A comparative study of mitochondrial respiration in circulating blood cells and skeletal muscle fibers in women.

Skeletal muscle mitochondrial respiration is thought to be altered in obesity, insulin resistance, and type 2 diabetes; however, the invasive nature of tissue biopsies is an important limiting factor for studying mitochondrial function. Recent findings suggest that bioenergetics profiling of circulating cells may inform on mitochondrial function in other tissues in lieu of biopsies. Thus, we sought to determine whether mitochondrial respiration in circulating cells [peripheral blood mononuclear cells (PBMCs) and platelets] reflects that of skeletal muscle fibers derived from the same subjects. PBMCs, platelets, and skeletal muscle (vastus lateralis) samples were obtained from 32 young (25-35 yr) women of varying body mass indexes. With the use of extracellular flux analysis and high-resolution respirometry, mitochondrial respiration was measured in intact blood cells as well as in permeabilized cells and permeabilized muscle fibers. Respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets. In a subset of samples (n = 12-13) with permeabilized blood cells available, raw measures of substrate (pyruvate, malate, glutamate, and succinate)-driven respiration did not correlate between permeabilized muscle (per mg tissue) and permeabilized PBMCs (per 106 cells); however, complex I leak and oxidative phosphorylation coupling efficiency correlated between permeabilized platelets and muscle (Spearman's ρ = 0.64, P = 0.030; Spearman's ρ = 0.72, P = 0.010, respectively). Our data indicate that bioenergetics phenotypes in circulating cells cannot recapitulate muscle mitochondrial function. Select circulating cell bioenergetics phenotypes may possibly inform on overall metabolic health, but this postulate awaits validation in cohorts spanning a larger range of insulin resistance and type 2 diabetes status.

Notch and wingless signaling cooperate in regulation of dendritic cell differentiation.

Dendritic cell (DC) differentiation is regulated by stroma via a network of soluble and cell-bound factors. Notch is one of the major elements of this network. Its role in DC differentiation, however, is controversial. Here, we demonstrate that activation of Notch signaling in hematopoietic progenitor cells (HPCs) promoted differentiation of conventional DCs via activation of the canonical Wingless (Wnt) pathway. Inhibition of the Wnt pathway abrogated the effect of Notch on DC differentiation. The fact that activation of the Wnt pathway in Notch-1-deficient embryonic stem cells restored DC differentiation indicates that Wnt signaling is downstream of the Notch pathway in regulating DC differentiation. Notch signaling activated the Wnt pathway in HPCs via expression of multiple members of the Frizzled family of Wnt receptors, which was directly regulated by the CSL (RPB-Jkappa) transcription factor. Thus, these data suggest a model of DC differentiation via cooperation between Wnt and Notch pathways.

Mitochondrial respiratory capacity and coupling control decline with age in human skeletal muscle.

Mitochondrial health is critical to physiological function, particularly in tissues with high ATP turnover, such as striated muscle. It has been postulated that derangements in skeletal muscle mitochondrial function contribute to impaired physical function in older adults. Here, we determined mitochondrial respiratory capacity and coupling control in skeletal muscle biopsies obtained from young and older adults. Twenty-four young (28 ± 7 yr) and thirty-one older (62 ± 8 yr) adults were studied. Mitochondrial respiration was determined in permeabilized myofibers from the vastus lateralis after the addition of substrates oligomycin and CCCP. Thereafter, mitochondrial coupling control was calculated. Maximal coupled respiration (respiration linked to ATP production) was lower in muscle from older vs. young subjects (P < 0.01), as was maximal uncoupled respiration (P = 0.06). Coupling control in response to the ATP synthase inhibitor oligomycin was lower in older adults (P < 0.05), as was the mitochondria flux control ratio, coupled respiration normalized to maximal uncoupled respiration (P < 0.05). Calculation of respiratory function revealed lower respiration linked to ATP production (P < 0.001) and greater reserve respiration (P < 0.01); i.e., respiratory capacity not used for phosphorylation in muscle from older adults. We conclude that skeletal muscle mitochondrial respiratory capacity and coupling control decline with age. Lower respiratory capacity and coupling efficiency result in a reduced capacity for ATP production in skeletal muscle of older adults.

