VGF-peptides in the Siberian hamster

vgf is one of the few genes selectively induced in the hypothalamus of Siberian hamsters upon their typical change from an obese phenotype (long day adaptation, during summer) to a lean, catabolic phenotype (short day, or winter adaptation). In fact, the i.c.v. injection of the VGF-derived peptide TLQP-21 caused hypophagia and a decrease in body weight in long day hamster. Hence, we studied VGF multi-peptide profiles in brain cortex and hypothalamus of (male) Siberian hamsters, in the long day (LD) versus short day (SD) adapted state. Specific antisera were produced against short sequences at the C- or N-termini of VGF, and of several known/predicted VGF-derived products: TLQP, NERP-1, and PGH peptides, and used in immunohistochemistry (IHC) and ELISA. Hamsters were perfused with 4% paraformaldehyde (n= 4/group) for IHC or used for tissue sampling and extraction (n= 7/group). In IHC, VGF C- and N- terminus peptides were brightly labelled, as well as most abundant. They were found in both perikarya and axons, in different layers of brain cortex and in multiple hypothalamic areas, including the paraventricular (PVN), suprachiasmatic (SCN), supraoptic (SON) and arcuate nuclei, the lateral and anterior hypothalamic areas, and the median eminence (ME). TLQP peptides were largely restricted to SCN perikarya, and ME axons, while PGH and NERP-1 peptides were revealed in perikarya of the brain cortex, in ME axons, and certain perikarya of PVN and SON (NERP-1 only). Most VGF peptides studied were well represented in tissue extracts of hypothalamus and cortex, VGF C- and N- terminus peptides being again most abundant (hypothalamus: 1.8±0.3 and 10.9±0.6; cortex: 0.7±0.1 and 5±0.3 nmol/g, mean±SEM, C- and N-terminus, respectively, LD animals). A selective decrease in certain VGF peptides was revealed in SD animals, compared to LD ones, so that NERP-1 peptides were decreased in hypothalamus and cortex (61.3±12.7% and 45.8±11.1% of LD animals, respectively, mean±SEM, p<0.04), PGH peptides were reduced in hypothalamus (24.8±12.7% of LD group, mean±SEM, p<0.02), and both TLQP and N-terminus peptides in the brain cortex (31.8±10.9% and 41.5±10.8% of LD animals, respectively, mean±SEM, p<0.02). In conclusion, VGF peptides were well represented in the Siberian hamster brain, with a distinct, apparently selective modulation in the hypothalamus and brain cortex. A regionally specific, differential post-translational processing of the VGF precursor may be implicated. Supported by a RAS grant (Regione Autonoma Sardegna, PO FSE 2007-1013, L.R. 7/2007)

Public prevention plans to manage climate change and respiratory allergic diseases. Innovative models used in Campania Region (Italy): the twinning Aria implementation and the allergy safe tree: decalogue

In recent years, climate change has been influenced by air pollution, and this destructive combination has justifiably sounded an alarm for nations and many institutional bodies worldwide. Official reports state that the emission of greenhouse gases produced by human activity are growing, and consequently also the average temperature. The World Health Organization (WHO) believes that health effects expected in the future due to climate change will be dramatic, and has invited international groups to investigate potential remedies. A task force has been established by the Italian Society of Allergology, Asthma and Clinical Immunology (SIAAIC), with the aim to actively work on correlation between pollution and climate change. The Task Force provided prevention tools to suggest city leaders how to improve the health conditions of allergic people in public urban parks. The “Allergy Safe Tree Decalogue” suggests the preparation and maintenance of public low allergyimpact greenery. Through the Twinning ARIA project, the Division for the Promotion and Enhancement of Health Innovation Programs of Campania Region (Italy), sought to promote the implementation of the project in the regional Health System. The main objective will be to investigate the current use and usefulness of mobile phone Apps in the management of allergic respiratory disease, through Mobile Airways Sentinel networK (MASK), the Phase 3 of the ARIA initiative, based on the freely available MASK App (the Allergy Diary, Android and iOS platforms). The effects of these prevention activities will be registered and compared with monitoring efforts thanks to the Aerobiology Units, located throughout the Campania area. A joint effort between researchers and public administrations for the implementation of prevention plans coherently with the two models proposed in a specific area, i.e. the Decalogue for public administrations and the MASK Allergy Diary app for individual patients suffering from allergy, will be implemented as a pilot

Real-life effects of dupilumab in patients with severe type 2 asthma, according to atopic trait and presence of chronic rhinosinusitis with nasal polyps

