Inhaled recombinant GM-CSF reduces the need for whole lung lavage and improves gas exchange in autoimmune pulmonary alveolar proteinosis patients.

RATIONALE: Whole lung lavage (WLL) is a widely accepted palliative treatment for autoimmune pulmonary alveolar proteinosis (aPAP) but does not correct myeloid cell dysfunction or reverse the pathological accumulation of surfactant. In contrast, inhaled recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) is a promising pharmacological approach that restores alveolar macrophage functions including surfactant clearance. Here, we evaluate WLL followed by inhaled rGM-CSF (sargramostim) as therapy of aPAP. METHODS: 18 patients with moderate-to-severe aPAP were enrolled, received baseline WLL, were randomised into either the rGM-CSF group (receiving inhaled sargramostim) or control group (no scheduled therapy) and followed for 30 months after the baseline WLL. Outcome measures included additional unscheduled "rescue" WLL for disease progression, assessment of arterial blood gases, pulmonary function, computed tomography, health status, biomarkers and adverse events. Patients requiring rescue WLL were considered to have failed their assigned intervention group. RESULTS: The primary end-point of time to first rescue WLL was longer in rGM-CSF-treated patients than controls (30 versus 18 months, n=9 per group, p=0.0078). Seven control patients (78%) and only one rGM-CSF-treated patient (11%) required rescue WLL, demonstrating a 7-fold increase in relative risk (p=0.015). Compared to controls, rGM-CSF-treated patients also had greater improvement in peripheral arterial oxygen tension, alveolar-arterial oxygen tension difference, diffusing capacity of the lungs for carbon monoxide and aPAP biomarkers. One patient from each group withdrew for personal reasons. No serious adverse events were reported. CONCLUSIONS: This long-term, prospective, randomised trial demonstrated inhaled sargramostim following WLL reduced the requirement for WLL, improved lung function and was safe in aPAP patients. WLL plus inhaled sargramostim may be useful as combined therapy for aPAP.

A Nanoprobe Based on Gated Mesoporous Silica Nanoparticles for The Selective and Sensitive Detection of Benzene Metabolite t,t-Muconic Acid in Urine.

Benzene is a highly toxic aromatic hydrocarbon. Inhaling benzene can cause dizziness, vertigo, headaches, aplasia, mutations and, in the most extreme cases, cancer. Trans,trans-muconic acid (t,t-MA) is one of the metabolization products of benzene. Although different analytical methods have been reported for the determination of t,t-MA, these are often expensive, require trained personnel, are not suitable for on-site measurements, and use hazardous organic solvents. For these reasons, the development of reliable, selective and sensitive methods for rapid and in situ detection of t,t-MA are of importance. Addressing this challenge, a nanodevice for the selective and sensitive quantification of t,t-MA in urine is reported. The nanodevice used is achieved using mesoporous silica nanoparticles loaded with a dye reporter and capped with a dicopper(II) azacryptand. Pore opening and payload release is induced rapidly (10 min) and selectively with t,t-MA in urine, using a simple fluorimeter without sample pretreatment.

Highly Sensitive and Selective Molecular Probes for Chromo-Fluorogenic Sensing of Carbon Monoxide in Air, Aqueous Solution and Cells.

Optical sensing offers a low-cost and effective means to sense carbon monoxide in air and in solution. This contribution reports the synthesis of a new series of vinyl complexes [Ru(CH=CHR)Cl(CO)(TBTD)(PPh3 )2 ] (R=aryl, TBTD=5-(3-thienyl)-2,1,3-benzothiadiazole) and shows them to be highly sensitive and selective probes for carbon monoxide in both solution and air. Depending on the vinyl substituent, chromogenic and fluorogenic responses signalled the presence of this invisible, odourless, tasteless and toxic gas. Adsorbing the complexes on silica produced colorimetric probes for the 'naked eye' detection of CO in the gas phase with a limit of detection as low as 8 ppm in some cases, while the release of the TBTD fluorophore allowed detection at much lower concentrations through the fluorescence response. Structural data were obtained by single-crystal X-ray diffraction techniques, while the photophysical behaviour was explored computationally using TD-DFT experiments. The systems were also shown to be selective for CO over all other gases tested, including water vapour and common organic solvents. By introducing a poly(ethylene)glycol chain to the vinyl functionality, water compatibility was achieved and these non-cytotoxic complexes were employed in the sensing of CO in HeLa cells, offering a simple and rapid system for sensing this gasotransmitter in this challenging medium.