Elevated LDL-C, high blood pressure, and low peak V ˙ O2 associate with platelet mitochondria function in children-The Arkansas Active Kids Study.

Purpose: To evaluate the association of platelet (PL) mitochondria respiration with markers of cardiovascular health in children ages 7-10 years. Methods: PL mitochondrial respiration (n = 91) was assessed by high resolution respirometry (HRR): Routine (R) respiration, complex (C) I linked respiration (CI), and maximal uncoupled electron transport capacity of CII (CIIE) were measured. The respiratory control ratio (RCR) was calculated as the ratio of maximal oxidative phosphorylation capacity of CI and CI leak respiration (PCI/LCI). Peak V ˙ O2 (incremental bike test) and body composition (dual-energy X-ray absorptiometry) were measured. Multiple generalized linear regression analysis was used to model the association of measures by HRR with variables of interest: adiposity, low-density lipoprotein (LDL-C) and triglyceride (TG) status (normal vs. elevated) HOMA2-IR, blood pressure status (normal vs. high), and demographics. Results: R and CI-linked respiration positively associated with adiposity, high blood pressure (HBP), and peak V ˙ O2. R and CI-linked respiration had inverse association with age and elevated LDL-C. CIIE was higher in children with elevated LDL-C (log-ß = -0.54, p = 0.010). HBP and peak V ˙ O2 interacted in relation to RCR (log-ß = -0.01, p = 0.028). Specifically, RCR was lowest among children with HBP and low aerobic capacity (i.e., mean peak V ˙ O2 -1SD). HOMA2-IR did not associate with measures of PL mitochondria respiration. Conclusion: In PL, R and CI-linked mitochondrial respiration directly associate with adiposity, peak V ˙ O2 and HBP. Elevated LDL-C associates with lower CI-linked respiration which is compensated by increasing CII respiration. PL bioenergetics phenotypes in children associate with whole-body metabolic health status.

ChatGPT and Bard exhibit spontaneous citation fabrication during psychiatry literature search.

ChatGPT (Generative Pre-Trained Transformer) is a large language model (LLM), which comprises a neural network that has learned information and patterns of language use from large amounts of text on the internet. ChatGPT, introduced by OpenAI, responds to human queries in a conversational manner. Here, we aimed to assess whether ChatGPT could reliably produce accurate references to supplement the literature search process. We describe our March 2023 exchange with ChatGPT, which generated thirty-five citations, two of which were real. 12 citations were similar to actual manuscripts (e.g., titles with incorrect author lists, journals, or publication years) and the remaining 21, while plausible, were in fact a pastiche of multiple existent manuscripts. In June 2023, we re-tested ChatGPT's performance and compared it to that of Google's GPT counterpart, Bard 2.0. We investigated performance in English, as well as in Spanish and Italian. Fabrications made by LLMs, including erroneous citations, have been called "hallucinations"; we discuss reasons for which this is a misnomer. Furthermore, we describe potential explanations for citation fabrication by GPTs, as well as measures being taken to remedy this issue, including reinforcement learning. Our results underscore that output from conversational LLMs should be verified.

Linguistic correlates of suicidal ideation in youth at clinical high-risk for psychosis.

