Aline S.C. Fabricio

Human Amniotic Fluid Stem Cell Preconditioning Improves Their Regenerative Potential

Human amniotic fluid stem (hAFS) cells, a novel class of broadly multipotent stem cells that share characteristics of both embryonic and adult stem cells, have been regarded as promising candidate for cell therapy. Taking advantage by the well-established murine model of acute kidney injury (AKI), we studied the proregenerative effect of hAFS cells in immunodeficient mice injected with the nephrotoxic drug cisplatin. Infusion of hAFS cells in cisplatin mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. Human AFS cells engrafted injured kidney predominantly in peritubular region without acquiring tubular epithelial markers. Human AFS cells exerted antiapoptotic effect, activated Akt, and stimulated proliferation of tubular cells possibly via local release of factors, including interleukin-6, vascular endothelial growth factor, and stromal cell-derived factor-1, which we documented in vitro to be produced by hAFS cells. The therapeutic potential of hAFS cells was enhanced by cell pretreatment with glial cell line-derived neurotrophic factor (GDNF), which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartment. By in vitro studies, GDNF increased hAFS cell production of growth factors, motility, and expression of receptors involved in cell homing and survival. These findings indicate that hAFS cells can promote functional recovery and contribute to renal regeneration in AKI mice via local production of mitogenic and prosurvival factors. The effects of hAFS cells can be remarkably enhanced by GDNF preconditioning.

A novel strategy to enhance mesenchymal stem cell migration capacity and promote tissue repair in an injury specific fashion.

Mesenchymal stem cells (MSCs) of bone marrow origin appear to be an attractive candidate for cell-based therapies. However, the major barrier to the effective implementation of MSC-based therapies is the lack of specific homing of exogenously infused cells and overall the inability to drive them to the diseased or damaged tissue. In order to circumvent these limitations, we developed a preconditioning strategy to optimize MSC migration efficiency and potentiate their beneficial effect at the site of injury. Initially, we screened different molecules by using an in vitro injury–migration setting, and subsequently, we evaluated the effectiveness of the different strategies in mice with acute kidney injury (AKI). Our results showed that preconditioning of MSCs with IGF-1 before infusion improved cell migration capacity and restored normal renal function after AKI. The present study demonstrates that promoting migration of MSCs could increase their therapeutic potential and indicates a new therapeutic paradigm for organ repair.

A comparative study of the antipyretic effects of indomethacin and dipyrone in rats

Abstract. Objective: Compare the antipyretic effects of dipyrone and indomethacin.¶Materials and methods: Fever was induced in rats by i.v. LPS or i.c.v. interleukins (IL), prostaglandins (PG), arachidonic acid (AA), pre-formed pyrogenic factor (PFPF), tumour necrosis factor-α (TNF-α) or corticotrophin releasing hormone (CRH). Dipyrone and indomethacin were administered i.p., arginine vasopressin V1-receptor antagonist, d(CH2)5 Tyr(Me)AVP, into the ventral septal area. Cyclooxygenase (COX-1/-2) blocking activity was assessed in transfected COS-7 cells. CRH release from isolated hypothalami was determined by ELISA.¶Results: Indomethacin or dipyrone reduced LPS, IL-1β, IL-6 or TNF-α induced fever and CRH release from rat hypothalamus. Only dipyrone inhibited IL-8, PFPF or PGF2α fever. Only indomethacin inhibited fever induced by AA or IL-1β plus AA. Neither antipyretic affected fever caused by PGE2 or CRH. d(CH2)5Tyr(Me)AVP only blocked antipyresis induced by indomethacin. Dipyrone at a very high concentration (10 mM) inhibited only COX-1, while indomethacin (0.1 μM) blocked COX-1 and COX-2 in COS-7 cells.¶Conclusion: The antipyretic effect of dipyrone differs from that of indomethacin in that it does not depend on AVP release or inhibition of PG synthesis.

Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats

1 The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and endothelin‐1 (ET‐1) was assessed in conscious rats. 2 Intravenous (i.v.) LPS (5.0 μg kg−1) markedly increased rectal temperature to a peak of 1.30°C over baseline at 2.5 h. Pretreatment with the mixed endothelin ETA/ETB receptor antagonist bosentan (10 mg kg−1, i.v.) or the selective endothelin ETB receptor antagonist BQ‐788 (N‐cis‐2,6‐dimethyl‐piperidinocarbonyl‐L‐γ‐methylleucyl‐D‐1‐methoxycarboyl‐D‐norleucine; 3 pmol, into a lateral cerebral ventricle–i.c.v.) reduced the peak response to LPS to 0.90 and 0.75°C, respectively. The selective endothelin ETA receptor antagonist BQ‐123 (cyclo[D‐Trp‐D‐Asp‐Pro‐D‐Val‐Leu]; 3 pmol, i.c.v.) was ineffective. 3 Increases in temperature caused by IL‐1β (180 fmol, i.c.v.), TNF‐α (14.4 pmol, i.c.v.) or IL‐1β (150 pmol kg−1, i.v.) were unaffected by BQ‐788 (3 pmol, i.c.v.). 4 Central injection of endothelin‐1 (0.1 to 3 fmol, i.c.v.) caused slowly‐developing and long‐lasting increases in rectal temperature (starting 2 h after administration and peaking at 4–6 h between 0.90 and 1.15°C) which were not clearly dose‐dependent. The response to endothelin‐1 (1 fmol, i.c.v.) was prevented by BQ‐788, but not by BQ‐123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo‐oxygenase inhibitor indomethacin (2 mg kg−1), which partially reduced LPS‐induced fever, did not modify the hyperthermic response to endothelin‐1 (3 fmol, i.c.v.). 5 Therefore, central endothelin(s) participates importantly in the development of LPS‐induced fever, via activation of a prostanoid‐independent endothelin ETB receptor‐mediated mechanism possibly not situated downstream from IL‐1β or TNF‐α in the fever cascade.

Endothelin-1 as a central mediator of LPS-induced fever in rats.

Fever induced by E. coli lipopolysaccharide (LPS) in rats is substantially reduced by blockade of central endothelin ET(B) receptors. This study explores the role of endothelin-1 as a central mediator of fever in rats, by investigating the effect of a pyrogenic dose of LPS on the levels of big endothelin-1 and endothelin-1 in the cerebrospinal fluid (CSF) and endothelin-1 in the plasma. We further assessed whether the increase in body temperature caused by central injection of endothelin-1 constitutes solely a hyperthermia or a true integrated febrile response. LPS (5 mug kg(-1), i.v.) induced fever which peaked at 1.16 +/- 0.24 degrees C within 2 h and remained stable up to 5 h. CSF levels of immunoreactive (ir) big endothelin-1 decreased to undetectable levels at 3 h after LPS, returning only partially at 5 h post-injection. CSF ir-endothelin-1 levels were undetectable in saline-treated animals, but reached 21.9 +/- 5.2 fmol ml(-1) at 3 h after LPS treatment. Plasma ir-endothelin-1 levels were unchanged after saline or LPS. Central injection of endothelin-1 (1 pmol, i.c.v.) caused long-lasting increases in body temperature (0.81 +/- 0.17 degrees C, 3 h), but simultaneously decreased tail skin temperature (-1.10 +/- 0.26 degrees C), indicating cutaneous vasoconstriction. Moreover, endothelin-1 induced fever (1.0 +/- 0.3 degrees C, 3 h) when injected into the preoptic area of the anterior hypothalamus (100 fmol), but not i.v. (1 or 10 pmol). These data suggest that endothelin-1 is produced in the brain and acts centrally as a mediator of LPS-induced fever.

The antipyretic effect of dipyrone is unrelated to inhibition of PGE2 synthesis in the hypothalamus

BACKGROUND AND PURPOSE Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)‐dependent and the other, PG‐independent and involving endothelin‐1 (ET‐1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE2 content in plasma, CSF and hypothalamus induced by either LPS or ET‐1.

