A hydrolytically‐tunable photocrosslinked PLA‐PEG‐PLA/PCL‐PEG‐PCL dual‐component hydrogel that enhances matrix deposition of encapsulated chondrocytes

In this study, a series of photocrosslinked hydrogels were designed composed of both poly(lactide)‐poly(ethylene glycol)‐poly(lactide) (PEL) and poly(ε‐caprolactone)‐poly(ethylene glycol)‐poly(ε‐caprolactone) (PEC) macromers. The PEL/PEC hydrogels at ratios of 100:0, 75:25, 50;50, 25:75 and 0:100 were studied for their degradation characteristics and their ability to support chondrogenesis of encapsulated chondrocytes. Difference in hydrolytic susceptibility between copolymers led to different degradation patterns where higher PEC content correlated with slower degradation. Increased chondrogenic gene expression was observed in chondrocyte‐laden hydrogels within a 4‐week culture period. Biochemical and histological evaluations revealed significant accumulation of extracellular matrix proteins such as glycosaminoglycans and collagen in the 50/50 hydrogel owing to appropriate tuning of hydrogel degradation. These results demonstrate that the dual‐component photocrosslinked hydrogel system is suitable for use as scaffold to support chondrogenesis and, moreover, the tunability of these systems opens up possibilities for use in different cell culturing applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Falcipain‐2 inhibitors

Malaria, particularly that one caused by Plasmodium falciparum, remains a serious health problem in Africa, South America, and many parts of Asia where it afflicts about 500 million people and is responsible for the death of more than one million children each year. The main reasons for the persistence of malaria are the emergence of resistance to common antimalarial drugs, inadequate control of mosquito vectors, and the lack of effective vaccines. Therefore, the identification and characterization of new targets for antimalarial chemotherapy are of urgent priority. This review is focused on inhibitors of falcipain‐2, a cysteine protease from P. falciparum, which represents one of the most promising targets for antimalarial drug design. Falcipain‐2 is a key enzyme in the life cycle of P. falciparum since it degrades hemoglobin, at the early trophozoite stage, and cleaves ankyrin and protein 4.1, the cytoskeletal elements vital to the stability of red cell membrane, at the schizont stage. The main classes of falcipain‐2 inhibitors are peptides or peptidomimetics bearing the most popular pharmacophores of cysteine protease inhibitors, such as vinyl sulfones, halomethyl ketones, and aldehydes. Furthermore, many other chemotypes have been identified as inhibitors of falcipain‐2, such as isoquinolines, thiosemicarbazones, and chalcones. These inhibitors represent all classes, which, to the best of our knowledge, have been disclosed in journal articles to date. © 2009 Wiley Periodicals, Inc. Med Res Rev, 30, No. 1, 136–167, 2010     

Evidence that phospholipase A2 (PLA2) inhibitors can selectively block PLA2-mediated contractions of guinea pig lung pleural strips.

We have demonstrated previously that porcine pancreatic phospholipase A2 (PLA2)-induced contractions of guinea pig lung pleural strips can be abated by inhibitors of cyclooxygenase and 5-lipoxygenase pathways, suggesting the liberation of arachidonic acid. To validate further the involvement of a PLA2-related mechanism, the effects of three known inhibitors of PLA2 were evaluated. Manoalogue, an irreversible inhibitor of PLA2, parabromophenacyl bromide, an irreversible, active site directed inhibitor and a transition-state analog, a competitive inhibitor of PLA2 were used. Transition-state analog (3-30 microM), added to the tissues for 30 min before PLA2, shifted the PLA2 curves to the right in a concentration-related manner. In contrast, the reported inactive enantiomer of transition-state analog failed to alter the PLA2 curves. Manoalogue and para-bromophenacyl bromide, at concentrations up to 40- and 50-fold higher than the enzyme concentration, respectively, were incubated in Krebs' buffer with the enzyme for 24 hr before challenging the tissues. Under these conditions, the PLA2-induced contractions were suppressed markedly. In contrast, neither reduced nor methylated manoalogue, incubated at a 20-fold molar excess with PLA2 for 24 hr, suppressed the maximal PLA2-induced responses. These results demonstrate that inhibitors of secretory PLA2, acting by different mechanisms, can alter the contractile responses induced by PLA2 on pleural strips from guinea pig lung.     

