Antigen specificity shapes antibody functions in tuberculosis.

Tuberculosis (TB) is the number one infectious disease cause of death worldwide in part due to an incomplete understanding of immunity. Emerging data highlight antibody functions as correlates of protection and disease across human TB. However, little is known about how antibody functions impact Mycobacterium tuberculosis (Mtb) , the causative agent. Here, we use antigen specificity to understand how antibodies mediate host- Mtb interactions. We focus on Mtb cell wall and ESAT-6 & CFP-10, critical bacterial structural and secreted virulence proteins. In polyclonal IgG from TB patients, we observe that antigen specificity alters IgG subclass and glycosylation that drives Fc receptor binding and effector functions. Through in vitro models of Mtb macrophage infection we find that Mtb cell wall IgG3, sialic acid, and fucose increase opsonophagocytosis of extracellular Mtb and bacterial burden, suggesting that some polyclonal IgG enhance disease. In contrast, ESAT-6 & CFP-10 IgG1 inhibits intracellular Mtb , suggesting that antibodies targeting secreted virulence factors are protective. We test this hypothesis by generating a mAb that reacts to ESAT-6 & CFP-10 and show that it alone inhibits intracellular Mtb . Understanding which antigens elicit antibody mediated disease enhancement and or protection will be critical in appreciating the many roles for antibodies in TB.

Effect of PCSK9 inhibition on plasma levels of small dense low density lipoprotein-cholesterol and 7-ketocholesterol.

BACKGROUND: Oxidized forms of cholesterol (oxysterols) are implicated in atherogenesis and can accumulate in the body via direct absorption from food or through oxidative reactions of endogenous cholesterol, inducing the formation of LDL particles loaded with oxidized cholesterol. It remains unknown whether drastic reductions in LDL-cholesterol (LDL-C) are associated with changes in circulating oxysterols and whether small dense LDL (sdLDL) are more likely to carry these oxysterols and susceptible to the effects of PCSK9 inhibition (PCSK9i). OBJECTIVE: We investigate the effect of LDL-C reduction accomplished via PCSK9i on changes in plasma levels of sdLDL-cholesterol (sdLDL-C) and a common, stable oxysterol, 7-ketocholesterol (7-KC), among 134 patients referred to our Preventive Cardiology clinic. METHODS: Plasma lipid panel, sdLDL-C, and 7-KC measurements were obtained from patients before and after initiation of PCSK9i. RESULTS: The intervention caused a significant lowering of LDL-C (-55.4 %). The changes in sdLDL-C levels (mean reduction 51.4 %) were highly correlated with the reductions in LDL-C levels (R = 0.829, p < 0.001). Interestingly, whereas changes in plasma free 7-KC levels with PCSK9i treatment were much smaller than (-6.6 %) and did not parallel those of LDL-C and sdLDL-C levels, they did significantly correlate with changes in triglycerides and very low-density lipoprotein-cholesterol (VLDL-C) levels (R = 0.219, p = 0.025). CONCLUSION: Our findings suggest a non-preferential clearance of LDL subparticles as a consequence of LDL receptor upregulation caused by PCSK9 inhibition. Moreover, the lack of significant reduction in 7-KC with PCSK9i suggests that 7-KC may be in part carried by VLDL and lost during lipoprotein processing leading to LDL formation.

Vertical jump impulse deficits persist from six to nine months after ACL reconstruction.

Later-stage rehabilitation following anterior cruciate ligament (ACL) reconstruction (ACLR) provides a valuable opportunity to target performance deficits before return to sport. This study aimed to: (1) evaluate bilateral counter-movement jump (CMJ) phase-specific impulse and isokinetic strength inter-limb asymmetry progression from 6 to 9 months post-ACLR; and (2) examine the extent to which individual changes in strength asymmetry could explain changes in impulse asymmetry. Male athletes (n = 44) with a hamstring tendon or bone-patellar tendon-bone autograft were tested 6 and 9 months post-ACLR. Two-way mixed-model ANOVAs were used to identify inter-session and inter-graft differences in CMJ phase-specific impulse asymmetries and knee isokinetic flexor and extensor strength asymmetries, as well as in absolute impulse and strength values of independent (ACLR/uninvolved) limbs. Linear regression models were used to assess the relationship between changes in impulse asymmetry and strength asymmetry. Reductions in strength asymmetry arose from improved ACLR-limb performance, whereas concentric impulse asymmetry reduced consequent to decreased uninvolved-limb performance and eccentric deceleration impulses decreased bilaterally. Graft type did not modulate findings. Changes in strength asymmetry had little or no ability to explain changes in impulse asymmetry. Consideration of approaches that may influence persisting deficits observed bi-laterally throughout vertical jumping performance post-ACLR may enhance rehabilitation practice.

