In Vitro Evaluation of the Biological Availability of Hyaluronic Acid Polyethylene Glycols-Cross-Linked Hydrogels to Bovine Testes Hyaluronidase.

During last years, hyaluronic acid- (HA-) based dermal fillers have grown rapidly and continuously, as reported by the American Society of Aesthetic Plastic Surgery (ASAPS). In fact, HA fillers are considered the gold standard technique for soft tissue augmentation, deep skin hydration, and facial recontouring, playing a key role as an alternative to plastic surgery. HA fillers are less invasive, more biocompatible, and safer and with a more natural and immediate result if compared to plastic surgery. Hence, the safety of HA-based dermal fillers plays a crucial role, mostly in terms of biocompatibility and adjustability in case of unpleasant results and side effects such as, tyndall effect, edema, or granulomas. Hyaluronidase is a naturally occurring enzyme, present in the human body, and can degrade HA fillers avoiding more severe complications. In this article, we analyzed the bioavailability of hyaluronidase degradation of five fillers of Neauvia® hydrogels line (MatexLab SA, Lugano, CH), composed of pure hyaluronic acid and based on PEGDE cross-linking (polyethylene glycol) technology that guarantees a higher biocompatibility and an optimal biointegration and rheological characteristics. The performed in vitro testing is based on the colorimetric determination of the N-acetyl-D-glucosamine (NAG) present in solution after incubation with hyaluronidase, determined at different time points in order to assess the kinetic of each product degradation (1h, 3h, 6h, 24h, 48h, 72h, 120h, and 168h). The aim of this study was to assess, in vitro, how the difference in HA content and PEGDE concentration of the analyzed fillers can influence the product biocompatibility, intended as product enzymatic clearance and duration in time. The results demonstrated that the method was reproducible and easy to perform and that all the analyzed fillers are naturally immediately available for hyaluronidase-mediated degradation.

Effects of mutations in the post-translational modification sites on the trafficking of hyaluronan synthase 2 (HAS2).

Vesicular trafficking of hyaluronan synthases (HAS1-3) from endoplasmic reticulum (ER) through Golgi to plasma membrane (PM), and either back to endosomes and lysosomes, or out into extracellular vesicles, is important for their activities. We studied how post-translational modifications affect the trafficking of HAS2 by mutagenesis of the sites of ubiquitination (K190R), phosphorylation (T110A) and O-GlcNAcylation (S221A), using Dendra2- and EGFP-HAS2 transfected into COS1 cells. Confocal microscopy showed HAS2 wild type (wt) and its K190R and S221A mutants in ER, Golgi and extracellular vesicles, while the T110A mutant remained mostly in the ER. HA synthesis was reduced by S221A, while completely blocked by K190R and T110A. Cell-surface biotinylation indicated that T110A was absent from PM, while S221A was close to the level of wt, and K190R was increased in PM. TIRF microscopy analysis gave similar results. Rab10 silencing increased HA secretion by HAS2, likely by inhibiting endocytosis of the enzyme from PM, as reported before for HAS3. Green-to-red photo-conversion of Dendra2-HAS2 constructs suggested slower decay of K190R and S221A than HAS2 wt, while T110A was barely degraded at all. S221D and S221E, the phosphomimetic mutants of this site, decayed faster and blocked hyaluronan synthesis, suggesting alternative O-GlcNAc/-PO4 substitution to regulate the stability of the enzyme. Probing the role of dynamic O-GlcNAcylation at S221 by adding glucosamine increased the half-life of only HAS2 wt. The Dendra2·HAS2 disappearance from Golgi was slower for K190R. Of the two inactive constructs, K190R co-transfected with HAS2 wt suppressed, whereas T110A had no effect on HA synthesis. Interestingly, the HAS2-stimulated shedding of extracellular vesicles was dependent on HAS residence in PM but independent of HA synthesis. The results indicate that post-translational modifications control the trafficking of HAS2, and that trafficking is an integral part of the post-translational regulation of HAS2 activity.

Human ß2-defensin in oral lichen planus expresses the degree of inflammation.

