absolute cDNA standards for absolute quantitative real-time PCR (AQ-PCR), and measured transcript copy numbers for three major APP isoforms (APP770, APP751, and APP695), in SAT from C57BL/6 mice fed either a normal or high-fat diet. Expression of all three major APP isoforms was increased in diet-induced obese mice. Transcript copy numbers of APP770 and APP695 correlated with plasma insulin and CCL2 gene expression. The ratios of APP770 and APP751 to APP695 gradually decreased with aging, and correlated with plasma glucose levels. In addition, APP770 was significantly decreased in thiazolidinedione-treated mice. We describe quantification of APP isoform transcripts by AQ-PCR, which allows for direct comparison of gene copy number across isoforms, between experiments, and across studies conducted by independent research groups, which relative quantitative PCR does not allow. Our results suggest a possible role of differential expression of APP isoforms in the development of obesity-related insulin resistance and adipose tissue inflammation. In addition, it is important to determine if altered ratios of APP isoforms in SAT contribute to higher circulating Aß peptides and increased risk of abnormalities in obesity.

Mutan: A mixed linkage α-[(1,3)- and (1,6)]-d-glucan from Streptococcus mutans, that induces osteoclast differentiation and promotes alveolar bone loss.

Mutan is an extracellular polysaccharide of Streptococcus mutans (S. mutans) that consists of α-(1,3)-linked glucose residues in main chains and α-(1,6) bonds in side chains. In the present study, mutan was isolated from S. mutans, and its structural characteristics were determined using Fourier-transform infrared spectroscopy (FT-IR) and (13)C nuclear magnetic resonance (NMR) spectroscopy. The effects of mutan on RANKL-induced osteoclast differentiation in RAW 264.7 cells were examined. Furthermore, microCT and morphometric analyses were used to determine the contribution of mutan to alveolar bone loss in the maxilla of a rat periodontitis model. Mutan increased (more than 2-fold) RANKL-induced osteoclast differentiation in a dose-dependent manner. Mutan also enhanced the alveolar bone loss in the rat maxilla 2.3-fold. In mutan-treated rats, the bone mineral density, bone volume, trabecular number, and trabecular thickness decreased, whereas trabecular separation significantly increased. In addition, mutan and lipopolysaccharide (LPS) induced similar microarray profiles in RAW 264.7 cells. A total of 43 genes related to osteoclastogenesis were differentially expressed after either mutan or LPS treatment. Five-fold increases in the expression of several genes, including IL-1ß, IL-1α, IL-6, and chemokine ligands, were observed in mutan-treated RAW 264.7 cells. These results suggest a molecular mechanism for the inflammation induced by S. mutans during the establishment of periodontal disease.

Effects of Pueraria lobata Root Ethanol Extract on Adipogenesis and Lipogenesis During 3T3-L1 Differentiation into Adipocytes.

We evaluated the inhibitory effect of Pueraria lobata root ethanol extract (PLREE) on lipid accumulation during 3T3-L1 differentiation to adipocytes by measuring the intracellular expression of adipogenic, lipogenic, and lipolytic markers and lipid accumulation. The total polyphenol and flavonoid content of PLREE were 47 and 29 mg/g, respectively. The electron donating capacity of PLREE at 1,000 µg/mL was 48.8%. Treatment of 3T3-L1 preadipocytes with 100, 250, or 500 µg/mL PLREE for 8 days dose-dependently promoted the differentiation of 3T3-L1 cells. In contrast, the lipid content of PLREE-treated cells was significantly reduced by 7.8% (p < 0.05), 35.6% (p < 0.001), and 42.2% (p < 0.001) following treatment with 100, 250, and 500 µg/mL PLREE, respectively, as compared to differentiated control cells. PLREE upregulated peroxisome proliferator-activated receptor γ mRNA and protein, and sterol regulator element-binding protein-1c mRNA levels, but did not affect CCAAT/enhancer binding-protein ß and α mRNA levels. PLREE also downregulated acetyl-CoA carboxylase mRNA and protein, fatty acid synthase (FAS) protein, and leptin mRNA levels, but did not affect FAS mRNA expression. PLREE upregulated adipose triglyceride lipase mRNA and protein expression, and hormone-sensitive lipase (HSL) protein expression, but did not affect HSL mRNA expression. In conclusion, we found that PLREE enhanced adipogenesis, but reduced lipogenesis, resulting in decreased lipid accumulation in 3T3-L1 cells.

The effect of long-term aerobic exercise on maximal oxygen consumption, left ventricular function and serum lipids in elderly women.

The purpose of this study was to investigate the changes of maximal oxygen consumption, left ventricular function and serum lipids after 36 weeks of aerobic exercise in elderly women without the influence of drugs. Eight elderly women were studied by M-mode and Doppler echocardiography to assess left ventricular size, mass and function. Maximal oxygen consumption (VO(2)max) was determined for each subject by administering a treadmill exercise test. The training intensity was decided by heart rate reserve. Subjects performed exercise for 40 minutes a day, 3 days a week at 50-60% of the heart rate reserve during the 36 weeks. Exercise capacity was assessed by VO(2)max with a graded exercise test of the treadmill. Weight and % body fat decreased after training. Cardiorespiratory function improved because of the increase in VO(2)max and VO(2)max normalized for body weight after training. Systolic blood pressure significantly decreased. There are no significant difference in all left ventricular's parameters (end-diastolic dimension, end-systolic dimension, end-diastolic volume, end-systolic volume, stroke volume, cardiac output, ejection fraction, fractional shortening) after 36 weeks. Exercise training did not induce left ventricular (LV) enlargement as evidence of an absence of increase in left ventricular end-diastolic volume. The total cholesterol level and triglyceride level decreased after training. High density lipoprotein-cholesterol significantly increased and low density lipoprotein-cholesterol significantly decreased, atherogenic index (AI) significantly decreased and apolipoprotein A-I increased and apolipoprotein B decreased after training. In conclusion, although there was no significant change in left ventricular function, aerobic training showed a positive influence on body composition, maximal oxygen consumption and serum lipids.