Genetic reversal of the globin switch concurrently modulates both fetal and sickle hemoglobin and reduces red cell sickling.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCLshmiR, median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects.

Ionophore-mediated swelling of erythrocytes as a therapeutic mechanism in sickle cell disease.

Sickle cell disease (SCD) is characterized by sickle hemoglobin (HbS) which polymerizes under deoxygenated conditions to form a stiff, sickled erythrocyte. The dehydration of sickle erythrocytes increases intracellular HbS concentration and the propensity of erythrocyte sickling. Prevention of this mechanism may provide a target for potential SCD therapy investigation. Ionophores such as monensin can increase erythrocyte sodium permeability by facilitating its transmembrane transport, leading to osmotic swelling of the erythrocyte and decreased hemoglobin concentration. In this study, we treated 13 blood samples from patients with SCD with 10 nM of monensin ex vivo. We measured changes in cell volume and hemoglobin concentration in response to monensin treatment, and we perfused treated blood samples through a microfluidic device that permits quantification of blood flow under controlled hypoxia. Monensin treatment led to increases in cell volume and reductions in hemoglobin concentration in most blood samples, though the degree of response varied across samples. Monensin-treated samples also demonstrated reduced blood flow impairment under hypoxic conditions relative to untreated controls. Moreover, there was a significant correlation between the improvement in blood flow and the decrease in hemoglobin concentration. Thus, our results demonstrate that a reduction in intracellular HbS concentration by osmotic swelling improves blood flow under hypoxic conditions. Although the toxicity of monensin will likely prevent it from being a viable clinical treatment, these results suggest that osmotic swelling should be investigated further as a potential mechanism for SCD therapy.

Effects of High-Intensity Interval and Moderate-Intensity Continuous Exercise on Physical Activity and Sedentary Behavior Levels in Inactive Obese Males: A Crossover Trial.

A total of 17 participants (30.2 ± 4.8 years; 35.4 ± 4 kg/m2; 38.2 ± 3.6 % body fat) were enrolled in this randomized crossover trial to analyze the effects of a single session of high-intensity interval (HIIE) and moderate-intensity continuous exercise (MICE) on the physical activity and sedentary behavior levels in inactive obese males. The participants performed two exercise sessions and one control session (no exercise): i) low-volume HIIE (10 x 60 s at 90% of maximal aerobic velocity [MAV] interspaced by 60 s at 30% of MAV); ii) MICE (20 min at 70% of maximum heart rate); and iii) control (25 min in a seated position). After all sessions, the physical activity and sedentary behavior levels were monitored by accelerometer over seven consecutive days. No differences in the physical activity (activity counts, and time spent at light, moderate, and vigorous intensities) and sedentary behavior (time spent at sedentary behavior, breaks, and bouts) levels were found among the sessions (HIIE, MICE and control) (p > 0.05). In summary, a single session of HIIE and MICE does not change the physical activity and sedentary behavior levels in inactive obese males. Therefore, low-volume of both high- and moderate-intensity exercise should be considered for inactive obese males given that it does not reduce the physical activity level or increase the time spent at sedentary behavior.

Effect of Acute Dietary Nitrate Supplementation on the Post-Exercise Ambulatory Blood Pressure in Obese Males: A Randomized, Controlled, Crossover Trial.

This study aimed to verify the effect of beetroot juice on post-exercise ambulatory blood pressure (BP) in obese individuals. Fourteen non-hypertensive obese males were randomly assigned to three experimental sessions: 1) Beetroot juice with exercise (BJE, 200ml with ≈ 800mg nitrate and 40 minutes of moderate-intensity aerobic exercise at an intensity of 50% of the heart rate reserve), 2) fruit soda with exercise (FSE, 200ml of a low-nitrate drink and the same exercise session) and 3) control (CON, 200ml of water, an insignificant nitrate drink without exercise). The concentration of total nitrites and nitrates in plasma (NOx) after the drinks and the 24-hour ambulatory BP were evaluated. A two-way (condition vs. time) ANOVA for repeated measures, with a Bonferroni post hoc was used to analyze variables. The plasma NOx concentration increased significantly after ingestion of beetroot juice (from 9.9 ± 8.4 µM to 47.0 ± 16.9 µM, p < 0.001) and remained elevated until 1 hour post-intervention (54.7 ± 10.1 µM, p < 0.001), while it did not change in FSE and CON groups. The BJE session decreased ambulatory systolic BP in 5.3 mmHg (IC95%, -10.1 to -0.6, p = 0.025) in the period of 1-6 h after the BJE session compared to the CON session and reduction of 3.8 mmHg (IC95%, -7.5 to -0.007, p = 0.05) compared to the FSE session. No significant changes were observed for ambulatory diastolic BP (p > 0.05). BJE enhanced the reduction of systolic ambulatory BP up to 6 hours following a moderate-intensity aerobic exercise in obese individuals with an elevated cardiovascular risk profile.

Response of an oscillatory differential delay equation to a periodic stimulus.

Periodic hematological diseases such as cyclical neutropenia or cyclical thrombocytopenia, with their characteristic oscillations of circulating neutrophils or platelets, may pose grave problems for patients. Likewise, periodically administered chemotherapy has the unintended side effect of establishing periodic fluctuations in circulating white cells, red cell precursors and/or platelets. These fluctuations, either spontaneous or induced, often have serious consequences for the patient (e.g. neutropenia, anemia, or thrombocytopenia respectively) which exogenously administered cytokines can partially correct. The question of when and how to administer these drugs is a difficult one for clinicians and not easily answered. In this paper we use a simple model consisting of a delay differential equation with a piecewise linear nonlinearity, that has a periodic solution, to model the effect of a periodic disease or periodic chemotherapy. We then examine the response of this toy model to both single and periodic perturbations, meant to mimic the drug administration, as a function of the drug dose and the duration and frequency of its administration to best determine how to avoid side effects.