Suicidal ideation (SI) is prevalent among individuals at clinical high-risk for psychosis (CHR). Natural language processing (NLP) provides an efficient method to identify linguistic markers of suicidality. Prior work has demonstrated that an increased use of "I", as well as words with semantic similarity to "anger", "sadness", "stress" and "lonely", are correlated with SI in other cohorts. The current project analyzes data collected in an SI supplement to an NIH R01 study of thought disorder and social cognition in CHR. This study is the first to use NLP analyses of spoken language to identify linguistic correlates of recent suicidal ideation among CHR individuals. The sample included 43 CHR individuals, 10 with recent suicidal ideation and 33 without, as measured by the Columbia-Suicide Severity Rating Scale, as well as 14 healthy volunteers without SI. NLP methods include part-of-speech (POS) tagging, a GoEmotions-trained BERT Model, and Zero-Shot Learning. As hypothesized, individuals at CHR for psychosis who endorsed recent SI utilized more words with semantic similarity to "anger" compared to those who did not. Words with semantic similarity to "stress", "loneliness", and "sadness" were not significantly different between the two CHR groups. Contrary to our hypotheses, CHR individuals with recent SI did not use the word "I" more than those without recent SI. As anger is not characteristic of CHR, findings have implications for the consideration of subthreshold anger-related sentiment in suicidal risk assessment. As NLP is scalable, findings suggest that language markers may improve suicide screening and prediction in this population.

Differential Expression of Anomalous Self-Experiences in Spontaneous Speech in Clinical High-Risk and Early-Course Psychosis Quantified by Natural Language Processing.

BACKGROUND: Basic self-disturbance, or anomalous self-experiences (ASEs), is a core feature of the schizophrenia spectrum. We propose a novel method of natural language processing to quantify ASEs in spoken language by direct comparison to an inventory of self-disturbance, the Inventory of Psychotic-Like Anomalous Self-Experiences (IPASE). We hypothesized that there would be increased similarity in open-ended speech to the IPASE items in individuals with early-course psychosis (PSY) compared with healthy individuals, with clinical high-risk (CHR) individuals intermediate in similarity. METHODS: Open-ended interviews were obtained from 170 healthy control participants, 167 CHR participants, and 89 PSY participants. We calculated the semantic similarity between IPASE items and "I" sentences from transcribed speech samples using S-BERT (Sentence Bidirectional Encoder Representation from Text). Kolmogorov-Smirnov tests were used to compare distributions across groups. A nonnegative matrix factorization of cosine similarity was performed to rank IPASE items. RESULTS: Spoken language of CHR individuals had the greatest semantic similarity to IPASE items when compared to both healthy control (s = 0.44, p < 10-14) and PSY (s = 0.36, p < 10-6) individuals, while IPASE scores were higher among PSY than CHR group participants. In addition, the nonnegative matrix factorization approach produced a data-driven domain that differentiated the CHR group from the others. CONCLUSIONS: We found that open-ended interviews elicited language with increased semantic similarity to the IPASE by participants in the CHR group compared with patients with psychosis. This demonstrates the utility of these methods for differentiating patients from healthy control participants. This complementary approach has the capacity to scale to large studies investigating phenomenological features of schizophrenia and potentially other clinical populations.

Comparison of Arterial-Venous Balance and Tracer Incorporation Methods for Measuring Muscle Fractional Synthesis and Fractional Breakdown Rates.

Loss of muscle mass in response to injury or immobilization impairs functional capacity and metabolic health, thus hindering rehabilitation. Stable isotope techniques are powerful in determining skeletal muscle protein fluxes. Traditional tracer incorporation methods to measure muscle protein synthesis and breakdown are cumbersome and invasive to perform in vulnerable populations such as children. To circumvent these issues, a two-bolus stable isotope amino acid method has been developed; although, measured rates of protein synthesis and breakdown have not been validated simultaneously against an accepted technique such as the arterial-venous balance method. The purpose of the current analysis was to provide preliminary data from the simultaneous determination of the arteriovenous balance and two-bolus tracer incorporation methods on muscle fractional synthesis and breakdown rates in children with burns. Five were administered a primed-constant infusion of L-[15N]Threonine for 180 minutes (Prime: 8 µmol/kg; constant: 0.1 µmol·kg-1·minute-1). At 120 and 150 minutes, bolus injections of L-[ring-13C6]Phenylalanine and L-[15N]Phenylalanine (50 µmol/kg each) were administered, respectively. Blood and muscle tissue samples were collected to assess mixed muscle protein synthesis and breakdown rates. The preliminary results from this study indicate that there is no difference in either fractional synthesis rate (mean ± SD; arteriovenous balance: 0.19 ± 0.17 %/h; tracer incorporation: 0.14 ± 0.08 %/h; P = .42) or fractional breakdown rate (arteriovenous balance: 0.29 ± 0.22 %/h; tracer incorporation: 0.23 ± 0.14 %/h; P = .84) between methods. These data support the validity of both methods in quantifying muscle amino acid kinetics; however, the results are limited and adequately powered research is still required.

