European consensus proposal for immunoglobulin therapies

The use of immunoglobulin (Ig) preparations (intravenous, IVIg, subcutaneous, SCIg) for replacement and immunomodulation therapy worldwide has tripled in the past 20 years and represents an ever‐increasing cost factor for healthcare organizations. The limited access to the starting material of this essential medicinal product is currently the driving force for human plasma collection. Increasing awareness and improved diagnosis of human primary immunodeficiencies and a broadening of immunomodulatory indications are responsible for this development, and on a longer run might lead to plasma supply shortages. Consensus recommendations for the optimal use of Ig in clinical practice, including priority rankings for the most urgent indications, are therefore urgently needed. During a recent meeting in Kreuth, Germany, expert nominees from 36 Council of Europe states, together with colleagues from observer countries and regulatory agencies came up with this consensus statement.

     

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.     

Infrared LED light therapy influences the expression of fibronectin and tenascin in skin wounds of malnourished rats—A preliminary study

The aim of this investigation was to evaluate the effect of infrared (λ 846 ± 20 nm) LED irradiation on the expression profile of the extracellular matrix protein components, tenascin and fibronectin on skin wounds induced in well nourished and malnourished rats. Eighteen albino rats (21 days old) were randomly divided into a well-nourished group (standard diet) and a malnourished group (regional basic diet). After receiving the diet for 70 days, skin wounds were created and the animals were subdivided into three groups: well-nourished control (n = 6), malnourished control (n = 6), and malnourished + LED irradiated (λ 846 ± 20 nm, 100 mW, 4 J/cm²) (n = 6). The animals were sacrificed 3 and 7 days after injury and histological sections were immunostained for both proteins. They were examined for the presence, intensity, distribution and pattern of immunolabeling. At 3 days, the distribution of tenascin was shown to be greater in the wound bed of malnourished animals compared to the well-nourished group. The intensity and distribution of tenascin was shown to be lower in the malnourished LED irradiated group compared to the malnourished control. There was a significant difference regarding the presence of fibronectin in the malnourished and well-nourished groups after 7 days (p = 0.03). The intensity of fibronectin was slight (100%) in the irradiated group and moderate to intense in the malnourished control group. The results of the present study indicate that infrared LED irradiation modulates positively the expression of tenascin and particularly fibronectin.     

Best practice for PTEN gene and protein assessment in anatomic pathology

There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p < 0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p < 0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.     

Rutin-modified silver nanoparticles as a chromogenic probe for the selective detection of Fe3+ in aqueous medium

The development of nanoprobes for selective detection of metal ions in solution has attracted great attention due to their impact on living organisms. As a contribution to this field, this paper reports the synthesis of silver nanoparticles modified with rutin in the presence of ascorbic acid and their successful use as a chromogenic probe for the selective detection of Fe³⁺ in aqueous solution. Limits of detection and quantification were found to be 17 nmol L⁻¹ and 56 nmol L⁻¹, respectively. The sensing ability is proposed to proceed via an iron-induced nanoparticle growth/aggregation mechanism. A practical approach using image analysis for quantification of Fe³⁺ is also described.

The use of rutin-modified silver nanoparticles for selective detection and sensitive quantification of Fe³⁺ in aqueous solution is described.