Prevalence and associated risk factors of chronic kidney disease: A case study within SIME clinics in Quito, Ecuador 2019-2021.

Background: Ecuador has been experiencing an epidemiological transition due to its demographic and lifestyle changes, where non-communicable diseases are the leading cause of death, including chronic kidney disease (CKD). Quito, Ecuador's capital city, is one of the cities burdened by CKD, yet it is unknown the factors that contribute to the rising incidence of this disease. The purpose of this study was to estimate the prevalence of CKD among non-institutionalized adults in Quito between 2019 and 2021, and to examine its associations with various risk factors. Methods: For the analysis of prevalence, the Kidney Disease: Improving Global Outcomes guidelines were used, where an estimated glomerular filtration rate (eGFR) of < 60 ml/min/1.73 m2 was counted as a presumed case of CKD. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to calculate eGFR. Multiple linear regression models were used to determined associations between blood pressure, blood glucose, sex, and zone with eGFR. A t-test of independence was used to determine difference in means between sex and zone and eGFR. Results: A prevalence of 7.2% was found, in which almost 45% of the participants were classified within stages 2-4 of this disease. The risk factors that were significantly associated with eGFR were systolic blood pressure (ß = -0.43, p < 0.001), sex, and zone (p < 0.001). Conclusions: Overall a high prevalence of CKD was found among adults who visited SIME clinics in Quito. Associations between main risk factors and eGFR were found, yet further research is needed to explore CKD in Ecuador and its main cities.

Biomechanical asymmetries differ between autograft types during unplanned change of direction after ACL reconstruction.

Nine months after anterior cruciate ligament (ACL) reconstruction, athletes who undergo surgery using a bone-patellar-tendon-bone (BPTB) autograft demonstrate higher loading asymmetries during vertical jumping than those with a hamstring tendon (HT) autograft. These asymmetries may transfer into sporting movements with a greater ACL injury risk. The aim of this study was to compare between-limb asymmetries in knee mechanics and task performance during an unplanned 90° change-of-direction (CoD) task in male field sport athletes reconstructed with BPTB or HT autografts. Seventy-eight male multidirectional field sport athletes with either a BPTB (n = 39) or HT (n = 39) autograft completed maximal unplanned CoD trials in a three-dimensional motion capture laboratory at approximately 9 months post-surgery. A mixed-model 2x2 ANOVA (autograft type x limb) was used to compare variables related to ACL injury risk (e.g., internal knee moments) and performance (e.g., completion time) between autografts and limbs. Statistical parametric mapping was used for a waveform comparison throughout stance, supplemented with a discrete point analyses of peak knee moments and performance variables. Interaction effects were found at the knee joint, with BPTB demonstrating greater asymmetries than HT in knee extension moment (p < 0.001); resultant ground reaction force (p < 0.001); peak knee external rotation moment (p = 0.04); and knee adduction (p = 0.05), medial rotation (p < 0.001), and flexion (p < 0.001) angles. No differences were found between autografts for any performance variable. BPTB demonstrated greater lower-limb biomechanical asymmetries than HT during CoD, which may influence knee loading and longer-term outcomes and should thus be targeted during rehabilitation prior to return to play.

Association between biological maturation and anterior cruciate ligament injury risk factors during cutting.

