Anti‐atherogenic peptide Ep1.B derived from apolipoprotein E induces tolerogenic plasmacytoid dendritic cells

Tolerogenic dendritic cells (DCs) play a critical role in the induction of regulatory T cells (T_regs), which in turn suppress effector T cell responses. We have previously shown the induction of DCs from human and mouse monocytic cell lines, mouse splenocytes and human peripheral blood monocytes by a novel apolipoprotein E (ApoE)‐derived self‐peptide termed Ep1.B. We also showed that this C‐terminal region 239–252 peptide of ApoE has strong anti‐atherogenic activity and reduces neointimal hyperplasia after vascular surgery in rats and wild‐type as well as ApoE‐deficient mice. In this study, we explored the phenotype of DC subset induced by Ep1.B from monocytic cell lines and from the bone marrow‐derived cells. We found Ep1.B treatment induced cells that showed characteristics of plasmacytoid dendritic cells (pDC). We explored in‐vitro and in‐vivo effects of Ep1.B‐induced DCs on antigen‐specific T cell responses. Upon in‐vivo injection of these cells with antigen, the subsequent ex‐vivo antigen‐specific proliferation of lymph node cells and splenocytes from recipient mice was greatly reduced. Our results suggest that Ep1.B‐induced pDCs promote the generation of T_reg cells, and these cells contribute to the induction of peripheral tolerance in adaptive immunity and potentially contribute its anti‐atherogenic activity.     

Evaluation of R‐ (−) and S‐ (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC‐TQ‐MS

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol‐fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S‐ (+) and R‐ (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC‐TQ‐MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5–800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.     

A multicentre phase II study of vorinostat in patients with relapsed or refractory indolent B‐cell non‐Hodgkin lymphoma and mantle cell lymphoma

Although initial rituximab‐containing chemotherapies achieve high response rates, indolent B‐cell non‐Hodgkin lymphoma (B‐NHL), such as follicular lymphoma (FL), is still incurable. Therefore, new effective agents with novel mechanisms are anticipated. In this multicentre phase II study, patients with relapsed/refractory indolent B‐NHL and mantle cell lymphoma (MCL) received vorinostat 200 mg twice daily for 14 consecutive days in a 21‐d cycle until disease progression or unacceptable toxicity occurred. The primary endpoint was overall response rate (ORR) in FL patients and safety and tolerability in all patients. Secondary endpoints included progression‐free survival (PFS). Fifty‐six eligible patients were enrolled; 50 patients (39 with FL, seven with other B‐NHL, and four with MCL) were evaluable for ORR, and 40 patients had received rituximab‐containing prior chemotherapeutic regimens. For the 39 patients with FL, the ORR was 49% [95% confidence interval (CI): 32·4, 65·2] and the median PFS was 20 months (95% CI: 11·2, 29·7). Major toxicities were manageable grade 3/4 thrombocytopenia and neutropenia. Vorinostat offers sustained antitumour activity in patients with relapsed or refractory FL with an acceptable safety profile. Further investigation of vorinostat for clinical efficacy is warranted.     

When Genetic Load Does Not Correlate with Phenotypic Spectrum: Lessons from the GnRH Receptor (GNRHR)

Context: A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations. Objective: The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR. Subjects: Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes. Results: The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P < 0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes. Conclusions: In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.     

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1β, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-β without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1β, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.