Interstitial Outburst of Angiogenic Factors During Skeletal Muscle Regeneration After Acute Mechanical Trauma

Angiogenesis is a key event during tissue regeneration, but the intimate mechanisms controlling this process are still largely unclear. Therefore, the cellular and molecular interplay along normal tissue regeneration should be carefully unveiled. To this matter, we investigated by xMAP assay the dynamics of some angiogenic factors known to be involved in tissue repair, such as follistatin (FST), Placental Growth Factor‐2 (PLGF‐2), epidermal growth factor (EGF), betacellulin (BTC), and amphiregulin (AREG) using an animal model that mimics acute muscle contusion injuries. In situ immunofluorescence was used for the evaluation and tissue distribution of their cellular sources. Tissue levels of explored factors increased significantly during degeneration and inflammatory stage of regeneration, peaking first week postinjury. However, except for PLGF‐2 and EGF, their levels remained significantly elevated after the inflammatory process started to fade. Serum levels were significantly increased only after 24 h for AREG and EGF. Though, for all factors except FST, the levels in injured samples did not correlate with serum or contralateral tissue levels, excluding the systemic influence. We found significant correlations between the levels of EGF and AREG, BTC, FST and FST and AREG in injured samples. Interstitial cells expressing these factors were highlighted by in situ immunolabeling and their number correlated with measured levels dynamics. Our study provides evidence of a dynamic level variation along the regeneration process and a potential interplay between selected angiogenic factors. They are synthesized, at least partially, by cell populations residing in skeletal muscle interstitium during regeneration after acute muscle trauma. Anat Rec, 298:1864–1879, 2015. © 2015 Wiley Periodicals, Inc.     

7‐keto‐DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field

7‐keto‐DHEA (3β‐hydroxy‐androst‐5‐ene‐7,17‐dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7‐keto‐DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst‐5‐ene‐7,17‐dione) and 7‐keto‐DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro‐, and sulpho‐conjugated steroids extracted from urine samples obtained before and after the administration of 7‐keto‐DHEA were analyzed by different gas chromatographic (GC)–mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7‐keto‐DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7‐keto‐DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used.     

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC‐C‐IRMS

Prednisone and prednisolone are two anti‐inflammatory steroidal drugs listed by the World Anti‐Doping Agency (WADA) within the class of glucocorticoids, which are prohibited “in competition” and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC‐C‐IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ¹³C value reference range (?28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC‐C‐IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro‐11‐deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC‐C‐IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.     

Effect of copper exposure and recovery period in reared Diplodus sargus

The aquaculture growth can be followed by the occurrence of more and new pathogenic agents, since the production leads to higher fish densities in confined areas more appropriate to the appearance and propagation of pathologies. Copper sulfate has been widely used in preventing and controlling fish parasites. The objective of this study is to investigate the effects of copper treatments in the fish tissues (bioaccumulation and histological changes in different organs), mortality and evaluate what happens during the recovery period. White sea bream (Diplodus sargus) were exposed to copper sulfate (0.25 and 0.5 mg L⁻¹) during 60 days followed with a 75-day recovery period. The results showed that the concentration of copper in fish liver was significantly higher in the 0.5 mg L⁻¹ treatment than in the 0.25 mg L⁻¹ treatment. Conversely, copper load in the muscle did not differ significantly between treatments and control. Copper levels in muscle, and especially in liver, increased during copper exposure (up to 60 days). In summary, at higher concentrations copper sulfate treatment (0.5 mg L⁻¹) might be toxic to fish, which showed histological alterations and copper accumulation in their tissues, mainly in the liver. Nevertheless, individuals returned to their original state after a 75-day recovery period and the tested copper concentrations does not represents risk for food safety.

     

Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.     

Phase II study of sorafenib in patients with relapsed or refractory lymphoma

The safety and activity of the multikinase inhibitor sorafenib were investigated in patients with relapsed or refractory lymphoproliferative disorders who received sorafenib (400 mg) twice daily until disease progression or appearance of significant clinical toxicity. The primary endpoint was overall response rate (ORR). Biomarkers of sorafenib activity were analysed at baseline and during treatment. Thirty patients (median age, 61 years; range, 18-74) received a median of 4 months of therapy. Grade 3-4 toxicities included hand/foot skin reactions (20%), infections (12%), neutropenia (20%) and thrombocytopenia (14%). Two patients achieved complete remission (CR), and two achieved partial remission (PR) for an ORR of 13%. Stable disease (SD) and progressive disease (PD) was observed in 15 (50%) and 11 patients (37%), respectively. The median overall survival (OS) for all patients was 16 months. For patients who achieved CR, PR and SD, the median time to progression and OS was 5 and 24 months, respectively. Compared with patients with PD, responsive patients had significantly higher baseline levels of extracellular signal-regulated kinase phosphorylation and autophagy and presented a significant reduction of these parameters after 1 month of therapy. Sorafenib was well tolerated and had a clinical activity that warrants development of combination regimens.