BACKGROUND: Adolescent females are particularly susceptible to suffering anterior cruciate ligament (ACL) injuries, likely influenced by well-established maturational changes. This study investigated ACL biomechanical injury risk factors and their association with biological maturation in females. METHODS: Thirty-five adolescent females (15±1 year) completed a series of maximum-effort 90° unanticipated cutting maneuvers. Established biomechanical ACL injury risk factors (including external knee abduction moments, knee abduction, hip abduction, knee flexion, ground reaction force) were derived from an optoelectronic motion analysis system and force platforms, with inter-limb asymmetries in these risk factors also computed. Biological maturation (percentage of predicted adult stature) was assessed using validated regression equations, incorporating anthropometric measures of participants and their biological parents. RESULTS: Significant bilateral asymmetries were observed with higher peak external knee abduction moments, higher ground reaction forces and less knee flexion (from 0-18% and 30-39% of contact) during the non-dominant vs. dominant cuts (effect sizes =0.36, 0.63 and 0.50, respectively). Maturation did not appear to influence these asymmetries; however, less hip abduction was observed (e.g., 21-51% of contact for dominant cuts) in more biologically-mature females. CONCLUSIONS: These results highlight a potential maturation-related change in cutting technique that may explain the apparent heightened ACL injury risk in this population. As females mature, training targeted at neuromuscular control of hip abductor (e.g. gluteal) muscle groups could potentially mitigate ACL injury risk.

Hepatic Sensing Loop Regulates PCSK9 Secretion in Response to Inhibitory Antibodies.

BACKGROUND: Monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9i) lower LDL-C by up to 60% and increase plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels by 10-fold. OBJECTIVES: The authors studied the reasons behind the robust increase in plasma PCSK9 levels by testing the hypothesis that mechanisms beyond clearance via the low-density lipoprotein receptor (LDLR) contribute to the regulation of cholesterol homeostasis. METHODS: In clinical cohorts, animal models, and cell-based studies, we measured kinetic changes in PCSK9 production and clearance in response to PCSK9i. RESULTS: In a patient cohort receiving PCSK9i therapy, plasma PCSK9 levels rose 11-fold during the first 3 months and then plateaued for 15 months. In a cohort of healthy volunteers, a single injection of PCSK9i increased plasma PCSK9 levels within 12 hours; the rise continued for 9 days until it plateaued at 10-fold above baseline. We recapitulated the rapid rise in PCSK9 levels in a mouse model, but only in the presence of LDLR. In vivo turnover and in vitro pulse-chase studies identified 2 mechanisms contributing to the rapid increase in plasma PCSK9 levels in response to PCSK9i: 1) the expected delayed clearance of the antibody-bound PCSK9; and 2) the unexpected post-translational increase in PCSK9 secretion. CONCLUSIONS: PCSK9 re-entry to the liver via LDLR triggers a sensing loop regulating PCSK9 secretion. PCSK9i therapy enhances the secretion of PCSK9, an effect that contributes to the increased plasma PCSK9 levels in treated subjects.

Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches.

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.

Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22.

Abnormalities of the peripheral myelin protein 22 (PMP22) gene, including duplication, deletion and point mutations are a major culprit in Type 1 Charcot-Marie-Tooth (CMT) diseases. The complete absence of PMP22 alters cholesterol metabolism in Schwann cells, which likely contributes to myelination deficits. Here, we examined the subcellular trafficking of cholesterol in distinct models of PMP22-linked neuropathies. In Schwann cells from homozygous Trembler J (TrJ) mice carrying a Leu16Pro mutation, cholesterol was retained with TrJ-PMP22 in the Golgi, alongside a corresponding reduction in its plasma membrane level. PMP22 overexpression, which models CMT1A caused by gene duplication, triggered cholesterol sequestration to lysosomes, and reduced ATP-binding cassette transporter-dependent cholesterol efflux. Conversely, lysosomal targeting of cholesterol by U18666A treatment increased wild type (WT)-PMP22 levels in lysosomes. Mutagenesis of a cholesterol recognition motif, or CRAC domain, in human PMP22 lead to increased levels of PMP22 in the ER and Golgi compartments, along with higher cytosolic, and lower membrane-associated cholesterol. Importantly, cholesterol trafficking defects observed in PMP22-deficient Schwann cells were rescued by WT but not CRAC-mutant-PMP22. We also observed that myelination deficits in dorsal root ganglia explants from heterozygous PMP22-deficient mice were improved by cholesterol supplementation. Collectively, these findings indicate that PMP22 is critical in cholesterol metabolism, and this mechanism is likely a contributing factor in PMP22-linked hereditary neuropathies. Our results provide a basis for understanding how altered expression of PMP22 impacts cholesterol metabolism.