Breastfeeding duration modifies the association between maternal weight status and offspring dietary palmitate oxidation.

BACKGROUND: Offspring of obese rodents develop a metabolic phenotype that favors fat deposition. Data regarding the impact of maternal obesity programing of offspring fuel usage in humans is scarce. OBJECTIVE: The objective of this study was to explore the association between maternal weight status and dietary palmitate oxidation (DPO) in 2-y-old offspring, taking into consideration potential confounders and modifiers. METHODS: Women (n = 56) were enrolled by the first trimester of gestation. Maternal physical activity (PA; measured with accelerometers) at enrollment and gestational weight gain (GWG) were measured. Offspring sex, race, and breastfeeding (BF) duration were self-reported. Human milk (HM) composition was determined at 6 mo postpartum. At age 2 y, dietary quality [healthy eating index (HEI)] and parental feeding practices [Child Feeding Questionnaire (CFQ)] were assessed. DPO in 2-y-olds (2-yo-DPO) was measured using deuterated palmitic acid. Generalized linear regression analysis was used to model the associations of 2-yo-DPO with maternal weight status [normal weight (NW), BMI <25 (in kg/m2) compared with excessive weight (EW), BMI ≥25]. RESULTS: DPO was higher in offspring of women with EW compared with NW (2.1 ± 1.2%/h compared with 1.4 ± 0.7%/h, P = 0.03). Maternal weight status interacted with BF duration in association with 2-yo-DPO (log ß: 0.05, P = 0.04). Specifically, 2-yo-DPO was higher in the EW compared with NW group if BF duration was ≥9 mo. HM insulin (log ß: 0.35, P = 0.002) and HM leptin (log ß: 0.81, P = 0.001) concentrations directly associated with 2-yo-DPO. PA (log ß: 0.06, P = 0.013), parental feeding restriction (log ß: 0.05, P < 0.0001), and male sex (log ß: 0.54, P < 0.001) were positively associated with 2-yo-DPO. HEI was negatively associated with 2-yo-DPO (log ß:-0.03, P < 0.0001). CONCLUSIONS: Higher 2-yo-DPO in offspring of women with EW compared with NW were driven by BF duration. Higher HM insulin and leptin concentrations in women with EW may explain these finding. More studies are needed to confirm these results. This trial was registered at clinicaltrials.gov as NCT03281850.

Real-World Data of Palbociclib in Combination With Endocrine Therapy for the Treatment of Metastatic Breast Cancer in Men.

This report examined the benefits and risks of palbociclib plus endocrine therapy (ET) in men with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC). Palbociclib was evaluated using three independent data sources: real-world data from pharmacy and medical claims, a de-identified real-world data source derived from electronic health records (EHRs), and a global safety database. From medical and pharmacy records, 1,139 men with MBC were identified; in the first-line setting, median duration of treatment was longer with palbociclib plus ET (n = 37, 8.5 months, 95% confidence interval (CI), 4.4-13.0) than ET alone (n = 214, 4.3 months, 95% CI, 3.0-5.7) and specifically, was longer with palbociclib plus letrozole (n = 26, 9.4 months, 95% CI, 4.4-14.0) than letrozole alone (n = 63, 3.0 months, 95% CI, 1.8-4.8). In the EHR-derived database, 59 men received treatment for MBC; real-world response across all lines of therapy in the metastatic setting was reported in 4 of 12 patients (33.3%) in the palbociclib plus ET group vs. 1 of 8 (12.5%) patients in the ET group. Review of the global safety database did not identify any new safety signals in palbociclib-treated men. Real-world data indicated that men with MBC benefit from palbociclib plus ET, with a safety profile consistent with previous observations in women with MBC. Collective data on palbociclib in women and men in this report, including clinical trial data, real-world data, and a well-established risk/benefit profile, led to US approval of an expansion of the palbociclib indication to include men with MBC.