Metal ions are key species in nature due to their essential functions in living organisms.^1,2 On the other hand, heavy metals as well as essential metals at abnormally high levels are toxic.² Iron, for instance, in addition to its popular use in industry and construction, is essential to the human body and active in biological processes. Although the trivalent form of iron is particularly important for oxygen transport in blood and the mitochondrial respiratory chain, high levels of this cation are associated with important pathologies.^3,4 The detection of metal ions in aqueous solution is traditionally performed by methods including atomic absorption spectrometry,⁵ electrochemical measurements,^6,7 and inductively coupled plasma techniques,⁸ among others. However, these techniques have important drawbacks, notably the need for sophisticated instrumentation, in addition to being time-consuming and requiring laborious procedures. To overcome these issues, the development of chromogenic and fluorogenic chemosensors for the selective detection of metal-targets has attracted great attention, especially due to the possibility of fast, sensitive and non-expensive analysis.^9,10 In the last decade, nanoscaled materials have been reported as selective probes for metal ions, including Fe³⁺.^11–24Silver nanoparticles (AgNPs) are of particular interest because of the affordable price of starting materials, ease of controlling size and morphology, possibility to functionalize their surface with organic molecules, and optical properties that enable detection of a variety of analytes via simple UV-vis spectroscopy and digital image analysis. Furthermore, applications of AgNPs are also biotechnologically relevant due to the possibility of green synthetic protocols, including the use of plant extracts,²⁵ natural sources,²⁶ glycerol,²⁷ among others.Flavonoids are secondary metabolites naturally found in fruits and other vegetables with relevant roles due to their nutritional, pharmaceutical and medicinal properties.²⁸ Because of their adequate structural features, flavonoids are candidates to be employed in the synthesis of AgNPs.²⁶ Rutin (RU), a sugar-based flavonoid, may be employed as reducing agent in the synthesis of AgNPs along with a stabilizer such as polyvinylpyrrolidone (PVP)²⁶ or used as crude plant extract component.^29,30This paper reports the use of RU-modified AgNPs (RU-AgNPs) as a chromogenic probe for Fe³⁺ in aqueous medium in the presence of ascorbic acid (AA). Sensing ability of RU-AgNPs for the selective detection of Fe³⁺ toward other metal cations was investigated with UV-vis spectroscopy analysis. These data and transmission electronic microscopy (TEM) results allowed a mechanistic proposal involved in the selective detection of Fe³⁺ by RU-AgNPs. Furthermore, a practical approach based on correlation of images of solutions obtained with a conventional smartphone and chemometrics was employed for a simpler quantification of Fe³⁺ in aqueous medium.Initially, the order of reagent combination was investigated in the synthesis of RU-AgNPs. Concentration of RU ranged from 0.10 to 0.50 mmol L⁻¹, while concentrations of other components were fixed at 0.20 mmol L⁻¹ AgNO₃, 0.10 mmol L⁻¹ AA and 0.10 M NaOH. Water was used as solvent in all cases. Narrower surface plasmon resonance (SPR) bands were obtained from adding a solution of AA and NaOH to a solution containing RU and AgNO₃ (Fig. 1a) against the addition of RU, AA, and NaOH to AgNO₃ solution (Fig. S1a – ESI^†), or the addition of RU and NaOH to a solution of AA and AgNO₃ (Fig. S1b^†). RU-AgNPs obtained from the condition presented in Fig. 1a are small (4.1 nm average diameter) and considerably polydisperse (standard deviation of 4.7 nm), however, presenting only one population (Fig. 1b and S2^†). A study of the influence of RU concentration (0.10 to 0.50 mmol L⁻¹) on the stability of RU-AgNPs over time indicated that 0.10 mmol L⁻¹ RU generates more stable RU-AgNPs (Fig. S3^†). Next, a study on the influence of pH indicated that RU-AgNPs are only stable under strong alkaline conditions (pH 12.5 or higher) (Fig. S4^†).Open in a separate windowFig. 