Unusual responses to PCSK9 inhibitors in a clinical cohort utilizing a structured follow-up protocol.

OBJECTIVE: To characterize unusual responses to PCSK9 inhibitor (PCSK9i) therapy in a real-world setting, given their extremely low prevalence in clinical trials. METHODS: A retrospective study of patients seen in a structured academic PCSK9i clinic who had LDL-C measurements before and after initiation of PCSK9i (up to 12 months). Unusual response was defined as: (1) no response: no changes in LDL-C level at all time points; (2) delayed response: <30% LDL-C reduction by the third dose, but achieving this threshold at a later time; (3) reduced response: <30% LDL-C reduction at all time points; and (4) lost response: ≥30% LDL-C reduction by the third dose, but displaying <30% reduction at a later time. RESULTS: Of the 411 patients meeting inclusion criteria, 54 were initially classified as unusual responders. After excluding those not adherent to prescribed interventions, 31 patients (7.5%) were classified as true unusual responders. These included: 2 with no response, 12 with delayed response, 3 with reduced response, 6 with delayed or reduced response, 4 with lost response, and 4 with delayed and lost response. Response to PCSK9i therapy at all time points revealed higher on-treatment LDL-C values (94-100 vs. 47-51 â€‹mg/dL, p â€‹< â€‹0.001) and lower degree of percent reduction in LDL-C (23.3-34% vs. 61.1-64.5%, p â€‹< â€‹0.001) in the unusual versus usual responders. Lipoprotein (a) (Lp[a]) values were consistently higher in the unusual responders (81-92.5 vs. 28.5-52 â€‹mg/dL, p â€‹< â€‹0.01). Fold change in post-versus pre-treatment PCSK9 plasma results was similar between the two cohorts (p â€‹> â€‹0.05), suggesting that unusual responses were not due to insufficient plasma PCSK9 blockade. Multiple logistic regression analysis identified clinical FH (OR 2.9, 95% CI 1.27-7.24) and no ezetimibe therapy (OR 0.334, 95% CI 0.150-0.728) as factors related to true unusual response. CONCLUSIONS: Unusual responses to PCSK9i in a clinical cohort are more common than reported in clinical trials. Of the suspected unusual responders, nearly half were the result of adherence issues, and thus careful medication reconciliation should be the first step in diagnosing an unusual response.

A neutral lipid-enriched diet improves myelination and alleviates peripheral nerve pathology in neuropathic mice.

Charcot-Marie-Tooth (CMT) diseases comprise a genetically heterogeneous group of hereditary peripheral neuropathies. Trembler J (TrJ) mice carry a spontaneous mutation in peripheral myelin protein 22 (PMP22) and model early-onset, severe CMT type 1E disease. Recent studies indicate that phospholipid substitution, or cholesterol-enriched diet, benefit myelinated nerves, however such interventions have not been tested in early-onset dysmyelinating neuropathies. Here, we examined the lipid profile of peripheral nerves from 6-month-old TrJ mice with advanced neuropathy and tested the impact of a 6-week-long neutral lipid-enriched high-fat diet (HFD) on neuropathy progression in young, newly-weaned mice. Oil Red O staining showed pronounced neutral lipid accumulation in nerves from 6-month-old TrJ mice, along with elevated levels of key cholesterol and triglyceride transport proteins including apoE, LRP1 and ABCA1, compared with wild type (Wt). In young mice, the short-term HFD intervention increased serum cholesterol levels without impacting triglycerides, or body and liver weights. Tissue samples from neuropathic TrJ mice showed improvements in the maintenance of myelinated axons after the 6-week-long dietary intervention, and this effect was evident both in the sciatic and phrenic nerves. Concomitantly, aberrant Schwann cell proliferation was attenuated, as detected by reduction in mitotic markers and in c-Jun expression. Nerves from HFD-fed TrJ mice contained fewer macrophages, with a normalized count of CD11b + cells. In addition, we detected an increase in neutral lipids in the nerve endoneurium and a trend toward normalization of apoE, LRP1, and ABCA1 expression after the HFD feeding. Together, these results demonstrate the beneficial influence of a short-term neutral lipid-enriched diet on neuropathy progression in young TrJ mice and support further work in investigating the potential benefits of dietary lipids on hereditary neuropathies.