Mechanism regulating reactive oxygen species in tumor-induced myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network described in cancer and many other pathological conditions. Recent studies have demonstrated that one of the major mechanisms of MDSC-induced immune suppression is mediated by reactive oxygen species (ROS). However, the mechanism of this phenomenon remained unknown. In this study, we observed a substantial up-regulation of ROS by MDSC in all of seven different tumor models and in patients with head and neck cancer. The increased ROS production by MDSC is mediated by up-regulated activity of NADPH oxidase (NOX2). MDSC from tumor-bearing mice had significantly higher expression of NOX2 subunits, primarily p47(phox) and gp91(phox), compared with immature myeloid cells from tumor-free mice. Expression of NOX2 subunits in MDSC was controlled by the STAT3 transcription factor. In the absence of NOX2 activity, MDSC lost the ability to suppress T cell responses and quickly differentiated into mature macrophages and dendritic cells. These findings expand our fundamental understanding of the biology of MDSC and may also open new opportunities for therapeutic regulation of these cells in cancer.

Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

A Phase I trial of talazoparib in patients with advanced hematologic malignancies.

AIM: The objective of this study was to establish the maximum tolerated dose (MTD), safety, pharmacokinetics, and anti-leukemic activity of talazoparib. PATIENTS & METHODS: This Phase I, two-cohort, dose-escalation trial evaluated talazoparib monotherapy in advanced hematologic malignancies (cohort 1: acute myeloid leukemia/myelodysplastic syndrome; cohort 2: chronic lymphocytic leukemia/mantle cell lymphoma). RESULTS: Thirty-three (cohort 1: n = 25; cohort 2: n = 8) patients received talazoparib (0.1-2.0 mg once daily). The MTD was exceeded at 2.0 mg/day in cohort 1 and at 0.9 mg/day in cohort 2. Grade ≥3 adverse events were primarily hematologic. Eighteen (54.5%) patients reported stable disease. CONCLUSION: Talazoparib is relatively well tolerated in hematologic malignancies, with a similar MTD as in solid tumors, and shows preliminary anti leukemic activity.Clinical trial registration: NCT01399840 (ClinicalTrials.gov).

Antibody ligation of murine Ly-6G induces neutropenia, blood flow cessation, and death via complement-dependent and independent mechanisms.

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.

Neonatal diet impacts liver mitochondrial bioenergetics in piglets fed formula or human milk.

BACKGROUND: Neonatal diet impacts many physiological systems and can modify risk for developing metabolic disease and obesity later in life. Less well studied is the effect of postnatal diet (e.g., comparing human milk (HM) or milk formula (MF) feeding) on mitochondrial bioenergetics. Such effects may be most profound in splanchnic tissues that would have early exposure to diet-associated or gut microbe-derived factors. METHODS: To address this question, we measured ileal and liver mitochondrial bioenergetics phenotypes in male piglets fed with HM or MF from day 2 to day 21 age. Ileal and liver tissue were processed for mitochondrial respiration (substrate only [pyruvate, malate, glutamate], substrate + ADP, and proton "leak" post-oligomycin; measured by Oroboros methods), mitochondrial DNA (mtDNA) and metabolically-relevant gene expression analyses. RESULTS: No differences between the diet groups were observed in mitochondrial bioenergetics indices in ileal tissue. In contrast, ADP-dependent liver Complex I-linked OXPHOS capacity and Complex I + II-linked OXPHOS capacity were significantly higher in MF animals relative to HM fed piglets. Interestingly, p53, Trap1, and Pparß transcript abundances were higher in MF-fed relative to HM-fed piglets in the liver. Mitochondrial DNA copy numbers (normalized to nuclear DNA) were similar within-tissue regardless of postnatal diet, and were ~ 2-3 times higher in liver vs. ileal tissue. CONCLUSION: While mechanisms remain to be identified, the data indicate that neonatal diet can significantly impact liver mitochondrial bioenergetics phenotypes, even in the absence of a change in mtDNA abundance. Since permeabilized liver mitochondrial respiration was increased in MF piglets only in the presence of ADP, it suggests that formula feeding led to a higher ATP turnover. Specific mechanisms and signals involved with neonatal diet-associated differences in liver bioenergetics remain to be elucidated.