1(a) UV-vis analysis of RU-AgNPs under strong alkaline condition (pH > 12.5) based on the order of adding reagents (RU, 0.10 to 0.50 mmol L⁻¹; AgNO₃, 0.20 mmol L⁻¹; AA, 0.10 mmol L⁻¹; NaOH, 0.10 mmol L⁻¹); (b) TEM image of RU-AgNPs under the selected condition.The ability of RU-AgNPs to sense metal cations was investigated by both naked-eye and UV-vis spectroscopy analysis (Fig. 2). The separate addition of 10 μmol L⁻¹ of several metal cations (Fe³⁺, Co²⁺, Zn²⁺, Sr²⁺, Cu²⁺. Al³⁺, Ba²⁺, Cd²⁺, Pb²⁺, Ni²⁺, Mg²⁺, Hg²⁺, Cu⁺ and Cr³⁺) to solutions of RU-AgNPs (prepared according to Fig. 1a) indicated that only Fe³⁺ induces a significant colorimetric change in the final aspect of solution after 50 minutes (Fig. 2a).Open in a separate windowFig. 2Naked-eye (a) and UV-vis (b) analysis of RU-AgNPs in absence (control, C) and presence of 10 μmol L⁻¹ of selected metal cations (Fe³⁺, Co²⁺, Zn²⁺, Sr²⁺, Cu²⁺. Al³⁺, Ba²⁺, Cd²⁺, Pb²⁺, Ni²⁺, Mg²⁺, Hg²⁺, Cu⁺ and Cr³⁺) after 50 min; (c) calibration curve obtained by the addition of different amounts (0 to 10 μmol L⁻¹) of Fe³⁺ to solutions of RU-AgNPs; (d) TEM image of RU-AgNPs after addition of Fe³⁺ (10 μmol L⁻¹). In all experiments, RU-AgNPs were prepared in the presence of ascorbic acid.The results presented in Fig. 2a are consistent with the UV-vis spectroscopy analysis (Fig. 2b). Co²⁺ ions also induce some change in the system, however at a considerably smaller extension than Fe³⁺. In this study, AA played a crucial role in the selective detection of Fe³⁺ by the referred nanoprobe. In the absence of AA, Co²⁺ (mainly) as well as other cations induce stronger colorimetric and spectral changes (Fig. S5a and b,^† respectively) in the analysis of solutions of RU-AgNPs when compared to the system containing AA. This selectivity may arise from two possible reasons: (i) preservation of RU by avoiding its oxidation in the reduction of Ag⁺ ions; (ii) coordination of ascorbate anion to cations other than Fe³⁺.Interaction of RU-AgNPs and Fe³⁺ (10 μmol L⁻¹) stabilizes after approximately 40 minutes (Fig. S6^†). Next, a calibration curve was built from the direct relationship between the absorbance at 396 nm and the concentration of Fe³⁺ (Fig. 2c), presenting a good correlation (R² = 0.9929). The limits of detection and quantification were found to be 17 nmol L⁻¹ and 56 nmol L⁻¹, respectively, which is very satisfactory.^13,14 The influence of other cations in the detection of Fe³⁺ was investigated by UV-vis spectroscopy. Fig. S7^† clearly demonstrates that there are only small changes when a second cation (30 μmol L⁻¹) is added together with Fe³⁺ to the RU-AgNPs solution.Mechanistically, the detection of Fe³⁺ by RU-AgNPs in aqueous medium proceeds via a growth/aggregation-combined process. This proposal is first evidenced by UV-vis analysis due the suppression of SPR band (Fig. 2b), a behavior consistent with the literature.^31,32 Interestingly, TEM analysis clearly shows a growth in AgNPs size after addition of Fe³⁺ to the solution (Fig. 2d), resulting in a final single AgNPs population with average diameter of 14.7 nm ± 8.9 nm. Due to the strong alkaline medium, the main specie responsible for the behavior of NPs is likely to be Fe(OH)₃. A schematic illustration of the mechanism involving aggregation of RU-AgNPs induced by the addition of Fe³⁺ is presented in Fig. 3. AgNPs are initially formed by adsorption of anionic RU to the silver surface via deprotonated 5-hydroxychromen-4-one moiety. This is supported by the literature³³ and confirmed by alteration in the 1800–1500 cm⁻¹ region of the RU infrared spectra before and after coordination with silver (Fig. S9^†). Afterwards, the addition of Fe³⁺ induces the formation of a coordination complex through an anionic catechol group, in which at least 2 : 1 ligand–Fe³⁺ stoichiometry is required for an aggregated effect.Open in a separate windowFig. 3Mechanistic proposal for growth/aggregation of RU-AgNPs in the presence of Fe³⁺. Insert: binding model for RU-AgNPs.Due to increasing interest in image processing as an analytical tool for many purposes,^34,35 Multiple Linear Regression was employed to verify the capacity of the RU-AgNPs to probe Fe³⁺ standards at distinct concentrations, as presented in Fig. 4. The curve was obtained by plotting the color absorbances RGB-based values versus the concentrations of Fe³⁺ standards after RU-AgNPs interaction. Predicted iron is a vector based on RGB values that were then extracted from the filtered images and inserted in the equation described by Beer–Lambert law in order to generate the absorbances for the construction of the analytical curve. A linear behavior between the predicted response and the measured concentrations was observed (R² = 0.9806).Open in a separate windowFig. 