Elevated Lipoprotein(a) Levels Lower ABCA1 Cholesterol Efflux Capacity.

CONTEXT: Elevated serum lipoprotein(a) [Lp(a)] levels are associated with increased cardiovascular disease risk. ABCA1-mediated cholesterol efflux from macrophages may be an antiatherogenic process. Plasminogen (PLG) is a driver of ABCA1-mediated cholesterol efflux, and its action is inhibited by purified human Lp(a). OBJECTIVE: To determine the effects of Lp(a) in human serum on ABCA1 cholesterol efflux. METHODS: Cholesterol efflux capacity (CEC) was measured with two different cell-culture models using serum from 76 patients with either low (<50 mg/dL) or high (>50 mg/dL) Lp(a) levels. RESULTS: Using cAMP-stimulated J774 macrophages or baby hamster kidney fibroblasts overexpressing human ABCA1, we show that CEC was lower in patients with high Lp(a) levels compared with patients with low levels (-30.6%, P = 0.002 vs -24.1%, P < 0.001, respectively). Total-serum CEC negatively correlated with Lp(a) levels (r = -0.433, P = 0.0007 vs r = -0.505, P = 0.0011, respectively). These negative associations persisted after adjusting for serum cholesterol, age, sex, and statin use in a multiple linear regression model (adjusted R2 = 0.413 or 0.405, respectively) and were strengthened when further adjusting for the interaction between Lp(a) and PLG levels (adjusted R2 = 0.465 and 0.409, respectively). Total-serum and isolated Lp(a) from patients with high Lp(a) inhibited PLG-mediated ABCA1 cholesterol efflux. CONCLUSION: Total-serum CEC is reduced in patients with high Lp(a) levels. This is in part due to the inhibition of PLG-mediated ABCA1 cholesterol efflux by Lp(a). Our findings suggest an atherogenic role for Lp(a) through its ability to inhibit CEC.

PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux.

The absence of functional peripheral myelin protein 22 (PMP22) is associated with shortened lifespan in rodents and severe peripheral nerve myelin abnormalities in several species including humans. Schwann cells and nerves from PMP22 knock-out (KO) mice show deranged cholesterol distribution and aberrant lipid raft morphology, supporting an unrecognized role for PMP22 in cellular lipid metabolism. To examine the mechanisms underlying these abnormalities, we studied Schwann cells and nerves from male and female PMP22 KO mice. Whole-cell current-clamp recordings in cultured Schwann cells revealed increased membrane capacitance and decreased membrane resistance in the absence of PMP22, which was consistent with a reduction in membrane cholesterol. Nerves from PMP22-deficient mice contained abnormal lipid droplets, with both mRNA and protein levels of apolipoprotein E (apoE) and ATP-binding cassette transporter A1 (ABCA1) being highly upregulated. Despite the upregulation of ABCA1 and apoE, the absence of PMP22 resulted in reduced localization of the transporter to the cell membrane and diminished secretion of apoE. The absence of PMP22 also impaired ABCA1-mediated cholesterol efflux capacity. In nerves from ABCA1 KO mice, the expression of PMP22 was significantly elevated and the subcellular processing of the overproduced protein was aberrant. In wild-type samples, double immunolabeling identified overlapping distribution of PMP22 and ABCA1 at the Schwann cell plasma membrane and the two proteins were coimmunoprecipitated from Schwann cell and nerve lysates. Together, these results reveal a novel role for PMP22 in regulating lipid metabolism and cholesterol trafficking through functional interaction with the cholesterol efflux regulatory protein ABCA1.SIGNIFICANCE STATEMENT Understanding the subcellular events that underlie abnormal myelin formation in hereditary neuropathies is critical for advancing therapy development. Peripheral myelin protein 22 (PMP22) is an essential peripheral myelin protein because its genetic abnormalities account for ∼80% of hereditary neuropathies. Here, we demonstrate that in the absence of PMP22, the cellular and electrophysiological properties of the Schwann cells' plasma membrane are altered and cholesterol trafficking and lipid homeostasis are perturbed. The molecular mechanisms for these abnormalities involve a functional interplay among PMP22, cholesterol, apolipoprotein E, and the major cholesterol-efflux transporter protein ATP-binding cassette transporter A1 (ABCA1). These findings establish a critical role for PMP22 in the maintenance of cholesterol homeostasis in Schwann cells.