Alterations of Inflammatory and Matrix Production Indices in Celiac Disease With Low Bone Mass on Long-term Gluten-free Diet.

BACKGROUND: A clinically meaningful impairment of bone mass secondary to malabsorption is frequent in untreated celiac disease. In adult patients, a rigorous gluten-free diet (GFD) significantly improves, but does not always normalize, bone mineral density (BMD). The reason for this marginal response is unclear. Accordingly, we evaluated the role of both local and systemic factors for bone loss in celiac patients on long-term GFD. STUDY: In a prospective cohort, 22 patients with low lumbar and/or femoral BMD and 22 with normal BMD underwent bone and mineral metabolism evaluation: we tested calcium, phosphate, parathyroid hormone, and vitamin D; telopeptide of type I collagen, a bone resorption index; propeptide of type I procollagen, a bone neoformation index; receptor antagonist of NF-kB ligand, an osteoclast-stimulating factor; osteoprotegerin (OPG), a decoy receptor for RANKL. Sunlight exposure and physical exercise were measured. RESULTS: Patients with bone loss showed prevalently osteopenia, severe osteoporosis was rare. In comparison with normal BMD patients, they showed higher serum OPG, telopeptide, and lower serum propeptide, suggesting an increased bone turnover. Lumbar T-score was negatively correlated with OPG, telopeptide and RANKL and positively with propeptide. Propeptide was negatively correlated with OPG and telopeptide. OPG was positively correlated with telopeptide. CONCLUSIONS: The persistent activation of inflammation should be considered the main pathophysiological mechanism for bone defect in celiac disease patients with bone loss on long-term GFD. High levels of OPG, an attempt at protective mechanism, and low levels of propeptide of type I procollagen, reflecting an insufficient matrix production, characterize this subgroup of patients.

ϵ-Polylysine-Capped Mesoporous Silica Nanoparticles as Carrier of the C9h Peptide to Induce Apoptosis in Cancer Cells.

Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safranin O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safranin O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.

VSL#3 probiotic differently influences IEC-6 intestinal epithelial cell status and function.

The data here reported introduce the wound-healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi-strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non-transformed small-intestinal epithelial cell line, IEC-6. The effects on cellular morphology, viability, migration, and H2 O2 -induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA-sourced product ("USA-made") VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy-derived product ("Italy-made") VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with "USA-made" lysate led to a higher rate of wounded monolayer healing, while the addition of "Italy-made" bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from "USA-made" VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, "Italy-made" lysates had not similar effect. "USA-made" lysate was able to cause a total inhibition of H2 O2 -induced cytotoxic effect whereas "Italy-made" VSL#3 lysate was unable to protect IEC-6 cells from H2 O2 -induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of "USA-made" VSL#3 lysates, as well as the idea that "Italy-made" formulation was unable to prevent significantly the H2 O2 -induced oxidative stress.

Ex Vivo Tracking of Endogenous CO with a Ruthenium(II) Complex.

A two-photon fluorescent probe based on a ruthenium(II) vinyl complex is capable of selectively detecting carbon monoxide in cells and ex vivo using mice with a subcutaneous air pouch as a model for inflammation. This probe combines highly selective and sensitive ex vivo detection of endogenous CO in a realistic model with facile, inexpensive synthesis, and displays many advantages over the widely used palladium-based systems.

Enzyme-Controlled Nanodevice for Acetylcholine-Triggered Cargo Delivery Based on Janus Au-Mesoporous Silica Nanoparticles.

This work reports a new gated nanodevice for acetylcholine-triggered cargo delivery. We prepared and characterized Janus Au-mesoporous silica nanoparticles functionalized with acetylcholinesterase on the Au face and with supramolecular ß-cyclodextrin:benzimidazole inclusion complexes as caps on the mesoporous silica face. The nanodevice is able to selectively deliver the cargo in the presence of acetylcholine via enzyme-mediated acetylcholine hydrolysis, locally lowering the pH and opening the supramolecular gate. Given the key role played by ACh and its relation with Parkinson's disease and other nervous system diseases, we believe that these findings could help design new therapeutic strategies.

