Niemann-Pick type C2 protein supplementation in experimental non-alcoholic fatty liver disease

Background and aims Hepatic cholesterol deposition drives inflammation and fibrosis in non-alcoholic steatohepatitis (NASH). The Niemann-Pick type C2 (NPC2) protein plays an important role in regulating intracellular cholesterol trafficking and homeostasis. We hypothesized that intravenous NPC2 supplementation reduces cholesterol accumulation, hepatic inflammation and fibrogenesis in a nutritional NASH rat model. Methods: Rats were fed a high-fat, high-cholesterol (HFHC) diet for four weeks resulting in moderately severe NASH. Animals were treated with intravenous NPC2 or placebo twice weekly for either the last two weeks or the entire four weeks. End-points were liver/body- and spleen/body weight ratios, histopathological NASH scores, fibrosis, serum liver enzymes, cholesterol, lipoproteins, cytokines, and quantitative polymerase chain reaction derived hepatic gene expression related to cholesterol metabolism, inflammation, and fibrosis. Results: HFHC rats developed hepatomegaly, non-fibrotic NASH histopathology, elevated liver enzymes, serum cholesterol, and pro-inflammatory cytokines. Their sterol regulatory element binding factor 2 (SREBF2) and low-density lipoprotein receptor (LDL-R) mRNAs were down-regulated compared with rats on standard chow. NPC2 did not improve liver weight, histopathology, levels of serum liver enzymes or pro-inflammatory tumor necrosis factor-α (TNFα), Interleukin (IL)-6, or IL-1β in HFHC rats. Two weeks of NPC2 treatment lowered hepatic TNFα and COL1A1 mRNA expression. However, this effect was ultimately reversed following additional two weeks of treatment. Four weeks NPC2 treatment of rats raised ATP-binding cassette A1 (ABCA1) and low-density lipoprotein receptor (LDLR) mRNAs in the liver, concurrent with a strong tendency towards higher serum high-density lipoprotein (HDL). Furthermore, the peroxisome proliferator activated receptor-ɣ (PPARG) gene expression was reduced. Conclusions: NPC2 proved inefficient at modifying robust hepatic NASH end-points in a HFHC NASH model. Nonetheless, our data suggest that hepatic ABCA1 expression and reverse cholesterol transport were upregulated by NPC2 treatment, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism

Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification

The authors thank the financial support from Fundacao para a Ciencia e a Tecnologia through Grant PEst-C/EQB/LA0006/2011 and contracts no. PTDC/EBB-BIO/102163/2008, PTDC/EBB-BIO/098961/2008, PTDC/EBB-BIO/118317/2010, SFRH/BD/72650/2010 for V.L.D, and Santander Totta Bank - Universidade Nova de Lisboa for the Scientific Award 2009/2010. The authors are grateful to Dr. Abid Hussain and M. Telma Barroso (REQUIMTE, FCT-UNL, Portugal) for the preparation of the synthetic affinity ligands, to Lonza Biologics, U.K. (Dr. Richard Alldread), and the Animal Cell Technology Unit of ITQB-UNL/IBET (Dr. Paula M Alves and Dr. Ana Teixeira) for providing the cells and the culture bulks and to Mr. Filipe Cardoso and Prof. Paulo Freitas (INESC-MN, Lisbon, Portugal) for the help with the VSM measurements.Dextran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 10⁴ M⁻¹. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.proofpublishe

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 lg/ ml, mean 0.028 lg/ml while 10 % of both normals and patients with cancers had OLFM4 between 4 and 60 lg/ml, mean 15 lg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers

Le Scienze della Terra: Didattica e Laboratorio di Didattica. Riflessioni metodologico-disciplinari

Riflessioni metodologico-disciplinari sull'insegnamento della Didattica e del Laboratorio di Didattica delle Scienze della Terra per docenti in formazione e in servizio nella Scuola Secondaria di 2\ub0 grado

Hepatic histology scores.

<p>Hepatic histology scores graded semi-quantitatively according to Kleiner et. al. non-alcoholic fatty liver disease (NAFLD) activity score (NAS), steatosis (0–3), lobular inflammation (0–2), ballooning (0–2) and fibrosis (0–4) in isocaloric controls, high-fat, high-cholesterol (HFHC) controls, two-week NPC2 (treatment) and four-week NPC2 (prevention) animals. No animal had significant histological fibrosis on HE (Panel A) or Masson trichrome staining (Panel B). *: P < 0.05 compared with Isocaloric Controls. #: P < 0.05 compared with HFHC Controls.</p