(E)‐2‐benzylidene‐4‐phenyl‐1,3‐diselenole has antioxidant and hepatoprotective properties against oxidative damage induced by 2‐nitropropane in rats

The in vitro effect of (E)‐2‐benzylidene‐4‐phenyl‐1,3‐diselenole (BPD) was evaluated through iron/EDTA‐induced thiobarbituric acid reactive species (TBARS) and reactive species (RS) determinations as well as of the scavenging 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH) radical quantification. BPD at the concentrations of 10 and 50 μΜ decreased RS and TBARS levels, respectively. The antioxidant activity was not related to the scavenging DPPH radical mechanism. A second objective of this study was to investigate the hepatoprotective action of BPD, administered by oral route, against oxidative damage induced by 2‐nitropropane (2‐NP) (100 mg/kg of body weight) in liver of rats. At the dose of 50 mg/kg, BPD protected against the increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities induced by 2‐NP. BPD (10 and 50 mg/kg) protected against the increase in TBARS levels and alkaline phosphatase (ALP) activity. Sections of liver from 2‐NP‐exposed rats presented intense infiltration of inflammatory cells and loss of cellular architecture. BPD (10 and 50 mg/kg) attenuated 2‐NP‐induced hepatic histological alterations. The inhibition of δ‐aminolevulinic dehydratase (δ‐ALA‐D), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S‐transferase (GST) activities and the decreased GSH levels caused by 2‐NP were protected by BPD (50 mg/kg). Catalase activity and ascorbic acid levels were not altered by 2‐NP. These results demonstrated the antioxidant and hepatoprotective effects of BPD in liver of rats.     

Biological activity of extracellular matrix‐associated BMP‐2

The critical requirement for matrix‐associated bone morphogenetic proteins (BMPs) during induction of bone formation in vivo has long been recognized. However, the role of extracellular matrix (ECM) physisorbed BMPs in inducing the differentiation of resident mesenchymal stem cells into osteoblasts has been ill‐defined. We therefore used BMP‐responsive C2C12s to study the biological activity of collagen type I physisorbed BMP‐2. Fibrillar collagen type I scaffolds were loaded with 75 ng BMP‐2/µg collagen. Under cell culture conditions, 40% of loaded ¹²⁵I‐labelled BMP‐2 was released within 24 h, whereas the remaining BMP‐2 was stably physisorbed for > 7 days. Using these systems suggested that physisorbed BMP‐2 is more active than diffusible BMP‐2. Thus, the current clinical practice of immobilizing BMPs on collagen type I scaffolds not only prolongs local delivery of the morphogen but could also enhance biological activity at the cellular level. Copyright © 2009 John Wiley & Sons, Ltd.     

Dapsone‐induced agranulocytosis—possible involvement of low‐activity N‐acetyltransferase 2

Dapsone‐induced agranulocytosis is a rare but potentially fatal adverse drug reaction (ADR). A 45‐year‐old male Caucasian patient developed agranulocytosis caused by dapsone (diamino‐diphenyl sulfone), which he was prescribed for leukocytoclastic vasculitis. Patient's treatment consisted of termination of dapsone, antibiotic therapy, and granulocyte colony‐stimulating factor leading to prompt improvement of symptoms and normalization of laboratory blood values. Diagnostic evaluation revealed methemoglobinemia and excluded glucose‐6‐phosphate dehydrogenase deficiency. Pharmacogenetics testing showed that he was a carrier of NAT2 *5/*6 genotype, predisposing to low activity of the N‐acetyltransferase 2 enzyme. This was the first and only ADR to dapsone reported in Croatia. In total, there have been 73 ADR to dapsone recorded worldwide, including only four cases of agranulocytosis.     

