María Pilar Domingo

Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis.

The virulence factor gliotoxin (GT) and its inactive derivative, bis(methylthio)gliotoxin (bmGT), are produced by pathogens of the genus Aspergillus. Here we report the detection of GT and bmGT in serum of humans at risk of invasive aspergillosis (IA) as well as in cultures of fungal isolates derived from patients with proven infection with A. fumigatus. Although both compounds are readily recoverable from spiked human serum or plasma, only bmGT is retained in whole blood, indicating that bmGT may be the better marker for in vivo detection. Accordingly, bmGT was found more frequently than GT in samples from patients at risk of IA and incultures of clinical isolates of A. fumigatus. In some cases, bmGT was detected before mycologic evidence ofinfection was gained. Importantly, neither GT nor bmGT was found in serum from healthy donors or from neutropenic patients without any sign of infection. Thus, bmGT presence might provide a more reliable indicator of A. fumigatus infections than GT. Due to its simplicity and sensitivity, a diagnostic technology based on this test could be easily adopted in clinical laboratories to help in the diagnosis of this often fatal fungal infection.

Application of Thin-layer Chromatography with Fluorescence Scanning Densitometry for Analysing Saturates in Heavy Liquids Derived from Co-pyrolysis of Biomass and Plastics

SummaryTwo alternative methods, based on Thin-Layer Chromatography (TLC) with Fluorescence Scanning Densitometry have been developed for characterization of heavy liquids from copyrolysis of different kinds of biomass and plastics in autoclaves under inert atmosphere.A conventional TLC system, which includes a vertical developing tank, and a High Performance TLC (HPTLC) system, with a horizontal developing chamber and the use of HPTLC plates, have been used. The analytical method involves in both cases the measurement of two chromatograms per sample: the first, on a silica gel berberine-impregnated plate, for detection of saturates using the phenomenon of berberine-induced fluorescence; and the second, on a silica gel plate, for detection of aromatic-polars and polars, by native fluorescence.Although the HPTLC system is more sensitive and faster, both techniques represent an improvement with regard to current methods for analyzing these kinds of products. However their application depends on the particular solubility of each sample and on its slope of the fluorescent response-sample load regression.

FRET based quantification and screening technology platform for the interactions of Leukocyte Function-associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)

The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple ‘in solution’ steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (Kd) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.

Thin-Layer Chromatography for Hydrocarbon Characterization in Petroleum Middle Distillates

Petroleum is fractionated according to its boiling point by distillation methods. Depending on the boiling point range of the sample, fractionated products fall into three main categories: light, middle and heavy distillates. Middle distillates (from approximately 150°C to 400°C, which approximately corresponds to C9-C24) are mostly used for heating and transportation fuels (e.g., diesel, jet fuel, gas oil, aviation turbine oils, kerosene).

Hydrocarbon group-type analysis by thin layer chromatography and scanning densitometry

Hydrocarbon group-type analysis (HGTA) is a common technique for characterization of complex mixtures derived from raw materials such as coal, petroleum, or biomass. In these and other, related, samples, trying to achieve extensive separation of all the components would be very difficult at least, and most of the relevant properties of the samples can be related to the amounts of the different types of hydrocarbon. Groups of interest depend mainly on the nature of the sample, and some kind of liquid chromatography is usually involved in the most common HGTA methods. Thin-layer chromatography (TLC) can nowadays usually be used instead of HPLC, resulting in several advantages in terms of speed, cost, and general convenience. Detection and quantification of the different peaks might involve the use of special equipment, e.g. in TLC—flame ionization detection (FID) methods, although it can also be accomplished by means of UV and fluorescence scanning densitometry. In this paper, we describe a series of TLC-based HGTA methods developed for coal-, biomass-, and petroleum-derived products that give a reasonably general overview of the possibilities of TLC applied to HGTA.

Disseminated aspergillosis in an immunocompetent patient with detectable bis(methylthio)gliotoxin and negative galactomannan

BACKGROUND Disseminated invasive aspergillosis is an exceptional finding in immunocompetent hosts. As in immunocompromised patients, it has high mortality rates. Early diagnostic methods are required in order to properly manage the patient. Bis(methylthio)gliotoxin (bmGT) is a novel biomarker, useful in onco-hematological patients. CASE REPORT A 70-year-old male, with non-insulin dependent type II diabetes mellitus and a past surgery history of aortic valve replacement with coronary by-pass five years ago, was seen in the emergency department with blurred vision. Three days later, endogen endophthalmitis was diagnosed in the ophthalmology clinic. During admission for the vitrectomy, he suffered an ischemia of the right lower limb. A thoracic computed tomography revealed a mycotic aneurysm of the ascending thoracic aorta and parietal thrombus. The ascending aorta was replaced and abundant brittle material of infectious appearance, found between the aortic valve graft and the aneurysm, was removed. Aspergillus fumigatus sensu stricto grew in both vitreous and aorta cultures. BmGT was detected in two serum samples obtained prior to intravenous antifungal treatment, which was then reduced after voriconazole treatment was started. CONCLUSIONS Disseminated invasive aspergillosis is a severe disease regardless of the immune status of the patient. This case report suggests that bmGT could be a suitable early diagnostic biomarker, not only in neutropenic patients, but also in immunocompetent hosts.

A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level

The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.