Patellar and hamstring autografts are associated with different jump task loading asymmetries after ACL reconstruction.

After anterior cruciate ligament reconstruction (ACLR), there is a higher re-injury rate to the contralateral limb in athletes who undergo surgery using a bone-patellar-tendon-bone (BPTB) autograft than using a semitendinosus and gracilis hamstring tendon (HT) autograft. This may be influenced by differing lower-limb loading asymmetries present when athletes of each graft type return to play (RTP). The aim of this study was to compare bilateral countermovement jump (CMJ) phase-specific impulse asymmetries between athletes with BPTB and HT autografts 9 months post-ACLR, and to identify the relationship between impulse and isokinetic strength asymmetries. Male field sport athletes with a BPTB (n = 22) or HT (n = 22) autograft were tested approximately 9 months post-ACLR. An uninjured control group (n = 22) was also tested on a single occasion. Phase-specific bilateral absolute impulse asymmetries were calculated during the CMJ and compared between groups using Kruskal-Wallis and post-hoc testing. A linear regression model was used to assess the relationship between impulse asymmetries and isokinetic concentric knee extensor strength asymmetries. BPTB athletes demonstrated greater impulse asymmetries than HT athletes during the eccentric (P = 0.01) and concentric (P = 0.008) phases of the jump. Isokinetic strength asymmetry was a significant predictor of CMJ concentric impulse asymmetry in both BPTB (r2  = 0.39) and HT athletes (r2  = 0.18) but not eccentric impulse asymmetry in any group. The greater loading asymmetries demonstrated by BPTB than HT athletes 9 months after ACLR may contribute to the differing incidence rates of contralateral ACL injury. The findings suggest that graft-specific loading asymmetries should be targeted during rehabilitation prior to RTP.

Application of PCSK9 Inhibitors in Practice.

PCSK9i (protein convertase subtilisin/kexin type 9 inhibitors) are set to revolutionize the treatment of hypercholesterolemia in the management of atherosclerotic risk, but numerous reports have detailed unprecedented barriers to access for these drugs. To overcome these challenges, our group created a model to facilitate provision of this new therapy for patients who qualify according to Food and Drug Administration criteria. This report details the real-world follow-up experience of PCSK9i use in a large patient cohort structured to ensure rigor in data collection, analysis, and interpretation. The 271 patients approved and actively followed in our PCSK9i clinic between July 2015 and August 2018 represent a 97% approval rate from insurance, with 28% of prescriptions requiring at least one appeal. Over 50% of patients were statin intolerant. On average, there was a median lapse of 15 days between initial visit and insurance approval. PCSK9i therapy was affordable for most patients, with an average monthly out-of-pocket expense of $58.05 (median $0). Only 2.3% of patients were unable to initiate or continue therapy because of cost. Reductions from baseline in LDL (low-density lipoprotein) cholesterol and Lp(a) (lipoprotein [a])were comparable to published reports with median reductions of 60% and 23% at 1 year, respectively. PCSK9i therapy was well-tolerated overall, though 9% of patients reported adverse events, and 5% of patients discontinued due mostly to musculoskeletal and flu-like symptoms. Our practice model demonstrates that PCSK9i therapy can be accessed easily and affordably for the majority of eligible patients, resulting in dramatic improvement in lipid profile results. Moreover, our registry data suggest that results from the prospective clinical trials of PCSK9i on LDL and Lp(a) reduction and on tolerability are applicable to a real-world cohort.