Syndrome (BMS). Seventeen OLP patients, with a positive histopathologic diagnosis of the disease, were recruited into this study in order to measure the relative quantity of HBD-2 in their saliva and crevicular fluid. The values were compared with those collected from a group of 9 patients affected by the Burning Mouth Syndrome (BMS) and with a control group (CTRL) of 9 patients. There was no statistically significant difference between the groups (p=0.523; p=0.897). However, patients affected by OLP showed a dycotomic distribution of values: while 10 of them showed similar values to those found out in the other two groups, 7 patients expressed high levels of HBD-2 and 3500 pg/ml was the threshold to distinguish the subgroups. During the dental visit the clinician classified OLP patients into two groups according to the clinical presentation of the disease: reticular and hyperplastic (white OLP), atrophic and erosive forms (red OLP). There was a statistical significant correlation between the clinical and numeric classification of the patients (p=0.004; p=0.001), and the expression of HBD-2 was higher in the red OLP group than in the white OLP group (p=0.000; p=0.000). In conclusion, this study shows that HBD-2 represents an index to assess active inflammation and it is probably linked to the presence of the typical band-like CD8+ infiltrate in Oral Lichen Planus.

Emerging resistant bacteria strains in bloodstream infections of acute leukaemia patients: results of a prospective study by the Rete Ematologica Lombarda (Rel).

Multiresistant bacterial infections are a potentially life-threatening condition in acute leukaemia (AL) patients. We aimed to better define the very recent epidemiology and outcome of bloodstream infections (BSIs) in a real-life setting. We prospectively collected all consecutive febrile/infectious episodes occurring in AL patients admitted to 9 haematology units. In 293 AL patients, 433 BSIs were diagnosed. Gram-positive (GP) bacteria were isolated in 44.8 % BSI and Gram-negative (GN) in 38.3 %, while polymicrobial aetiology- or fungi-related events were identified in 15.7 and 1.1 % of the cases, respectively. GP was observed more frequently in patients not in complete remission (p = 0.04), while GN during consolidation cycles (p = 0.003). Extended spectrum ß-lactamase-producing strains accounted for 23.2 % of enterobacteria. They were associated with previous antibiotic exposure, including fluoroquinolones prophylaxis (p = 0.01). Carbapenem-resistant (CR) strains occurred in 9 % of enterobacteria. Among Pseudomonas aeruginosa strains, 21.6 % were multiresistant. Overall 30-day mortality was 8.5 %. CR GN and multiresistant P. aeruginosa BSIs were independent predictors of death (p = 0.002), as well as relapsed/resistant AL (18.3 %; p = 0.0002) and the presence of pulmonary infiltrates (26.6 %; p < 0.001). Although GP still predominate over GN BSI, the percentage of antibiotic resistant GN strains is considerable in AL patients and it is associated with poor prognosis.

Endometrial cancer cells can express fibrinogen: Immunohistochemistry and RT-PCR analysis.

We investigated whether endometrial cancer (EC) cells can express fibrinogen. Consecutive patients treated for EC were enrolled (cases). A control group of women who had hysterectomy for benign conditions was identified in a case:control ratio of 4:1. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were performed to identify the presence of fibrinogen and the mRNA of its three chains (α, ß, γ) in the tissue specimens from both cases and controls. Sixteen EC cases and 4 benign controls were included. Immunohistochemistry failed in one case of EC. In 12/15 (80%) cases versus 0 controls, a moderate-to-intense positivity for fibrinogen was observed (p = 0.09; OR: 32.1; 95%CI: 1.4-752.9). Six (37.5%) women among the cases versus 0 controls expressed RNA for at least one chain of fibrinogen (p = 0.25). All the cases (6/6, 100%) with positive RT-PCR had moderate-to-intense positive immunohistochemistry. Molecular and immunohistochemistry show that some cases of EC have the capability to express fibrinogen and the mRNA of at least one of its chains.

Pregnancy outcomes and the use of two standards to assess adequacy of maternal body mass index in early gestation.