The prevalence of protozoa in the gut of German cockroaches (Blattella germanica) with special reference to Lophomonas blattarum.

The German cockroach (Blattella germanica) is a common domestic pest, which produces allergens that have been associated with broncho-pulmonary disease. Various protozoan species have been identified in the intestine of this cockroach and it has been hypothesised that these protozoa, or their proteases, may contribute to the burden of cockroach-associated allergens and adjuvants present in domestic dust. The aim of this study was therefore to determine the prevalence of protozoan species in the intestine of Blattella germanica. German cockroaches were anesthetised and dissected and gut contents are used to produce wet slides for microscopy. Both, Giemsa and Papanicolaou stains were used to confirm correct identification of Lophomonas blattarum. Representatives of four genera of protozoa were identified in 110 cockroaches: Nyctoterus sp. was observed in 91.8% of cases, Gregarina sp. in 64.5%, Amoeba sp. in 25.4% and Lophomonas blattarum in 13.6%. Nyctoterus and Gregarina were statistically significantly more likely to be found in diseased cockroaches compared to Amoeba or Lophomonas. The prevalence of Lophomonas blattarum was similar to that in published studies of a different species of cockroach, Periplaneta americana. Further work is needed to assess the interplay between protozoa, cockroaches and broncho-pulmonary diseases.

Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process.

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S-nitroso-N-acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0-28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin-labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki-67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP-induced migration and proliferation were antagonized by the NO-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[-4,3-a]quinoxalin-1-one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP-induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR-ß antagonist (GSK0660) may interfere with NO-mediated wound healing process. J. Cell. Physiol. 231: 2185-2195, 2016. © 2016 Wiley Periodicals, Inc.

Polyglutamic Acid-Gated Mesoporous Silica Nanoparticles for Enzyme-Controlled Drug Delivery.

Mesoporous silica nanoparticles (MSNs) are highly attractive as supports in the design of controlled delivery systems that can act as containers for the encapsulation of therapeutic agents, overcoming common issues such as poor water solubility and poor stability of some drugs and also enhancing their bioavailability. In this context, we describe herein the development of polyglutamic acid (PGA)-capped MSNs that can selectively deliver rhodamine B and doxorubicin. PGA-capped MSNs remain closed in an aqueous environment, yet they are able to deliver the cargo in the presence of pronase because of the hydrolysis of the peptide bonds in PGA. The prepared solids released less than 20% of the cargo in 1 day in water, whereas they were able to reach 90% of the maximum release of the entrapped guest in ca. 5 h in the presence of pronase. Studies of the PGA-capped nanoparticles with SK-BR-3 breast cancer cells were also undertaken. Rhodamine-loaded nanoparticles were not toxic, whereas doxorubicin-loaded nanoparticles were able to efficiently kill more than 90% of the cancer cells at a concentration of 100 µg/mL.

Improving the outcome of fractional CO2 laser resurfacing using a probiotic skin cream: Preliminary clinical evaluation.

As known, fractional CO2 resurfacing treatments are more effective than non-ablative ones against aging signs, but post-operative redness and swelling prolong the overall downtime requiring up to steroid administration in order to reduce these local systems. In the last years, an increasing interest has been focused on the possible use of probiotics for treating inflammatory and allergic conditions suggesting that they can exert profound beneficial effects on skin homeostasis. In this work, the Authors report their experience on fractional CO2 laser resurfacing and provide the results of a new post-operative topical treatment with an experimental cream containing probiotic-derived active principles potentially able to modulate the inflammatory reaction associated to laser-treatment. The cream containing DermaACB (CERABEST™) was administered post-operatively to 42 consecutive patients who were treated with fractional CO2 laser. All patients adopted the cream twice a day for 2 weeks. Grades were given according to outcome scale. The efficacy of the cream containing DermaACB was evaluated comparing the rate of post-operative signs vanishing with a control group of 20 patients topically treated with an antibiotic cream and a hyaluronic acid based cream. Results registered with the experimental treatment were good in 22 patients, moderate in 17, and poor in 3 cases. Patients using the study cream took an average time of 14.3 days for erythema resolution and 9.3 days for swelling vanishing. The post-operative administration of the cream containing DermaACB induces a quicker reduction of post-operative erythema and swelling when compared to a standard treatment.

