44 research outputs found
Protective effects of the postbiotic deriving from cow's milk fermentation with L. paracasei CBA L74 against Rotavirus infection in human enterocytes
: Rotavirus (RV) is the leading cause of acute gastroenteritis-associated mortality in early childhood. Emerging clinical evidence suggest the efficacy of the postbiotic approach based on cow's milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric acute gastroenteritis, but the mechanisms of action are still poorly characterized. We evaluated the protective action of FM-CBAL74 in an in vitro model of RV infection in human enterocytes. The number of infected cells together with the relevant aspects of RV infection were assessed: epithelial barrier damage (tight-junction proteins and transepithelial electrical resistance evaluation), and inflammation (reactive oxygen species, pro-inflammatory cytokines IL-6, IL-8 and TNF-α, and mitogen-activated protein kinase pathway activation). Pre-incubation with FM-CBA L74 resulted in an inhibition of epithelial barrier damage and inflammation mediated by mitogen-activated protein kinase pathway activation induced by RV infection. Modulating several protective mechanisms, the postbiotic FM-CBAL74 exerted a preventive action against RV infection. This approach could be a disrupting nutritional strategy against one of the most common killers for the pediatric age
Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon
Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and β chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the ‘MHCII RNA operon’ in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5′- and 3′-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer
Tolerogenic effect elicited by protein fraction derived from different hypoallergic formulas in PBMCs from children with cow milk allergy
are available for the dietary treatment of cow’s milk
allergy (CMA). Safety and nutritional profile of these
formulas have been well evaluated, but the potential
tolerogenic activity elicited by their protein fraction is still largely
undefined. We aimed to comparatively evaluate the tolerogenic effect
elicited by protein fraction derived from different hypoallergenic
formulas available for the dietary treatment of CMA
METHODS: Four hypoallergenic formulas were compared: extensively
whey formula (EHWF), extensively hydrolyzed casein formula (EHCF),
hydrolyzed rice formula (RHF), amino acid based formula (AAF).
Formulas were reconstituted in water according to manufacturer’s
instructions, and subjected to in vitro infant gut simulated digestion
using a sequential gastric and duodenal static model. Resulting protein
fractions were purified using C18 reversed phase pre-packed
cartridges (Sep-Pak, Waters, Milford, MA, USA),recovered in 70%
acetonitrile/0.1% trifluoroacetic acid and finally vacuum-dried.
Tolerogenic effects were was evaluated in peripheral blood
mononuclear cells (PBMCs) from 6 patients, with challenge-proven
IgE-mediated CMA (age range 1-5 yrs, all Caucasians), stimulated with
different doses of digested protein fractions (from 0.25 to 250 μg/ml)
or -lactoglobulin (BLG;100μg/ml) or bovine serum albumin
(BSA;100μg/ml) as positive and negative control respectively. The
production of Th2 (IL-4, IL-5, IL-13) and Th1 (IL-10, IFN-γ) cytokines
were assessed by ELISA. Modulatory action was also evaluated on
immune (IL-33) and non-immune tolerogenic factors (mucin 5AC,
tight-junction proteins ZO-1 and occludin) in human enterocytes
(Caco-2 cells) by ELISA and Real Time PCR, respectively.
RESULTS: Th2 cytokines were unaffected by the exposure to protein
fraction from all study formulas, whereas only protein fraction from
EHCF was able to positively modulate IL-10, IL-33, mucin 5AC, ZO-1
and occludin expression. All protein fraction from study formulas were
able to increase INF-γ expression in PBMCs.
CONCLUSION: The results suggest a different regulatory action on
immune and non-immune tolerogenic mechanisms elicited by protein
fraction from different hypoallergenic formulas
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Tristetraprolin/ZFP36 Regulates the Turnover of Autoimmune-Associated HLA-DQ mRNAs.