The maternal weight gain chart proposed by Rosso and Mardones (RM) was subsequently modified by Atalah et al. (AEA). Both charts are widely used in Latin America. The purpose of this study was to compare birth length (BL) and birth weight (BW) outcomes of both charts. A prospective study of pregnant women and their offspring's was performed in Santiago, Chile. From a total sample of 27,613 pregnant women a sub-sample of 11,465 term healthy singleton pregnant women was selected for additional analyses. κ statistics was used to study the degree of agreement of both charts in the diagnosis of maternal nutritional status. Obese and underweight women were classified using both standards at the beginning of pregnancy and compared in terms of BL4250 g proportions. Sensitivity and specificity values of at risk newborns, whose categories were considered as gold standard, were obtained for obese and underweight women of each chart. There was a moderate agreement in the nutritional classification of these charts. Proportions of BL4250 g were similar at each nutritional category; however, absolute figures for at risk newborns were much higher in the RM underweight and obese women. The RM chart showed higher sensitivity values than the AEA chart. The higher sensitivity of the RM chart would support its use for prevention purposes. This chart is advisable for Latin American countries and also for most developing countries.

Correlation between unstimulated salivary flow, pH and streptococcus mutans, analysed with real time PCR, in caries-free and caries-active children.

AIM: Evaluate the correlations between unstimulated salivary flow, pH and level of S. mutans, analysed through real time PCR, in caries-free and caries-active children. MATERIALS AND METHODS: Thirty healthy children were divided into 2 groups: test group (DMFT/dmft ≥ 3 and at least 1 active caries lesion) and control group (DMFT/dmft=0). Un-stimulated saliva was collected, pH was measured and S. mutans and total bacterial amount were evaluated with real-time PCR analysis. RESULTS: Unstimulated salivary flow in the test group was significantly lower (p = 0.0269) compared to group control. The level of S. mutans was higher in the test group (p = 0.176), and an inverse correlation was recorded between total bacterial amount and un-stimulated salivary flow (p = 0.063). In the control group a positive relationship was found between total bacterial amount and S. mutans (p = 0.045) and an inverse correlation between pH and S. mutans (p = 0.088). A t-test and a linear regression analysis were performed. CONCLUSION: A higher salivary flow and an increased salivary pH seem to represent protective factors against caries in children, while high levels of S. mutans are correlated with caries active lesions. Caries risk assessment should be performed considering all parameters involved in the development of the disease.

Dynamic interplay between breast cancer cells and normal endothelium mediates the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells.

BACKGROUND: Breast cancer-endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells. METHODS: To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties. RESULTS: BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome ß5 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC. CONCLUSIONS AND GENERAL SIGNIFICANCE: BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Impact of respiratory pattern on lung mechanics and interstitial proteoglycans in spontaneously breathing anaesthetized healthy rats.

AIM: The aim of this study was to investigate the effect of different pattern of spontaneous breathing on the respiratory mechanics and on the integrity of the pulmonary extracellular matrix. METHODS: Experiments were performed on adult healthy rats in which different spontaneously breathing pattern was elicited through administration of two commonly used anaesthetic mixtures: pentobarbital/urethane (P/U) and ketamine/medetomidine (K/M). The animals (five per group) were randomized and left to spontaneously breath for 10 min (P/U-sham; K/M-sham) or for 4h (P/U-4h; K/M-4h), targeting the anaesthesia level to obtain a tidal volume of about 8 mL kg(-1) body wt. At the end of the experiment, lung matrix integrity was assessed through determination of the glycosaminoglycans (GAGs) content in the lung parenchyma. RESULTS: Compared with K/M, anaesthesia with P/U cocktail induced: (1) a higher respiratory rate and minute ventilation attained with lower P(a) CO(2) ; (2) a higher pressure-time-product and work of breathing per minute; (3) a lower static lung compliance; (4) an increased activation of lung tissue metalloproteases; and (5) greater extraction of pulmonary interstitial GAGs. CONCLUSIONS: This study suggests that the breathing pattern induced by the different anaesthetic regimen may damage the pulmonary interstitium even during spontaneous breathing at physiological tidal volumes.

Glycosaminoglycans as key molecules in atherosclerosis: the role of versican and hyaluronan.