PET of Adoptively Transferred Chimeric Antigen Receptor T Cells with 89Zr-Oxine.

Chimeric antigen receptor (CAR) T cell therapy is a promising clinical approach for reducing tumor progression and prolonging patient survival. However, improvements in both the safety and the potency of CAR T cell therapy demand quantitative imaging techniques to determine the distribution of cells after adoptive transfer. The purpose of this study was to optimize 89Zr-oxine labeling of CAR T cells and evaluate PET as a platform for imaging adoptively transferred CAR T cells. Methods: CAR T cells were labeled with 0-1.4 MBq of 89Zr-oxine per 106 cells and assessed for radioactivity retention, viability, and functionality. In vivo trafficking of 89Zr-oxine-labeled CAR T cells was evaluated in 2 murine xenograft tumor models: glioblastoma brain tumors with intracranially delivered IL13Rα2-targeted CAR T cells, and subcutaneous prostate tumors with intravenously delivered prostate stem cell antigen (PSCA)-targeted CAR T cells. Results: CAR T cells were efficiently labeled (75%) and retained more than 60% of the 89Zr over 6 d. In vitro cytokine production, migration, and tumor cytotoxicity, as well as in vivo antitumor activity, were not significantly reduced when labeled with 70 kBq/106 cells. IL13Rα2-CAR T cells delivered intraventricularly were detectable by PET for at least 6 d throughout the central nervous system and within intracranial tumors. When intravenously administered, PSCA-CAR T cells also showed tumor tropism, with a 9-fold greater tumor-to-muscle ratio than for CAR-negative T cells. Conclusion:89Zr-oxine can be used for labeling and imaging CAR T cells while maintaining cell viability and function. On the basis of these studies, we conclude that 89Zr-oxine is a clinically translatable platform for real-time assessment of cell therapies.

Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of hepatic low-density lipoprotein (LDL) receptors (LDLR), thereby, decreasing hepatocyte LDL-cholesterol (LDL-C) uptake. However, it is unknown whether PCSK9 has effects on atherogenesis that are independent of lipid changes. The present study investigated the effect of human (h) PCSK9 on plasma lipids, hepatic lipogenesis, and atherosclerotic lesion size and composition in transgenic mice expressing hPCSK9 (hPCSK9tg) on wild-type (WT), LDLR⁻/⁻, or apoE⁻/⁻ background. METHODS AND RESULTS: hPCSK9 expression significantly increased plasma cholesterol (+91%), triglycerides (+18%), and apoB (+57%) levels only in WT mice. The increase in plasma lipids was a consequence of both decreased hepatic LDLR and increased hepatic lipid production, mediated transcriptionally and post-transcriptionally by PCSK9 and dependent on both LDLR and apoE. Despite the lack of changes in plasma lipids in mice expressing hPCSK9 and lacking LDLR (the main target for PCSK9) or apoE (a canonical ligand for the LDLR), hPCSK9 expression increased aortic lesion size in the absence of apoE (268 655 ± 97 972 µm² in hPCSK9tg/apoE⁻/⁻ vs. 189 423 ± 65 700 µm(2) in apoE⁻/⁻) but not in the absence of LDLR. Additionally, hPCSK9 accumulated in the atheroma and increased lesion Ly6C(hi) monocytes (by 21%) in apoE⁻/⁻ mice, but not in LDLR⁻/⁻ mice. CONCLUSIONS: PCSK9 increases hepatic lipid and lipoprotein production via apoE- and LDLR-dependent mechanisms. However, hPCSK9 also accumulate in the artery wall and directly affects atherosclerosis lesion size and composition independently of such plasma lipid and lipoprotein changes. These effects of hPCSK9 are dependent on LDLR but are independent of apoE.

Functional imaging of human epidermal growth factor receptor 2-positive metastatic breast cancer using (64)Cu-DOTA-trastuzumab PET.

UNLABELLED: Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. METHODS: Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). RESULTS: Liver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from (64)Cu-DOTA-trastuzumab was similar to (18)F-FDG. CONCLUSION: (64)Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a (64)Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. (64)Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.