HLA class II genes encode highly polymorphic heterodimeric proteins functioning to present antigens to T cells and stimulate a specific immune response. Many HLA genes are strongly associated with autoimmune diseases as they stimulate self-antigen specific CD4+ T cells driving pathogenic responses against host tissues or organs. High expression of HLA class II risk genes is associated with autoimmune diseases, influencing the strength of the CD4+ T-mediated autoimmune response. The expression of HLA class II genes is regulated at both transcriptional and post-transcriptional levels. Protein components of the RNP complex binding the 3'UTR and affecting mRNA processing have previously been identified. Following on from this, the regulation of HLA-DQ2.5 risk genes, the main susceptibility genetic factor for celiac disease (CD), was investigated. The DQ2.5 molecule, encoded by HLA-DQA1*05 and HLA-DQB1*02 alleles, presents the antigenic gluten peptides to CD4+ T lymphocytes, activating the autoimmune response. The zinc-finger protein Tristetraprolin (TTP) or ZFP36 was identified to be a component of the RNP complex and has been described as a factor modulating mRNA stability. The 3'UTR of CD-associated HLA-DQA1*05 and HLA-DQB1*02 mRNAs do not contain canonical TTP binding consensus sequences, therefore an in silico approach focusing on mRNA secondary structure accessibility and stability was undertaken. Key structural differences specific to the CD-associated mRNAs were uncovered, allowing them to strongly interact with TTP through their 3'UTR, conferring a rapid turnover, in contrast to lower affinity binding to HLA non-CD associated mRNA
Endoglin, a novel biomarker and therapeutical target to prevent malignant peripheral nerve sheath tumor growth and metastasis.
PURPOSE
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGF-β coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs.
EXPERIMENTAL DESIGN
ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models.
RESULTS
ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis.
CONCLUSIONS
Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.We apologize to those authors whose work could not be cited due to size limitations. We thank Dr. Eduard Serra, Dr. Conxi Lázaro and Dr. David Lyden for their support in the project. We also thank Héctor Tejero for his help in analyzing RNA-seq data. Dr. Peinado laboratory is funded by US Department of Defense (W81XWH-16-1-0131), Agencia Estatal de Investigación/Ministerio de Ciencia e Innovación (AEI/MCIN) (PID2020-118558RB-I00/AEI/10.13039/501100011033),
Fundación Proyecto Neurofibromatosis, European Union’s Horizon 2020 research and innovation programme “proEVLifeCycle” under the Marie Skłodowska-Curie grant agreement No 860303, and Fundación Científica AECC. We are also grateful for the support of the Ministerio de Universidades (Programa de Formación de Profesorado Universitario (FPU)) for the fellowship FPU016/05356 awarded to T. González-Muñoz and to the Translational NeTwork for the CLinical application of Extracellular VesicleS (TeNTaCLES) RED2018-102411-T(AEI/10.13039/501100011033). A. Di Giannatale was supported during this work by a research gran Nuovo-Soldati Foundation. The CNIO, certified as Severo Ochoa Excellence Centre, is supported by the Spanish Government through the Instituto de Salud Carlos III.N
Tristetraprolin/ZFP36 Regulates the Turnover of Autoimmune-Associated HLA-DQ mRNAs
HLA class II genes encode highly polymorphic heterodimeric proteins functioning to present antigens to T cells and stimulate a specific immune response. Many HLA genes are strongly associated with autoimmune diseases as they stimulate self-antigen specific CD4+ T cells driving pathogenic responses against host tissues or organs. High expression of HLA class II risk genes is associated with autoimmune diseases, influencing the strength of the CD4+ T-mediated autoimmune response. The expression of HLA class II genes is regulated at both transcriptional and post-transcriptional levels. Protein components of the RNP complex binding the 3′UTR and affecting mRNA processing have previously been identified. Following