The Mucosal Innate Immune Response in Primary Human Papillomavirus Infection: A Pilot Study.

OBJECTIVES: The purpose of the study was to evaluate the mucosal immune response in women affected by primary human papillomavirus (HPV) infection, in comparison with HPV-negative women with no previous history of HPV. METHODS: A case-control study comparing the activity of myeloperoxidase (MPO) and lactoferrin (LF) between 19 HPV-positive and 19 HPV-negative women matched for age. Plasmatic and cervicovaginal levels of polymorphonuclear neutrophils (PMN) exhibiting MPO and LF receptors were measured using cytofluorimetric analysis and expressed as mean of percentages. RESULTS: Cervicovaginal levels of MPO-/LF- PMN were lower among HPV-negative women, with a mean rate of 18.81% (SD, 21.38), as opposed to a mean rate of 35.56% (SD, 21.02) (P = 0.020) in HPV-positive women. A similar significant difference was not proven in plasma. The mean rates of plasmatic levels of MPO-/LF- PMN were 36.21% (SD, 16.87) and 36.93% (SD, 10.54) (P = 0.875) in cases and controls, respectively. All patients were evaluated 1 year later, and only 6 cases became negative. CONCLUSIONS: The presence of MPO-/LF- PMN has been considered as a marker of lower rate of apoptosis of HPV-infected cells. This could explain why HPV-positive women are less capable to deal with a primary infection.

In vitro evaluation of different methods of handling human liposuction aspirate and their effect on adipocytes and adipose derived stem cells.

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue-derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10-20-30 min) with the centrifugation technique at different speeds (90-400-1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, "middle layer", and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45-) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available.

Immunophenotypic characterization of human glioblastoma stem cells: correlation with clinical outcome.

Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. ß-tubulin III, nestin, CD133, GFAP, and SOX-2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX-2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P = 0.000) and progression-free survival (PFS) (P = 0.001). In addition, a very poor OS (P = 0.000) and PFS (P = 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX-2 may be useful to identify patients at risk of disease progression.

Caspase 3 Targeted Cargo Delivery in Apoptotic Cells Using Capped Mesoporous Silica Nanoparticles.

Excessive apoptotic cell death is at the origin of several pathologies, such as degenerative disorders, stroke or ischemia-reperfusion damage. In this context, strategies to improve inhibition of apoptosis and other types of cell death are of interest and may represent a pharmacological opportunity for the treatment of cell-death-related disorders. In this scenario new peptide-containing delivery systems (solids S1 -P1 and S1 -P2 ) are described based on mesoporous silica nanoparticles (MSNs) loaded with a dye and capped with the KKGDEVDKKARDEVDK (P1 ) peptide that contains two repeats of the DEVD target sequence that are selectively hydrolyzed by caspase 3 (C3). This enzyme plays a central role in the execution-phase of apoptosis. HeLa cells electroporated with S1 -P1 are able to deliver the cargo in the presence of staurosporin (STS), which induces apoptosis with the consequent activation of the cytoplasmic C3 enzyme. Moreover, the nanoparticles S1 -P2 , containing both a cell-penetrating TAT peptide and P1 also entered in HeLa cells and delivered the cargo preferentially in cells treated with the apoptosis inducer cisplatin.

Gated mesoporous silica nanoparticles for the controlled delivery of drugs in cancer cells.

In recent years, mesoporous silica nanoparticles (MSNs) have been used as effective supports for the development of controlled-release nanodevices that are able to act as multifunctional delivery platforms for the encapsulation of therapeutic agents, enhancing their bioavailability and overcoming common issues such as poor water solubility and poor stability of some drugs. In particular, redox-responsive delivery systems have attracted the attention of scientists because of the intracellular reductive environment related to a high concentration of glutathione (GSH). In this context, we describe herein the development of a GSH-responsive delivery system based on poly(ethylene glycol)- (PEG-) capped MSNs that are able to deliver safranin O and doxorubicin in a controlled manner. The results showed that the PEG-capped systems designed in this work can be maintained closed at low GSH concentrations, yet the cargo can be delivered when the concentration of GSH is increased. Moreover, the efficacy of the PEG-capped system in delivering the cytotoxic agent doxorubicin in cells was also demonstrated.