A carbohydrate beverage with additional protein calories produced significant improvements in time to fatigue and reductions in muscle damage in endurance athletes. Further research is necessary to determine whether these effects were the result of higher total caloric content of the CHO+P beverage or due to specific protein-mediated mechanisms.

We validated whole body composition estimates from dual-energy X-ray absorptiometry (DEXA) against estimates from a four-component model to determine whether accuracy is affected by gender, race, athletic status, or musculoskeletal development in young adults. Measurements of body density by hydrostatic weighing, body water by deuterium dilution, and bone mineral by whole body DEXA were obtained in 172 young men (n = 91) and women (n = 81). Estimates of body fat (%Fat) from DEXA (%FatDEXA) were highly correlated with estimates of body fat from the four-component model [body density, total body water, and total body mineral (%Fatd,w,m); r = 0.94, standard error of the estimante (SEE) = 2.8% body mass (BM)] with no significant difference between methods [mean of the difference +/- SD of the difference = -0.4 +/- 2.9 (SD) % BM, P = 0.10] in women and men. On the basis of the comparison with %Fatd,w,m, estimates of %FatDEXA were slightly more accurate than those from body density (r = 0.91, SEE = 3.4%; mean of the difference +/- SD of the difference = -1.2 +/- 3.4% BM). Differences between %FatDEXA and %Fatd,w,m were weakly related to body thickness, as reflected by BMI (r = -0.34), and to the percentage of water in the fat-free mass (r = -0.51), but were not affected by race, athletic status, or musculoskeletal development. We conclude that body composition estimates from DEXA are accurate compared with those from a four-component model in young adults who vary in gender, race, athletic status, body size, musculoskeletal development, and body fatness.

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.nanobody | CXCR4 antagonist | chemotaxis | HIV | stem cell mobilization

Background: Although caffeine supplementation improves performance, the ergogenic effect is variable. The cause(s) of this variability are unknown. A (C/A) single nucleotide polymorphism at intron 1 of the cytochrome P450 (CYP1A2) gene influences caffeine metabolism and clinical outcomes from caffeine ingestion. The purpose of this study was to determine if this polymorphism influences the ergogenic effect of caffeine supplementation. Methods: Thirty-five trained male cyclists (age = 25.0 ± 7.3 yrs, height = 178.2 ± 8.8 cm, weight = 74.3 ± 8.8 kg, VO 2 max = 59.35 ± 9.72 ml·kg -1 ·min -1 ) participated in two computer-simulated 40-kilometer time trials on a cycle ergometer. Each test was performed one hour following ingestion of 6 mg·kg -1 of anhydrous caffeine or a placebo administered in double-blind fashion. DNA was obtained from whole blood samples and genotyped using restriction fragment length polymorphism-polymerase chain reaction. Participants were classified as AA homozygotes (N = 16) or C allele carriers (N = 19). The effects of treatment (caffeine, placebo) and the treatment × genotype interaction were assessed using Repeated Measures Analysis of Variance. Results: Caffeine supplementation reduced 40 kilometer time by a greater (p < 0.05) magnitude in AA homozygotes (4.9%; caffeine = 72.4 ± 4.2 min, placebo = 76.1 ± 5.8 min) as compared to C allele carriers (1.8%; caffeine = 70.9 ± 4.3 min, placebo = 72.2 ± 4.2 min). Conclusions: Results suggest that individuals homozygous for the A allele of this polymorphism may have a larger ergogenic effect following caffeine ingestion.

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve “best-in-class” and broader neutralization capacity.

No differences in time to fatigue were observed between the beverages, despite lower total carbohydrate content in the CHOPA beverage. The CHOPA beverage attenuated postexercise muscle damage, as evidenced by CK and LDH values, compared with an isocaloric CHO beverage.

These findings suggest that at least some of the reported improvements in endurance with CHO+Pro beverages might be related to caloric differences between treatments. Postexercise improvements in markers of muscle disruption with CHO+Pro ingestion appear to be independent of carbohydrate and caloric content and were elicited with beverages consumed only during exercise.

Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor-β1 (TGF-β1). On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137-139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.