Cardiovascular disease is the largest cause of death in Western societies and it primarily results from atherosclerosis of large and medium-sized vessels. Atherosclerosis leads to myocardial infarction, when it occurs in the coronary arteries, or stroke, when it occurs in the cerebral arteries. Pathological processes involved in macrovascular disease include the accumulation of lipids which are retained by extracellular matrix (ECM) molecules, especially by the chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans (CS/DSPGs), such as versican, biglycan and decorin. The sulfation pattern of CS is a key player in protein interactions causing atherosclerosis. Several studies have shown that lipoproteins bind CSPGs via their glycosaminoglycan chains. Galactosaminoglycans, such as CS and DS, bind low density lipoproteins (LDL), affecting the role of these molecules in the arterial wall. In this article, the role of CS and versican in atherosclerosis and hyaluronan in atherogenesis as well as the up to date known mechanisms that provoke this pathological condition are presented and discussed.

Correlations between biochemical markers in the synovial fluid and severity of rotator cuff disease.

The role of biochemical factors in the onset and natural history of rotator cuff disease is not fully understood, but it is generally recognised that they could induce tendon damage in association with mechanical and vascular factors. In this study, 5 biochemical parameters were analysed (total protein concentration, matrix metalloproteinase (MMP)-2 or gelatinase A, MMP-9 or gelatinase B, type I collagen telopeptides, hyaluronic acid) in the synovial fluid (SF) aspirated from the gleno-humeral joint of 29 patients undergoing surgical therapy for rotator cuff lesions. Four different groups of patients were identified according to the severity of the lesion: partial tear of the rotator cuff, full thickness tear involving 1 tendon and cuff tear arthropathy (CTA). The total SF protein concentration progressively increased with loss of integrity of the rotator cuff, reaching the highest levels in CTA. The absolute enzymatic activity of gelatinases was greater in full thickness tears than in partial tears, while it decreased in CTA. Conversely, the ratio between gelatinases and total protein content reached the highest level in partial tears and then progressively decreased. Collagen I telopeptides were significantly increased in full thickness tears and CTA, whereas the levels of hyaluronic acid decreased with worsening of rotator cuff disease. These findings support the hypothesis that gelatinases, which are involved in physiological tendon remodelling, intervene in the evolution of rotator cuff disease, too. Increased levels of type I collagen telopeptides give evidence that tendon tears are associated with an anatomic loss of tendon tissue and not with simple tendon retraction.

Murine abortion is associated with enhanced hyaluronan expression and abnormal localization at the fetomaternal interface.

The remodelling of the endometrial architecture is fundamental to create a suitable environment for the establishment of pregnancy. During this process, substantial alterations in the composition of maternal extracellular matrix play an important role by providing a prosperous medium for implantation as well as modulating trophoblast invasion leading to the formation of a functional placental unit. Hyaluronan is a conspicuous component of the extracellular matrix, particularly in remodelling tissues undergoing regeneration and repair. During gestation, changes in HA deposition and distribution indicate that this molecule may participate in preparation of the endometrial stroma for reception and implantation of the embryo. However, little is known about the role of hyaluronan at the fetomaternal interface, specially regarding its influence in pregnancy outcome. In the present study we show increased decidual hyaluronan levels in spontaneous abortion compared with normal pregnancy mice on gestation day 7.5. Both in normal and pathologic pregnancies, high molecular size hyaluronan was found at the fetomaternal unit. However, hyaluronan metabolism (which results from the activity of hyaluronan synthases and hyaluronidases) seems to be altered in spontaneous abortion as shown by a decrease in Hyal-3 expression as well as by differences in hyaluronan molecular size spectrum. This alteration in hyaluronan metabolism in spontaneous abortion could explain its increased concentration observed in decidua and the abnormal distribution of hyaluronan around the embryo implantation crypt. Thus, increased decidual hyaluronan levels resulting from abnormal deposition and turn over may contribute to the pathogenesis of pregnancy failure.

C-reactive protein production in term human placental tissue.

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Development of lung edema: interstitial fluid dynamics and molecular structure.

Pulmonary interstitium is maintained dehydrated at subatmospheric pressure (-10 cmH(2)O) through low capillary permeability, low tissue compliance, and an efficient lymphatic drainage. Enzymatic degradation of proteoglycans disrupts the endothelial basal membrane and the matrix structure, triggering the development of pulmonary edema.

Pulmonary interstitial pressure and tissue matrix structure in acute hypoxia.