on from this, the regulation of HLA-DQ2.5 risk genes, the main susceptibility genetic factor for celiac disease (CD), was investigated. The DQ2.5 molecule, encoded by HLA-DQA1*05 and HLA-DQB1*02 alleles, presents the antigenic gluten peptides to CD4+ T lymphocytes, activating the autoimmune response. The zinc-finger protein Tristetraprolin (TTP) or ZFP36 was identified to be a component of the RNP complex and has been described as a factor modulating mRNA stability. The 3′UTR of CD-associated HLA-DQA1*05 and HLA-DQB1*02 mRNAs do not contain canonical TTP binding consensus sequences, therefore an in silico approach focusing on mRNA secondary structure accessibility and stability was undertaken. Key structural differences specific to the CD-associated mRNAs were uncovered, allowing them to strongly interact with TTP through their 3′UTR, conferring a rapid turnover, in contrast to lower affinity binding to HLA non-CD associated mRNA
Next generation sequencing identifies novel potential actionable mutations for grade I meningioma treatment
Meningiomas are common brain tumors that
arise from the meningeal membranes that envelope the
brain and spinal cord. The World Health Organization
classifies these tumors into three histopathological
grades. Because of tumor recurrence, treating
meningiomas may be challenging even in well-
differentiated grade I (GI) neoplasms. Indeed, around
5% of completely resected GI meningiomas relapse
within 5 years. Therefore, identifying driver mutations in
GI meningiomas through next generation sequencing
(NGS) assays is paramount. The aim of this study was to
validate the use of the 50-gene AmpliSeq Hotspot
Cancer Panel v2 to identify the mutational status of 23
GI meningioma, namely, 12 non recurrent and 11
recurrent. In 18 out of the 23 GI meningiomas analyzed,
we identified at least one gene mutation (78.2%). The
most frequently mutated genes were c-kit (39.1%), ATM
(26.1%), TP53 (26.1%), EGFR (26.1%), STK11 (21.7%),
NRAS (17.4%), SMAD4 (13%), FGFR3 (13%), and
PTPN11 (13%); less frequent mutations were SMARCB1
(8.7%), FLT3 (8.7%), KRAS (8.7%), FBWX7 (8.7%),
ABL1 (8.7%), ERBB2 (8.7%), IDH1 (8.7%), BRAF
(8.7%), MET (8.7%), HRAS (4.3%), RB1 (4.3%),
CTNNB1 (4.3%), PIK3CA (4.3%), VHL (4.3%), KDR
(4.3%), APC (4.3%), NOTCH1 (4.3%), JAK3 (4.3%),
and SRC (4.3%). To our knowledge, mutations in all of
these genes, except for TP53, STK11, SMARCB1,
PIK3CA, VHL, and BRAF, have never been described
before in meningiomas. Hence, these findings
demonstrate the viability of NGS to detect new genetic
alterations in GI meningiomas. Equally important, this
technology enabled us to detect possible novel
actionable mutations not previously associated with GI
and for which selective inhibitors already exis
Effect of Gliadin Stimulation on HLA-DQ2.5 Gene Expression in Macrophages from Adult Celiac Disease Patients
Macrophages play an important role in the pathogenesis of celiac disease (CD) because they are involved in both inflammatory reaction and antigen presentation. We analyzed the expression of CD-associated HLA-DQ2.5 risk alleles on macrophages isolated by two cohorts of adult patients, from the U.S. and Italy, at different stages of disease and with different genotypes. After isolating and differentiating macrophages from PBMC, we assessed the HLA genotype and quantified the HLA-DQ2.5 mRNAs by qPCR, before and after gliadin stimulation. The results confirmed the differences in expression between DQA1*05:01 and DQB1*02:01 predisposing alleles and the non-CD associated alleles, as previously shown on other types of APCs. The gliadin challenge confirmed the differentiation of macrophages toward a proinflammatory phenotype, but above all, it triggered an increase of DQA1*05:01 mRNA, as well as a decrease of the DQB1*02:01 transcript. Furthermore, we observed a decrease in the DRB1 genes expression and a downregulation of the CIITA transactivator. In conclusion, our findings provide new evidences on the non-coordinated regulation of celiac disease DQ2.5 risk genes and support the hypothesis that gliadin could interfere in the three-dimensional arrangement of chromatin at the HLA locus