Endothelium‐dependent and endothelium‐independent effects of 1‐nitro‐2‐propylbenzene on rat aorta

A group of nitro compounds contains a benzene ring in a short aliphatic chain with the NO₂ group, property that supposedly favors its vasodilator profile. In this study, we evaluated in isolated rat aorta the effects of 1‐nitro‐2‐propylbenzene (NPB), a nitro compound containing the NO₂ in the aromatic ring. In aorta precontracted with KCl, NPB (1‐3000 μm ) induced full endothelium‐independent relaxation. In endothelium‐intact preparations, phenylephrine‐induced contractions were fully relaxed by NPB, effect unaltered by N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). In the concentration range of 30–300 μm , NPB slightly but significantly potentiated the phenylephrine‐induced contraction. Such potentiation was unaltered by the thromboxane‐prostanoid receptor antagonist seratrodast, but was abolished by endothelium removal or by preincubation of endothelium‐intact preparations with L‐NAME, ODQ or by ruthenium red and HC‐030031, blockers of subtype 1 of ankyrin transient receptor potential (TRPA₁) channels. Verapamil exacerbated the potentiating effect of NPB. The potentiating effect was undetectable in preparations precontracted by 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (U‐46619). Relaxation was reduced by ruthenium red while it was enhanced by HC‐030031. In conclusion, NPB has vasodilator properties but with a mechanism of action distinct from its analogues. Contrary to other nitro compounds, its relaxing effects did not involve recruitment of the guanylyl cyclase pathway. NPB has also endothelium‐dependent potentiating properties on phenylephrine‐induced contractions, a phenomenon that putatively required a role of endothelial TRPA₁ channels. The present findings reinforce the notion that the functional group NO₂ in the aliphatic chain of these nitro compounds determines favorably their vasodilator properties.     

D‐ and L‐[123I]‐2‐I‐phenylalanine show a long tumour retention compared with D‐ and L‐[123I]‐2‐I‐tyrosine in R1M rhabdomyosarcoma tumour‐bearing Wag/Rij rats

The aim of this study was the comparison of the tumour uptake and the long‐term retention of [¹²³I]‐2‐I‐L ‐phenylalanine and [¹²³I]‐2‐I‐D ‐phenylalanine with those of [¹²³I]‐2‐I‐L ‐tyrosine and [¹²³I]‐2‐I‐D ‐tyrosine in R1M rhabdomyosarcoma tumour‐bearing rats. The biodistribution of the radioactivity as a function of time in R1M tumour‐bearing rats was measured by planar gamma camera imaging (dynamic and static). If dissection was applied, the activity in the tumours and tissues of interest was measured by gamma counting. [¹²³I]‐2‐iodo‐L ‐phenylalanine, [¹²³I]‐2‐iodo‐D ‐phenylalaine, [¹²³I]‐2‐I‐L ‐tyrosine showed a considerable tumour uptake reaching a maximum between 10 and 30 min. At 30 min p.i. the differential uptake ratio values of this uptake were, respectively, 2.1, 2.3, 2.5 and 1.7. The activity in the tumour was shown to be related to a tumour cell uptake and not to an increased blood pool activity. All the tracers showed a clearance from the blood to the bladder without renal retention. At longer times both L ‐ and D ‐ [¹²³I]‐2‐I‐tyrosine were cleared for a large part from the tumours and the body. [¹²³I]‐2‐I‐L ‐Phe and [¹²³I]‐2‐I‐D ‐Phe showed a considerable and equal retention in the tumours: as compared with 0.5 h, 91% at 24 h and 80% at 48 h. This was related to the longer retention of activity in the blood pool noticed for these compounds (81% at 24 h and 65% at 48 h). The tumour‐to‐background ratio increased with 25% at those longer times. At short times all the tracers were taken up to a considerable extent in the tumours. In the R1M‐bearing Wag/Rij rat model only [¹²³I]‐2‐I‐L ‐phenylalanine and [¹²³I]‐2‐I‐D ‐phenylalanine showed an especially high retention at long times without any significant difference between the enantiomers. Copyright © 2007 John Wiley & Sons, Ltd.     

Effects of transgene‐mediated endomorphin‐2 in inflammatory pain

We examined the analgesic properties of endomorphin‐2 expressed in DRG neurons transduced with a non‐replicating herpes simplex virus (HSV)‐based vector containing a synthetic endomorphin‐2 gene construct. HSV‐mediated endomorphin‐2 expression reduced nocisponsive behaviors in response to mechanical and thermal stimuli after injection of complete Freund's adjuvant (CFA) into the paw, and reduced peripheral inflammation measured by paw swelling after injection of CFA. The analgesic effect of the vector was blocked by either intraperitoneal or intrathecal administration of naloxone methiodide, blocking peripheral and central μ opioid receptors, respectively. Endomorphin‐2 vector injection also reduced spontaneous pain‐related behaviors in the delayed phase of the formalin test and in both CFA and formalin models suppressed spinal c‐fos expression. The magnitude of the vector‐mediated analgesic effect on the delayed phase of the formalin test was similar in naïve animals and in animals with opiate tolerance induced by twice daily treatment with morphine, suggesting that there was no cross‐tolerance between vector‐mediated endomorphin‐2 and morphine. These results suggest that transgene‐mediated expression of endomorphin‐2 in transduced DRG neurons in vivo acts both peripherally and centrally through mu opioid receptors to reduce pain perception.     

