Besides its metabolic and endocrine effects, growth hormone (GH)-releasing hormone (GHRH) is involved in the modulation of inflammation. Recently synthetized GHRH antagonist MIA-690 and MR-409, GHRH agonist, developed by us have shown potent pharmacological effects in various experimental paradigms. However, whether their administration modify resistance to chronic inflammatory stimuli in colon is still unknown. Ex vivo results demonstrated that MIA-690 and MR-409 inhibited production of pro-inflammatory and oxidative markers induced by lipopolysaccharide on isolated mouse colon specimens. In vivo, both MIA-690 and MR-409 have also been able to decrease the responsiveness to nociceptive stimulus, in hot plate test. Additionally, both peptides also induced a decreased sensitivity to acute and persistent inflammatory stimuli in male mice, in formalin test and dextran sodium sulfate (DSS)-induced colitis model, respectively. MIA-690 and MR-409 attenuate DSS-induced colitis with particular regard to clinical manifestations, histopathological damage and release of pro-inflammatory and oxidative markers in colon specimens. Respect to MR-409, MIA-690 showed higher efficacy in inhibiting prostaglandin (PG)E2, 8-iso-PGF2α and serotonin (5-HT) levels, as well as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nitric oxide synthase gene expression in colon specimens of DSS-induced colitis. Furthermore, MIA-690 decreased serum insulin-like growth factor (IGF)-1 levels in mice DSS-treated, respect to MR-409. Thus, our findings highlight the protective effects of MIA-690 and MR-409 on inflammation stimuli. The higher antinflammatory and antioxidant activities observed with MIA-690 could be related to decreased serum IGF-1 levels.

Phenylketonuria (PKU) is an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development.

The successful isolation and propagation of patient-derived keratinocytes from cervical lesions constitute a more appropriate model of cervical disease than traditional cervical cancer-derived cell lines such as SiHa and CaSki. Our aim was to streamline the growth of patient-obtained, cervical keratinocytes into a reproducible process. We performed an observational case series study with 60 women referred to colposcopy for a diagnostic biopsy. Main outcome measures were how many samples could be passaged at least once (n = 11), and where enough cells could be established, to precisely define their proliferation profile over time (n = 3). Altering cell culture conditions over those reported by other groups markedly improved outcomes. We were also successful in making freeze backs which could be resuscitated to successfully propagate multi-layered, organoids from cervical keratinocytes (n = 3). For best results, biopsy-intrinsic factors such as size and tissue digestion appear to be major variables. This seems to be the first systematic report with a well characterized and defined sample size, detailed protocol, and carefully assessed cell yield and performance. This research is particularly impactful for constituting a sample repository-on-demand for appropriate disease modelling and drug screening under the umbrella of personalized health.

Growth hormone-releasing hormone (GHRH) antagonist MIA-690 and GHRH agonist MR-409, previously synthesized and developed by us have demonstrated potent antitumor effects. However, little is known about the effects of these analogs on brain functions. We investigated the potential antinflammatory and antioxidant effects of GHRH antagonist MIA-690 and GHRH agonist MR-409, on isolated mouse prefrontal cortex specimens treated with lipopolysaccharide (LPS). Additionally, we studied their effects on emotional behavior after chronic in vivo treatment. Ex vivo, MIA-690 and MR-409 inhibited LPS-induced inflammatory and pro-oxidative markers. In vivo, both MIA-690 and MR-409 induced anxiolytic and antidepressant-like effects, increased norepinephrine and serotonin levels and decreased nuclear factor-kB, tumor necrosis factor-α and interleukin-6 gene expression in prefrontal cortex. Increased nuclear factor erythroid 2-related factor 2 expression was also found in mice treated with MIA-690 and MR-409. MIA-690 showed higher efficacy in inhibiting all tested inflammatory and oxidative markers. In addition, MR-409 induced a down regulation of the gene and protein expression of pituitary-type GHRH-receptor in prefrontal cortex of mice after 4 weeks of treatment at 5 µg/day. In conclusion, our results demonstrate anxiolytic and antidepressant-like effects of GHRH analogs that could involve modulatory effects on monoaminergic signaling, inflammatory and oxidative status.

Lung inflammation due to sarcoidosis is characterized by a complex cascade of immunopathologic events, including leukocyte recruitment and granuloma formation. α-melanocyte stimulating hormone (α-MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of α-MSH in a novel in vitro sarcoidosis model. An in vitro sarcoidosis-like granuloma model was developed by challenging peripheral blood mononuclear cells (PBMCs) derived from patients with confirmed treatment-naïve sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 μM α-MSH or saline as control. Cytokine concentrations in supernatants of culture and in cell extracts were measured using Illumina multiplex Elisa and western blot, respectively. Gene expression was analyzed using RNA-Seq and RT-PCR. Protein secretion and gene expression of IL-7, IL-7R, IFN-γ, MC1R, NF-κB, phosphorylated NF-κB (p-NF-κB), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-γ protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-γ protein expression was significantly reduced in developed granulomas after exposure to α-MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-κB and p-NF-κB were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with α-MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of α-MSH were blocked by specific p-CREB inhibition. α-MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.

Phenylketonuria (PKU) is a genetic deficiency of phenylalanine hydroxylase (PAH) in liver resulting in blood phenylalanine (Phe) elevation and neurotoxicity. A pegylated phenylalanine ammonia lyase (PEG-PAL) metabolizing Phe into cinnamic acid was recently approved as treatment for PKU patients. A potentially one-time rAAV-based delivery of PAH gene into liver to convert Phe into tyrosine (Tyr), a normal way of Phe metabolism, has now also entered the clinic. To understand differences between these two Phe lowering strategies, we evaluated PAH and PAL expression in livers of PAHenu2 mice on brain and liver functions. Both lowered brain Phe and increased neurotransmitter levels and corrected animal behavior. However, PAL delivery required dose optimization, did not elevate brain Tyr levels and resulted in an immune response. The effect of hyperphenylalanemia on liver functions in PKU mice was assessed by transcriptome and proteomic analyses. We observed an elevation in Cyp4a10/14 proteins involved in lipid metabolism and upregulation of genes involved in cholesterol biosynthesis. Majority of the gene expression changes were corrected by PAH and PAL delivery though the role of these changes in PKU pathology is currently unclear. Taken together, here we show that blood Phe lowering strategy using PAH or PAL corrects both brain pathology as well as previously unknown lipid metabolism associated pathway changes in liver.