Pulmonary interstitial pressure was measured via micropuncture in anesthetized rabbits in normoxia and after breathing 12% O(2). In normoxia [arterial PO(2) = 88 +/- 2 (SD) mmHg], pulmonary arterial pressure and pulmonary interstitial pressure were 16 +/- 8 and -9.6 +/- 2 cmH(2)O, respectively. After 6 h of hypoxia (arterial PO(2) = 39 +/- 16 mm Hg), the corresponding values were 30+/-8 and 3.5+/-2.5 cm H(2)O (P<0.05). Pulmonary interstitial proteoglycan extractability, evaluated by hexuronate assay after 0.4 M guanidinium hydrochloride extraction, was 12.3, 32.4, and 60.6 microg/g wet tissue in normoxia and after 3 and 6 h of hypoxia, respectively, indicating a weakening of the noncovalent bonds linking proteoglycans to other extracellular matrix components. Gel filtration chromatography showed an increased fragmentation of chondroitin sulfate- and heparan sulfate-proteoglycans during hypoxic exposure, accounting for a loss of extracellular matrix native architecture and basement membrane structure. Gelatin zymography demonstrated increased amounts of the proteolytically activated form of gelatinase B (matrix metalloproteinase-9) after hypoxic exposure, providing evidence that the activation of proteinases may play a role in hypoxia-induced lung injury.

Isolation of pulmonary interstitial fluid in rabbits by a modified wick technique.

Interstitial fluid protein concentration (C(protein)) values in perivascular and peribronchial lung tissues were never simultaneously measured in mammals; in this study, perivascular and peribronchial interstitial fluids were collected from rabbits under control conditions and rabbits with hydraulic edema or lesional edema. Postmortem dry wicks were implanted in the perivascular and peribronchial tissues; after 20 min, the wicks were withdrawn and the interstitial fluid was collected to measure C(protein) and colloid osmotic pressure. Plasma, perivascular, and peribronchial C(protein) values averaged 6.4 +/- 0.7 (SD), 3.7 +/- 0.5, and 2.4 +/- 0.7 g/dl, respectively, in control rabbits; 4.8 +/- 0.7, 2.5 +/- 0.6, and 2.4 +/- 0.4 g/dl, respectively, in rabbits with hydraulic edema; and 5.1 +/- 0.3, 4.3 +/- 0.4 and 3.3 +/- 0.6 g/dl, respectively, in rabbits with lesional edema. Contamination of plasma proteins from microvascular lesions during wick insertion was 14% of plasma C(protein). In control animals, pulmonary interstitial C(protein) was lower than previous estimates from pre- and postnodal pulmonary lymph; furthermore, although the interstitium constitutes a continuum within the lung parenchyma, regional differences in tissue content seem to exist in the rabbit lung.

The effect of glycosaminoglycans and proteoglycans on lipid peroxidation.

Recent investigations show that glycosaminoglycans (GAGs) and proteoglycans (PGs) have the ability to affect lipid peroxidation, one the best characterized forms of free radical mediated biological damage. A protective effect of these extracellular matrix (ECM) components has been demonstrated in various experimental systems, including fatty acids and liposomes, where oxidation was induced by transition metals, including copper and iron. The effect was specific and dependent on the type and structural features of GAGs and PGs. The mechanism of peroxidation inhibition was likely to be dependent, at least to a large extent, on the sequestration of transition metals by GAG chains. Thus, it is conceivable that GAGs in the ECM and in the pericellular space may contribute to protecting cells against free radical damage. It is of particular interest that in certain tissues (cornea and aorta) aging was associated with a decrease of content of the GAGs which were most effective as anti-oxidant. This suggests that age-induced modifications of ECM composition in certain tissues may increase the susceptibility to oxidative stress. The investigation on the effect of GAGs on lipoprotein oxidation led to apparently conflicting results. An interesting reconciliation is possible, according to which GAGs exerted their protective effect under experimental conditions not compatible with the formation of lipoprotein-GAG complexes; rather, lipoproteins exhibited increased susceptibility to metal-catalyzed oxidation (MCO), possibly due to structural modifications of the particle after binding to GAGs or PGs. This process is likely to occur in the intimal matrix of arteries.