Enkephalinase inhibitor potentiates substance P- and electrically induced contraction in ferret trachea

To determine the role of endogenous enkephalinase (EC 3.4.24.11) in regulating peptide-induced contraction of airway smooth muscle, we studied the effect of the enkephalinase inhibitor, leucine-thiorphan (Leu-thiorphan), on responses of isolated ferret tracheal smooth muscle segments to substance P (SP) and to electrical field stimulation (EFS). Leu-thiorphan shifted the dose-response curve to SP to lower concentrations. Atropine or the SP antagonist [D-Pro2,D-Trp7,9]SP significantly inhibited SP-induced contractions in the presence of Leu-thiorphan. Leu-thiorphan increased the contractile responses to EFS dose dependently, an effect that was significantly inhibited by the SP antagonist [D-Pro2,D-Trp7,9]SP. SP, in a concentration that did not cause contraction, increased the contractile responses to EFS. This effect was augmented by Leu-thiorphan dose dependently and was not inhibited by hexamethonium or by phentolamine but was inhibited by atropine. Because contractile responses to acetylcholine were not significantly affected by SP or by Leu-thiorphan, the potentiating effects of SP were probably on presynaptic-postganglionic cholinergic neurotransmission. Captopril, bestatin, or leupeptin did not augment contractions, suggesting that enkephalinase was responsible for the effects. These results suggest that endogenous tachykinins modulate smooth muscle contraction and endogenous enkephalinase modulates contractions produced by endogenous or exogenous tachykinins and tachykinin-induced facilitation of cholinergic neurotransmission.     

Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections

Neuropeptides such as substance P are implicated in inflammation mediated by sensory nerves (neurogenic inflammation), but the roles in disease of these peptides and the peptidases that degrade them are not understood. It is well established that inflammation is a prominent feature of several airway diseases, including viral infections, asthma, bronchitis, and cystic fibrosis. These diseases are characterized by cough, airway edema, and abnormal secretory and bronchoconstrictor responses, all of which can be elicited by substance P. The effects of substance P and other peptides that may be involved in inflammation are decreased by endogenous neutral endopeptidase (NEP; also called enkephalinase, EC 3.4.24.11), which is a peptidase that degrades substance P and other peptides. In the present study, we report that rats with histories of infections caused by common respiratory tract pathogens (parainfluenza virus type 1, rat corona-virus, and Mycoplasma pulmonis) not only have greater susceptibility to neurogenic inflammatory responses than do pathogen-free rats but also have a lower activity of NEP in the trachea. This reduction in NEP activity may cause the increased susceptibility to neurogenic inflammation by allowing higher concentrations of substance P to reach tachykinin receptors in the trachea. Thus decreased NEP activity may exacerbate some of the pathological responses in animals with respiratory tract infections.     

Cysteine-tailed class I-binding peptides bind to CpG adjuvant and enhance primary CTL responses

Immunostimulatory CpG motifs in synthetic oligonucleotides can be effective adjuvants for the priming of CTLs. We first observed that a single male-specific peptide (KCSRNRQYL) (HY2) was more efficient than another male-specific peptide (WMHHNMDLI) (HY1) at priming IFN-gamma-secreting CTLs in vivo when combined with lipid A and CpG and that it also visibly precipitated CpG. The addition of the six N-terminal residues (KCSRNR) from HY2 to HY1 yielded a peptide, KCSRNR-HY1, that both precipitated CpG and primed increased numbers of HY1-specific CTLs. We refer to this type of peptide as a primotope that includes a class I binding peptide tailed with amino acids that increase priming. Ala residues were substituted for the Arg/Lys residues (ACSANA-HY1), and these substitutions did not reduce in vivo priming potential. However, the substitution of Ala for Cys (KASRNR-HY1) resulted in the complete loss of priming, demonstrating the importance of Cys for in vivo priming when mixed with CpG. This result suggested that increased priming was based in disulfide bonding between Cys residues and internal phosphorothioate groups of synthetic CpG. The addition of Cys-bearing primotopes to radiolabeled CpG with a single thioate group resulted in the appearance of a new band that was inhibited by 1) Cys > Ala substitution and 2) reduction and alkylation of CpG. These results reveal a novel mechanism for complexing class I binding peptides and CpG adjuvant for development of new peptide-adjuvant combinations for vaccines for cancer and infectious diseases.     

Molecular testing of BRAF,RAS and TERT on thyroid FNAs with indeterminate cytology improves diagnostic accuracy

Objective

Liquid‐based (LB)‐FNA is widely recognized as a reliable diagnostic method to evaluate thyroid nodules. However, up to 30% of LB‐FNA remain indeterminate according to the Bethesda system. Use of molecular biomarkers has been recommended to improve its pathological accuracy but implementation of these tests in clinical practice may be difficult. Here, we evaluated feasibility and performance of molecular profiling in routine practice by testing LB‐FNA for BRAF, N/HRAS and TERT mutations.

Methods

We studied a large prospective cohort of 326 cases, including 61 atypia of undetermined significance, 124 follicular neoplasms, 72 suspicious for malignancy and 69 malignant cases. Diagnosis of malignancy was confirmed by histology on paired surgical specimen.

Results

Mutated LB‐FNAs were significantly associated with malignancy regardless of the cytological classification. Overall sensitivity was 60% and specificity 89%. Importantly, in atypia of undetermined significance and follicular neoplasm patients undergoing surgery according to the Bethesda guidelines, negative predictive values were 85.4% and 90% respectively. TERT promoter mutation was rare but very specific for malignancy (5.5%) suggesting that it could be of interest in patients with indeterminate cytology.

Conclusions

Mutation profiling can be successfully performed on thyroid LB‐FNA without any dedicated sample in a pathology laboratory. It is an easy way to improve diagnostic accuracy of routine LB‐FNA and may help to better select patients for surgery and to avoid unnecessary thyroidectomies.

     

Provision of nestboxes raises the breeding density of Great Tits Parus major equally in coniferous and deciduous woodland

Nestbox provision is a technique used to increase nest-site availability for secondary cavity-nesting birds. However, little is known about the demographic consequences of nestbox provision in different habitat types. To assess how nestbox provision affects the density of hole-nesting birds simultaneously in two contrasting habitats, we compared the breeding density of Great Tits along transects without nestboxes with that in transects where nestboxes were provided. Although the initial density of breeders was considerably higher in the deciduous habitat than in the coniferous habitat, provision of nestboxes increased density by a similar number of additional pairs in each habitat type. Thus, the provision of nestboxes in managed coniferous forests may be as effective in increasing the breeding opportunities of cavity nesters as in deciduous stands. Moreover, previous research showed that pairs in deciduous habitat with nestboxes have consistently lower breeding success than those in coniferous habitat with nestboxes. It is possible that the addition of nestboxes in the preferred habitat increased density to such an extent that density-dependent effects became apparent.