Electrospun fibrinogen–PLA nanofibres for vascular tissue engineering

Here we report on the development of a new type of hybrid fibrinogen–polylactic acid (FBG–PLA) nanofibres (NFs) with improved stiffness, combining the good mechanical properties of PLA with the excellent cell recognition properties of native FBG. We were particularly interested in the dorsal and ventral cell response to the nanofibres' organization (random or aligned), using human umbilical endothelial cells (HUVECs) as a model system. Upon ventral contact with random NFs, the cells developed a stellate‐like morphology with multiple projections. The well‐developed focal adhesion complexes suggested a successful cellular interaction. However, time‐lapse analysis shows significantly lowered cell movements, resulting in the cells traversing a relatively short distance in multiple directions. Conversely, an elongated cell shape and significantly increased cell mobility were observed in aligned NFs. To follow the dorsal cell response, artificial wounds were created on confluent cell layers previously grown on glass slides and covered with either random or aligned NFs. Time‐lapse analysis showed significantly faster wound coverage (within 12 h) of HUVECs on aligned samples vs. almost absent directional migration on random ones. However, nitric oxide (NO) release shows that endothelial cells possess lowered functionality on aligned NFs compared to random ones, where significantly higher NO production was found. Collectively, our studies show that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization. Copyright © 2016 John Wiley & Sons, Ltd.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

MR molecular imaging of HER‐2 in a murine tumor xenograft by SPIO labeling of anti‐HER‐2 affibody

In vivo molecular imaging is a rapidly growing research area both for basic and clinical science. Non‐invasive imaging of in vivo conditions at the molecular level increases understanding of the biological characteristics of normal and diseased tissues without the need for invasive surgical procedures. Among the various imaging modalities, magnetic resonance imaging (MRI) has garnered interest as a molecular imaging modality due to its high spatial resolution. Here, we have demonstrated that the combined use of HER‐2 targeting affibody, a small 7 kDa molecule that behaves similarly to antibodies, and superparamagnetic iron oxide (SPIO) can non‐invasively image HER‐2 expressing cells or tissues both in vitro and in vivo by MRI. This preliminary study demonstrates that affibody‐SPIO is a feasible, target‐specific contrast agent for in vivo MR molecular imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Periodontal regeneration using an injectable bone cement combined with BMP‐2 or FGF‐2

Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein‐2 (BMP‐2) or fibroblast growth factor‐2 (FGF‐2). After creating 30 periodontal defects in 15 Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP‐2 and CaP + FGF‐2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP‐2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP‐2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF‐2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP‐2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP + FGF‐2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP‐2 group. The combination of a topical application of FGF‐2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Clinical application of 18F‐fluoro‐2‐deoxy‐D‐glucose positron emission tomography/computed tomography in breast cancer

Positron emission tomography (PET)/computed tomography (CT) is not suited for primary diagnostics of breast tumours and it cannot replace sentinel lymph node technique in determining metastases to the axilla. PET/CT has a high sensitivity and specificity regarding the detection of loco‐regional recurrence and metastases to mediastinal and internal mammary lymph nodes, as well as distant metastases. Whether the method can replace conventional methods, or be a supplement when this is non‐conclusive, remains unresolved. PET/CT cannot be recommended for routine follow‐up but is recommended in patients with suspected relapse when conventional imaging has given equivocal results. PET/CT can be applied to confirm isolated loco‐regional relapse or metastatic lesion detected by conventional imaging. PET/CT has a high sensitivity for detecting response to treatment, but a low specificity calls for cautions. Further investigations into the use of PET/CT to predict and monitor response are warranted, before this approach may find its way into a clinical setting. In the future, PET/CT will probably find increasing use in treatment planning and evaluation of patients with breast cancer.