Concordance of real-world versus conventional progression-free survival from a phase 3 trial of endocrine therapy as first-line treatment for metastatic breast cancer.

There is growing interest in leveraging real-world data to complement knowledge gained from randomized clinical trials and inform the design of prospective randomized studies in oncology. The present study compared clinical outcomes in women with metastatic breast cancer who received letrozole as first-line monotherapy in oncology practices across the United States versus patients in the letrozole-alone cohort of the PALOMA-2 phase 3 trial. The real-world cohort (N = 107) was derived from de-identified patient data from the Flatiron Health electronic health record database. The clinical trial cohort (N = 222) comprised postmenopausal women in the letrozole-alone arm of PALOMA-2. Patients in the real-world cohort received letrozole monotherapy per labeling and clinical judgment; patients in PALOMA-2 received letrozole 2.5 mg/d, continuous. Real-world survival and response rates were based on evidence of disease burden curated from clinician notes, radiologic reports, and pathology reports available in the electronic health record. Progression-free survival and objective response rate in PALOMA-2 were based on Response Evaluation Criteria in Solid Tumors v1.1. Concordance of survival and response rates were retrospectively assessed using inverse probability of treatment weighting-adjusted Cox regression analysis. Inverse probability of treatment weighting-adjusted Cox regression results showed similar median progression-free survival in the real-world and PALOMA-2 cohorts (18.4 and 16.6 months, respectively): the hazard ratio using real-world data as reference was 1.04 (95% CI, 0.69-1.56). No significant difference was observed in response rates: 41.8% in the real-world cohort vs 39.4% in the PALOMA-2 cohort (odds ratio using real-world data as reference: 0.91 [95% CI, 0.57-1.44]). These findings indicate that data abstracted from electronic health records with proper quality controls can yield meaningful information on clinical outcomes. These data increase confidence in the use of real-world assessments of progression and response as efficacy endpoints. Trial registration NCT01740427; Funding: Pfizer.

Metabolic Effects of Late Dinner in Healthy Volunteers-A Randomized Crossover Clinical Trial.

CONTEXT: Consuming calories later in the day is associated with obesity and metabolic syndrome. We hypothesized that eating a late dinner alters substrate metabolism during sleep in a manner that promotes obesity. OBJECTIVE: The objective of this work is to examine the impact of late dinner on nocturnal metabolism in healthy volunteers. DESIGN AND SETTING: This is a randomized crossover trial of late dinner (LD, 22:00) vs routine dinner (RD, 18:00), with a fixed sleep period (23:00-07:00) in a laboratory setting. PARTICIPANTS: Participants comprised 20 healthy volunteers (10 male, 10 female), age 26.0 ± 0.6 years, body mass index 23.2 ±â€…0.7 kg/m2, accustomed to a bedtime between 22:00 and 01:00. INTERVENTIONS: An isocaloric macronutrient diet was administered on both visits. Dinner (35% daily kcal, 50% carbohydrate, 35% fat) with an oral lipid tracer ([2H31] palmitate, 15 mg/kg) was given at 18:00 with RD and 22:00 with LD. MAIN OUTCOME MEASURES: Measurements included nocturnal and next-morning hourly plasma glucose, insulin, triglycerides, free fatty acids (FFAs), cortisol, dietary fatty acid oxidation, and overnight polysomnography. RESULTS: LD caused a 4-hour shift in the postprandial period, overlapping with the sleep phase. Independent of this shift, the postprandial period following LD was characterized by higher glucose, a triglyceride peak delay, and lower FFA and dietary fatty acid oxidation. LD did not affect sleep architecture, but increased plasma cortisol. These metabolic changes were most pronounced in habitual earlier sleepers determined by actigraphy monitoring. CONCLUSION: LD induces nocturnal glucose intolerance, and reduces fatty acid oxidation and mobilization, particularly in earlier sleepers. These effects might promote obesity if they recur chronically.