Acute Effects of High-Intensity Interval and Moderate-Intensity Continuous Exercise on GLP-1, Appetite and Energy Intake in Obese Men: A Crossover Trial.

This study investigated the effect of high-intensity interval (HIIE) and moderate-intensity continuous exercise (MICE) on glucagon-like peptide 1 (GLP-1), appetite and energy intake (EI) in obese men. In a randomized crossover trial, 12 participants (28.4 ± 2.6 years, 35.5 ± 4.5 kg/m², 39.8 ± 2.2% body fat) performed: (I) Control (CON, no exercise); (II) MICE (20 min, 70% of maximal heart rate) and (III) HIIE (10 × 1 min at 90% of maximal heart rate with 1 min recovery). GLP-1 and appetite were assessed at: (I) PRE: pre-exercise; (II) POST: immediately post-exercise; (III) POST-1 h: 1 h post-exercise. EI was assessed after an ad libitum meal offered 1 h post-exercise and over 24 h. There was a significant time × condition interaction for GLP-1 (p = 0.035). Higher GLP-1 levels in MICE vs. CON (p = 0.024) and a trend for HIIE vs. CON (p = 0.069) POST-1h was found. Hunger was reduced immediately post-HIIE compared to CON (p < 0.01), but was not sustained POST-1 h (p > 0.05). EI did not differ between the sessions 1 h post-exercise or over 24H (p > 0.05). In summary, although MICE increased GLP-1 levels POST-1h and HIIE induced a transient reduction in hunger, both exercise protocols did not impact EI in obese men.

Effects of High-Intensity Interval and Moderate-Intensity Continuous Exercise on Inflammatory, Leptin, IgA, and Lipid Peroxidation Responses in Obese Males.

Purpose: To compare the effects of a single high-intensity interval exercise session (HIIE) with a moderate-intensity continuous exercise session (MICE) on the inflammatory profile, IgA levels, and lipid peroxidation in sedentary obese males. Methods: Ten sedentary obese men (age 28.5 ± 2.7 years; BMI 35.9 ± 4.9 kg/m2; body fat 40.6 ± 2.0%) performed three experimental sessions, on separate days with 1 week wash-out period between interventions, according to a randomized order: (1) HIIE: 10 × 60 s at 90% of the HRmax alternated by 60 s of active recovery; (2) MICE: 20 min at 70% of the HRmax; (3) Rest-control. Blood and saliva samples were collected before, immediately after and 60 min after the end of each session in order to analyse serum levels of cytokines, IgA, and lipoperoxidation markers. Results: Leptin levels decreased immediately after HIIE (P = 0.033) and was different from the MICE (P = 0.025). IFN-γ levels were reduced immediately after (P = 0.032) and 60 min after HIIE (P = 0.003) compared to baseline, and it also increased IL-4 levels immediately after exercise (P = 0.007) compared to resting values. MICE promoted an increase in IFN-γ levels immediately after exercise (P = 0.025) and 60 min after exercise (P = 0.004) in relation to baseline. Both exercise conditions increased IL-6 levels up to 60 min after exercise (P < 0.05). The IFN-γ/IL-4 ratio decreased immediately after (P = 0.002) and 60 min after HIIE (P = 0.005) in relation to pre-exercise. No changes were found for IgA-S and TBARS for any of the conditions. Conclusion: A single HIIE session is able to decrease IFN-γ/IL-4 ratio, indicating an anti-inflammatory response, without alterations in the function of the mucosal immune system and lipoperoxidation. On the other hand, a brief session of MICE induced changes in the pattern of cytokines associated with increased cellular immune function.

Measurement properties of the Human Activity Profile questionnaire in hospitalized patients.

OBJECTIVE: To test the measurement properties (reproducibility, internal consistency, ceiling and floor effects, and construct validity) of the Human Activity Profile (HAP) questionnaire in hospitalized patients. METHODS: This measurement properties study recruited one-hundred patients hospitalized for less than 48h for clinical or surgical reasons. The HAP was administered at baseline and after 48h in a test-retest design). The International Physical Activity Questionnaire (IPAQ-6) was also administered at baseline, aiming to assess the construct validity. We tested the following measurement properties: reproducibility (reliability assessed by type 2,1 intraclass correlation coefficient (ICC2,1)); agreement by the standard error of measurement (SEM) and by the minimum detectable change with 90% confidence (MDC90), internal consistency by Cronbach's alpha, construct validity using a chi-square test, and ceiling and floor effects by calculating the proportion of patients who achieved the minimum or maximum scores. RESULTS: Reliability was excellent with an ICC of 0.99 (95% CI=0.98-0.99). SEM was 1.44 points (1.5% of the total score), the MDD90 was 3.34 points (3.5% of the total score) and the Cronbach's alpha was 0.93 (alpha if item deleted ranging from 0.94 to 0.94). An association was observed between patients classified by HAP and by IPAQ-6 (χ2=3.38; p=0.18). Ceiling or floor effects were not observed. CONCLUSION: The HAP shows adequate measurement properties for the assessment of the physical activity/inactivity level in hospitalized patients.