CCL3/Macrophage inflammatory protein-1α induces fever and increases prostaglandin E2 in cerebrospinal fluid of rats: Effect of antipyretic drugs

The aim of this study was to investigate whether the increase in body temperature caused by intracerebroventricular (i.c.v.) injection of recombinant mouse CCL3/MIP1alpha [C-C (two adjacent conserved cysteines) ligand 3/macrophage inflammatory protein-1alpha] constitutes solely a hyperthermic response or a true integrated fever. Additionally, we examined the effects of systemic administration of different antipyretic drugs including the glucocorticoid dexamethasone, on cerebrospinal fluid (CSF) concentration of prostaglandin (PG) E2 and on febrile response induced by CCL3/MIP1alpha. I.c.v. administration of CCL3/MIP1alpha evokes an integrated fever accompanied by a reduction in tail skin temperature and an increase in PGE2 concentration in the CSF. Dexamethasone and indomethacin markedly reduced the fever and the elevation of CSF PGE2 concentration induced by lipopolysaccharide (LPS) whereas both response evoked by i.c.v. CCL3/MIP1alpha were insensitive to this steroid. Indomethacin only blocked the PGE2 increase in the CSF whereas ibuprofen and celecoxib each blocked the fever and the elevation of CSF PGE2. In this study, we have demonstrated for the first time that CCL3/MIP1alpha evokes an integrated febrile response accompanied by an increase of PGE2 levels in the CSF. These events are dissociated, especially in animals treated with indomethacin. If PGE2 does not participate in the febrile response evoked by CCL3/MIP1alpha, the inhibition of this response by celecoxib and ibuprofen indicates additional mechanisms to the well-known inhibition of COX enzymes by these drugs. Such mechanisms do not seem to depend on cytokine synthesis and subsequent COX-2 induction.

HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) as diagnostic tools for ovarian cancer in patients with a pelvic mass: An Italian multicenter study

OBJECTIVE This multicenter study aims to evaluate HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) performance in the differential diagnosis of epithelial ovarian cancer (EOC). METHODS A total of 405 patients referred to gynecological oncologist with suspicious pelvic mass requiring a surgery for identification of EOC were consecutively enrolled; 387 patients satisfied inclusion criteria: 290 benign diseases; 15 borderline neoplasia and 82 tumors (73 EOC). RESULTS Good diagnostic performance in discriminating benign from EOC patients was obtained for CA125, HE4 and ROMA when calculating optimal cut-off values: premenopause, specificity (SP) >86.6, sensitivity (SN) >82.6, area under the curves (AUC)≥0.894; postmenopause, SP>93.2, SN>82, AUC≥0.928. Fixing SP at 98%, performance indicators obtained for benign vs EOC patients were: premenopause, SN:65.2%, positive predictive value (+PV): 75%, positive likelihood ratio (+LR): 26.4 for CA125; SN:69.6%, +PV:76.2%, +LR:28.1 for HE4; SN:69.6%, +PV: 80%; +LR:35.1 for ROMA; postmenopause, SN:88%, +PV: 95.7%, +LR:38.7 for CA125; SN:78%, +PV:95.1%, +LR:34.3 for HE4; SN:88%, +PV:97.8%, +LR:77.4 for ROMA. When using routine cut-off thresholds, ROMA showed better well-balanced values of both SP and SN (premenopause, SN:87%, SP:86.1%; postmenopause, SN:90%; SP:94.3%). CONCLUSIONS Overall, ROMA showed well balanced diagnostic performance to differentiate EOC from benign diseases. Meaningful differences of +PVs and +LRs between HE4 and CA125 suggest that the two markers may play at least in part different roles in EOC diagnosis, with HE4 seeming to be more efficient than CA125 in ruling in EOC patients in the disease group, also in early stages tumors, both in pre and postmenopause.