Complement is an essential component of the immune response to adeno-associated virus vectors.

Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-kappaB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1beta (IL-1beta), IL-8, and MIP-1beta. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.

Overcoming immune tolerance against multiple myeloma with lentiviral calnexin-engineered dendritic cells.

The key to successful cancer immunotherapy is to induce an effective anticancer immunity that will overcome the acquired cancer-specific immune tolerance. In this study, we found that dendritic cells (DCs) from multiple myeloma (MM) patients suppressed rather than induced a cancer cell-specific immune response. We demonstrated that CD4(+)CD25(high) T cells from MM patients suppressed the proliferation of activated peripheral blood lymphocytes. Further analysis illustrated that MM cell lysates or MM-specific idiotype immunoglobulins (MM Id-Ig) specifically induced the expansion of peripheral CD4(+)CD25(high)FoxP3(high) T regulatory (Treg) cells in vitro. Supraphysiological expression of calnexin (CNX) using lentiviral (LV) vectors in DCs of MM patients overcame the immune suppression and enhanced MM-specific CD4 and CD8 T-cell responses. However, overexpression of CNX did not affect the peripheral expansion of Treg cells stimulated by MM antigens. Thus, the immune suppression effect of Treg cells in cancer patients may be overcome by improving antigen processing in DCs, which in turn may lower the activation threshold of the immune effector cells. This concept of modulating anticancer immunity by genetically engineering cancer patients' DCs may improve immunotherapeutic regimens in cancer treatment.

Treatment Patterns and Outcomes Associated With Palbociclib Plus Letrozole for Postmenopausal Women With HR+/HER2- Advanced Breast Cancer Enrolled in an Expanded Access Program.

PURPOSE: To evaluate treatment patterns and clinical outcomes of patients who received palbociclib in combination with letrozole in any line of therapy for treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer in an expanded access program (EAP) in the United States. PATIENTS AND METHODS: A retrospective chart review study was conducted of patients previously enrolled in the palbociclib EAP. Complete data from time of initial diagnosis of metastatic breast cancer until date of chart abstraction were obtained. Clinical outcomes as assessed by site investigators included clinical benefit rate, progression-free survival, and overall survival. Survival was descriptively assessed using Kaplan-Meier methods. RESULTS: Of 238 patients enrolled in the EAP, data from 126 patients were included. Median age was 62.5 years at EAP enrollment; 25% had de novo metastatic disease. Visceral disease was present in 71% of patients. The disease of most patients was heavily pretreated; nearly 60% of patients had received 3 or more prior lines for metastatic disease before initiating palbociclib + letrozole therapy. Most patients (87%) had received prior endocrine therapy, and 68% had received prior chemotherapy for metastatic disease. Patients with prior endocrine therapy for metastatic disease had a clinical benefit rate of 30%, while those with prior chemotherapy had a 26% clinical benefit rate. Patients receiving 2 or more prior lines had 6- and 12-month progression-free survival rates of 35% and 21%, respectively, and 12- and 24-month overall survival rates of 62% and 35%, respectively. CONCLUSION: Most patients derived benefit from palbociclib + letrozole treatment despite having received multiple prior treatment lines for metastatic disease.