BackgroundThe efficacy of dupilumab as biological treatment of severe asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) depends on its ability to inhibit the pathophysiologic mechanisms involved in type 2 inflammation.ObjectiveTo assess in a large sample of subjects with severe asthma, the therapeutic impact of dupilumab in real-life, with regard to positive or negative skin prick test (SPT) and CRSwNP presence or absence.MethodsClinical, functional, and laboratory parameters were measured at baseline and 24 weeks after the first dupilumab administration. Moreover, a comparative evaluation was carried out in relation to the presence or absence of SPT positivity and CRSwNP.ResultsAmong the 127 recruited patients with severe asthma, 90 had positive SPT, while 78 reported CRSwNP. Compared with the 6 months preceding the first dupilumab injection, asthma exacerbations decreased from 4.0 (2.0-5.0) to 0.0 (0.0-0.0) (p < 0.0001), as well as the daily prednisone intake fell from 12.50 mg (0.00-25.00) to 0.00 mg (0.00-0.00) (p < 0.0001). In the same period, asthma control test (ACT) score increased from 14 (10-18) to 22 (20-24) (p < 0.0001), and sino-nasal outcome test (SNOT-22) score dropped from 55.84 ± 20.32 to 19.76 ± 12.76 (p < 0.0001). Moreover, we observed relevant increases in forced expiratory volume in one second (FEV1) from the baseline value of 2.13 L (1.62-2.81) to 2.39 L (1.89-3.06) (p < 0.0001). Fractional exhaled nitric oxide (FeNO) values decreased from 27.0 ppb (18.0-37.5) to 13.0 ppb (5.0-20.0) (p < 0.0001). These improvements were quite similar in subgroups of patients characterized by SPT negativity or positivity, and CRSwNP absence or presence. No statistically significant correlations were detected between serum IgE levels, baseline blood eosinophils or FeNO levels and dupilumab-induced changes, with the exception of FEV1 increase, which was shown to be positively correlated with FeNO values (r = 0.3147; p < 0.01).ConclusionOur results consolidate the strategic position of dupilumab in its role as an excellent therapeutic option currently available within the context of modern biological treatments of severe asthma and CRSwNP, frequently driven by type 2 airway inflammation

TLQP-21 in <i>in vitro</i> experiments.

<p>TLQP-21 included in the medium of hypothalamic samples up-regulates the secretion of growth-hormone-releasing hormone (GHRH). Contr.: control medium analysed by ELISA without the addition of TLQP-21 <i>versus</i> the medium including the peptide (TLQP column).</p

Sephadex chromatography.

<p>Three major peaks were shown for TLQP peptides (left: a, b, c), corresponding to TLQP-62, TLQP-30 and TLQP-21 and found in all tissue extracts. Higher MW forms were seen, at approximately 10–12 kDa and in the void volume, or in the region overlapping the void volume in plasma. VGF C-terminus immunoreactivity was revealed in the void volume (mid-left: d), compatible with VGF precursor, and in a major broad peak in the approximately 10–15 kDa range (ibidem: e), while a lower peak (ibidem: f) appeared related to the TLQP “a” peak described above. The same peptides were mostly seen as low MW fragments in the ovary, while migrating as large forms in plasma (mid-left). The VGF N-terminus assay revealed abundant reactivity in the large MW fractions from plasma (mid-right: refer “g” to bottom panel), with lower levels at varied MW in the other tissue extracts. PGH peptides were revealed as different MW forms, especially in the hypothalamus while the largest MW forms eluting in the void volume were abundant in plasma (ibidem: i). Molecular weight markers are shown with the corresponding arrows. Pmol/f:picomoles/fraction; f:fraction.</p

VGF peptide localisation in Ovary.

<p>TLQP peptide immunoreactivity was found in the granulosa (A) as well as in the theca and interstitial cells (B), while PGH peptides were restricted to the oocyte (C). All VGF staining: Cy₃ red labeling. Scale bar: 50 um.</p

VGF peptide localisation in hypothalamus and pituitary.

<p><u>In the preoptic area</u> (A–D), PGH peptides (A) were immunostained within almost all neuronal terminals containing GnRH (B) conversely, VGF N-terminus peptides (C) were also revealed in most kisspeptin neuronal terminals (D): A, C: Cy3 red labeling; B, D: Cy₂ green labeling. <u>In the median eminence</u> (E–J), TLQP peptides (E) were selectively found in almost all somatostatin neuronal terminals and axons (F). Conversely, VGF C-terminus (G) and PGH (I) antibodies labeled GnRH neurone terminals (H and J, respectively): E, G, I: Cy3 red labeling; F, H, J: Cy₂ green labeling. In the <u>pituitary</u> (K–Q), the number of cells positive with both TLQP (K, M) and LH (L, N) antibodies was higher in diestrous (K, L) than in estrous phase (M, N). Conversely, the PGH peptides (O) were found in a large number of LH cells (P) and in a few PRL cells (Q, colocalised cells are identified by the arrows). Scale bars: A–B, E–Q: 100 um, C–D: 50 um.</p

Antisera and antibodies used in the study.

<p>C-term: VGF C-terminus; N-term: VGF N-terminus; FSH: follicle-stimulating hormone; GH: growth hormone; LH: luteinising hormone; PRL: prolactin; h. factor: hypothalamic factor, GHRH: growth hormone-releasing hormone; GnRH: gonadotrophin-releasing hormone; SRIF: somatostatin, Kissp: kisspeptin; Ref: references; E: ELISA; I: immunohistochemistry.</p><p>Antisera and antibodies used in the study.</p

ELISA characterization.

<p>Sequence differences versus rat VGF are underlined; IC50: dose producing 50% inhibition (pmol per well); CV1 and CV2: intra and inter assay variation, respectively, C-t and N-t: VGF C-terminus and N-terminus, respectively; ² “immunogen”: peptide used for conjugation to carrier protein and immunizations; used for plate coating and assay standards; R³: Arg-extended: with addition of Arginine residue at the peptide N- or C-terminus, respectively; R⁴ additional Arginine residue from adjacent cleavage site; “dY” and “C”: additional d-Tyrosine, or Cysteine residue used for conjugation; r: rat, h: human, m: mouse.</p><p>ELISA characterization.</p