Sirtuin 1 stabilization by HuR represses TNF-α- and glucose-induced E-selectin release and endothelial cell adhesiveness in vitro: relevance to human metabolic syndrome.

Chronic inflammation and hyperglycaemia, typical features of metabolic diseases, trigger endothelial damage and release of E-selectin, a marker of endothelial activation. In the present study, we investigated molecular pathways involved in the regulation of endothelial cell activation induced by tumour necrosis factor-α (TNF-α) and high glucose. In cultured human umbilical vein endothelial cells (HUVECs), we studied the role of HuR, an ELAV (embryonic lethal, abnormal vision, Drosophila) family RNA-binding protein, and Sirtuin 1 (SIRT1) on E-selectin release and cell adhesion at different glucose concentrations. HuR expression and binding to SIRT1 were also analysed ex vivo in peripheral blood mononuclear cells (PBMCs) of subjects with and without the metabolic syndrome (MS), by immunoprecipitation (IP) of the ribonucleoprotein (RNP) complex. We found that SIRT1 overexpression prevented TNF-α- and high-glucose-dependent nuclear factor-κB (NF-κB)-p65 acetylation, E-selectin promoter activity, E-selectin release and adhesion of THP-1 cells to HUVECs. The same was mimicked by HuR overexpression, which binds and stabilizes SIRT1 mRNA. Importantly, in PBMCs of individuals with MS compared with those without, SIRT1 expression was lower, and the ability of HuR to bind SIRT1 mRNA was significantly reduced, while plasma E-selectin was increased. We conclude that post-transcriptional stabilization of SIRT1 by HuR represses inflammation- and hyperglycaemia-induced E-selectin release and endothelial cell activation. Therefore, increasing SIRT1 expression represents a strategy to counter the accelerated vascular disease in metabolic disorders.

Evaluation of cell-free DNA in urine as a marker for bladder cancer diagnosis.

The diagnosis and follow-up of bladder cancer are mainly based on cystoscopy, an invasive method which could be negative in case of flat malignancies such as carcinoma in situ. Other noninvasive diagnostic methods have not yet given satisfactory results. There is a need for a reliable yet noninvasive method for the detection of bladder cancer. Our aim was to investigate whether cell-free DNA quantified in urine (ucf-DNA) could be a useful marker for the diagnosis of bladder cancer. A standard urine test was performed in 150 naturally voided morning urine samples that were processed to obtain a quantitative evaluation of ucf-DNA. Leukocyturia and/or bacteriuria were found in 18 subjects, who were excluded from the study. Statistical analysis was performed on 45 bladder cancer patients and 87 healthy subjects. Ucf-DNA was extracted from urine samples by a spin column-based method and quantified using four different methods: GeneQuant Pro (Amersham Biosciences, Pittsburg, PA, USA), Quant-iT DNA high-sensitivity assay kit (Invitrogen, Carlsbad, CA, USA), Real-Time PCR (Applied Biosystems, Foster City, CA, USA), and NanoDrop 1000 (NanoDrop Technologies, Houston, TX, USA). Median free DNA quantification did not differ statistically between bladder cancer patients and healthy subjects. A receiver-operating characteristic (ROC) curve was developed to evaluate the diagnostic performance of ucf-DNA quantification for each method. The area under the ROC curve was 0.578 for GeneQuant Pro, 0.573 for the Quant-iT DNA high-sensitivity assay kit, 0.507 for Real-Time PCR, and 0.551 for NanoDrop 1000, which indicated that ucf-DNA quantification by these methods is not able to discriminate between the presence and absence of bladder cancer. No association was found between ucf-DNA quantification and tumor size or tumor focality. In conclusion, ucf-DNA isolated by a spin column-based method and quantified by GeneQuant Pro, Quant-iT DNA high-sensitivity assay kit, Real-Time PCR or NanoDrop 1000 does not seem to be a reliable marker for the diagnosis of bladder cancer.