4Calibration curve for Fe³⁺ analysis showing the predicted iron (RGB) vs. iron(iii) concentration (1 to 8 μmol L⁻¹). Adjusted R² = 0.9806.Regression coefficients of the calibration model (using the R, G and B channels simultaneously) obtained by MLR method are shown in eqn (1):[Fe³⁺] = 44.5R − 4.9G − 16.4B + 5.11where Fe³⁺ concentration is the dependent variable (Predicted Iron), 5.1 is the intercept (β₀), 44.5, −4.9 and −16.4 are the regression coefficients of the independent variables (R, G and B channels, respectively).It is possible to observe an interesting performance of the method using the three RGB color channels allied with MLR to quantify the Fe³⁺ content, which presented reasonable deviations in its responses considering that it is simple and low-cost. The good linearity is similar to other colorimetric methods such as sodium determination in seawater and coconut water (R² > 0.91) by Moraes and coauthors,³⁶ or iron(ii) in simulated seawater (R² = 0.9993) by Gasparotto et al.³⁷Second order regression was applied to the dataset to obtain a better adjustment, resulting in an adjusted R-squared of 0.9955. RGB values were then inserted in eqn (2) in order to generate the construction of the correlation:[Fe³⁺] = 14.4R + 30.7G − 23.4B − 398RG − 348RB − 0.8BG − 978R² + 695G² + 26.9B² + 4.52This paper reports the use of AgNPs functionalized with RU as nanoprobes for selective detection and sensitive quantification of Fe³⁺ in aqueous solution. The synthesis of RU-AgNPs is reproducible, easily performed and requires no stabilizer agent other than RU. AA has a crucial role in the selectivity by either the avoidance of oxidation of RU by silver and/or coordination of ascorbate with other cations. The literature brings relevant examples of chromogenic and fluorogenic chemosensors for selective detection of Fe³⁺ in solution. Many of these artificial organic receptors present high selectivity and relevant limits of detection, requiring, however, very specific reagents and laborious synthetic procedures.^38–41 Metal-based nanoparticles have emerged as potential probes for detection of Fe³⁺.^11–16 Although effective in Fe³⁺ sensing, the synthesis of these nanoprobes require some toxic reagents, such as NaBH₄ or PVP, use plant extracts, which may lead to some drawbacks, such as the understanding of the sensing mechanism. In contrast, our method is based on commercially available, nontoxic, low-cost reagents. Fe³⁺ sensing performed satisfactorily in the 1–10 μmol L⁻¹ range, and the limit of detection obtained with this method (17 nmol L⁻¹) is comparable to the most sensitive methods reported in literature. A mechanism for the detection of Fe³⁺ by RU-AgNPs involves a combined growth/aggregation of the NPs. There is a still limited number of nanoscaled systems reported as being selective and sensitive in the detection of Fe³⁺, which reinforces the relevance of the method reported herein. The linearity range obtained by both UV-vis spectroscopy and image analysis comprises the maximum of residual Fe³⁺ in drinking water according to the European and US legislations.^15,42     

Using old liver grafts for liver transplantation: Where are the limits?

The scarcity of ideal liver grafts for orthotopic liver transplantation (OLT) has led transplant teams to investigate other sources of grafts in order to augment the donor liver pool. One way to get more liver grafts is to use marginal donors, a not well-defined group which includes mainly donors > 60 years, donors with hypernatremia or macrosteatosis > 30%, donors with hepatitis C virus or hepatitis B virus positive serologies, cold ischemia time > 12 h, non-heart-beating donors, and grafts from split-livers or living-related donations. Perhaps the most practical and frequent measure to increase the liver pool, and thus to reduce waiting list mortality, is to use older livers. In the past years the results of OLT with old livers have improved, mainly due to better selection and maintenance of donors, improvements in surgical techniques in donors and recipients, and intra- and post-OLT management. At the present time, sexagenarian livers are generally accepted, but there still exists some controversy regarding the use of septuagenarian and octogenarian liver grafts. The aim of this paper is to briefly review the aging process of the liver and reported experiences using old livers for OLT. Fundamentally, the series of septuagenarian and octogenarian livers will be addressed to see if there is a limit to using these aged grafts.