The impact of different antioxidant agents alone or in combination on reactive oxygen species,antioxidant enzymes and cytokines in a series of advanced cancer patients at different sites: correlation with disease progression

In the present study we tested the ability of different antioxidant agents, used alone or in combination, to reduce the reactive oxygen species (ROS) levels and to increase the glutathione peroxidase (GPx) activity. Moreover, we tested the ability of such antioxidant agents to reduce the serum levels of proinflammatory cytokines IL-6 and TNFalpha. Fifty-six advanced stage cancer patients with tumors at different sites were included in the study: they were mainly stage III (12.5%) and stage IV (82.1%). The study was divided into two phases. In the 1st phase 28 patients were divided into five groups and a single different antioxidant agent was administered to each group. The selected antioxidant agents were: alpha lipoic acid or carboxycysteine-lysine salt, amifostine, reduced glutathione, vitamin A plus vitamin E plus Vitamin C. In the 2nd phase of the study 28 patients were divided into five groups and a combination of two different antioxidant agents was administered to each group. The antioxidant treatment was administered for 10 consecutive days. The patients were studied at baseline and after antioxidant treatment. Our results show that all single antioxidants tested were effective in reducing the ROS levels and three of them in increasing GPx activity, too. Among the combinations of antioxidant agents, three were effective in reducing ROS, while three were effective in increasing GPx activity (arm 4 was effective in both instances). Comprehensively, the "antioxidant treatment" was found to be effective both on ROS levels and GPx activity. Moreover, the antioxidant treatment was able to reduce serum levels of IL-6 and TNFalpha. Furthermore, a correlation was shown between the Eastern Cooperative Oncology Group Performance Status of patients and blood levels of ROS, GPx activity, serum levels of proinflammatory cytokines.     

Modeled microgravity affects motility and cytoskeletal structures

The hypothesis to be tested is that reduced cell-cell interactions between T cells and monocytes are one of the reasons for the observed depression of the "in vitro" activation of human lymphocytes in microgravity. Locomotion is essential for cell-cell contacts. Lymphocytes in suspension are highly motile in microgravity, whereas no data are available so far on the motility of adherent monocytes. It can be argued that an impaired locomotion of monocytes and cytoskeletal changes, both linked to cell contacts, could be responsible for their reduced interaction with T lymphocytes. This study is aimed at revealing how locomotion as well as cytoskeletal structures of adherent monocytes are modified under modeled microgravity conditions using the Random Positioning Machine (RPM, Dutch-Space) as earth based model of spaceflight.     

Physical Factors Influencing Pleasant Touch during Passive Fingertip Stimulation

Objective

Tactile explorations with the fingertips provide information regarding the physical properties of surfaces and their relative pleasantness. Previously, we performed an investigation in the active touch domain and linked several surface properties (i.e. frictional force fluctuations and net friction) with their pleasantness levels. The aim of the present study was to investigate physical factors being important for pleasantness perception during passive fingertip stimulation. Specifically we were interested to see whether factors, such as surfaces'' topographies or their frictional characteristics could influence pleasantness. Furthermore, we ascertained how the stimulus pleasantness level was impacted by (i) the normal force of stimulus application (F_N) and (ii) the stimulus temperature (T_S).

Methods and Results

The right index fingertips of 22 blindfolded participants were stimulated using 27 different stimuli, which varied in average roughness (Ra) and T_S. A 4-axis robot moved the stimuli horizontally under participants'' fingertips with three levels of F_N. The robot was equipped with force sensors, which recorded the F_N and friction force (F_T) during stimulation. Participants rated each stimulus according to a three-level pleasantness scale, as very pleasant (scored 0), pleasant (scored 1), or unpleasant (scored 2). These ordinal pleasantness ratings were logarithmically transformed into linear and unidimensional pleasantness measures with the Rasch model. Statistical analyses were conducted to investigate a possible link between the stimulus properties (i.e. Ra, F_N, F_T, and T_S) and their respective pleasantness levels. Only the mean Ra and F_T values were negatively correlated with pleasantness. No significant correlation was detected between F_N or T_S and pleasantness.

Conclusion

Pleasantness perception, resulting from passive fingertip stimulation, seems to be influenced by the surfaces'' average roughness levels and average F_T occurring during fingertip stimulation.     

Candida Endocarditis in Patients with Candidemia: A Single-Center Experience of 14 Cases

Mycopathologia - A retrospective, single-center analysis of 14 cases of Candida endocarditis (from 355 candidemia cases during the years 2012–2019) revealed a high in-hospital mortality...     

Enzyme-assisted photosensitization activates different apoptotic pathways in Rose Bengal acetate treated HeLa cells

Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially powerful cytotoxic agent.     

Reactive oxygen species,antioxidant mechanisms and serum cytokine levels in cancer patients: impact of an antioxidant treatment

Objective. So far, it is not well established whether oxidative stress found in cancer patients results from an increased production of oxidants in the body or from a failure of physiological antioxidant systems. To further investigate this question we have assessed the blood levels of reactive oxygen species as a marker of free radicals producing oxidative stress and the most relevant of the physiological body enzymes counteracting reactive oxygen species, namely glutathione peroxidase and superoxide dismutase. Serum levels of proinflammatory cytokines and IL‐2 were also investigated. All these parameters were studied in relation to the clinically most important index of disease progression, namely Performance Status (ECOG PS). We also tested the reducing ability of different antioxidant agents on reactive oxygen species levels by measuring the increase in glutathione peroxidase activity, and the reduction of serum levels of IL‐6 and TNF. Design, setting and subjects. We carried out an open non randomized study on 28 advanced stage cancer patients (stage III, 10.7%, and stage IV, 89.3%) with tumours at different (8) sites: all were hospitalized in the Medical Oncology Dept, University of Cagliari Interventions. The patients were divided into 5 groups and a different antioxidant treatment was administered to each group. The selected antioxidants were: alpha lipoic acid 200 mg/day orally, N‐acetylcysteine 1800 mg/day i.v. or carboxycysteine‐lysine salt 2.7 g/day orally, amifostine 375 mg/day i.v., reduced glutathione 600 mg/day i.v., vitamin A 30000 IU/day orally plus vitamin E 70 mg/day orally plus Vitamin C 500 mg/day orally. The antioxidant treatment was administered for 10 consecutive days. Results. Our results show that all but one of the antioxidants tested were effective in reducing reactive oxygen species levels and 2 of them (cysteine‐containing compounds and amifostine) had the additional effect of increasing glutathione peroxidase activity. Comprehensively, the “antioxidant treatment” was found to have an effect both on reactive oxygen species levels and glutathione peroxidase activity. The antioxidant treatment also reduced serum levels of IL‐6 and TNF. Patients in both ECOG PS 0‐1 and ECOG PS 2‐3 responded to antioxidant treatment.     

A micro-anatomical model of the distribution of myocardial endomysial collagen

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.     

FcRav2, a gene with a ROGDI domain involved in Fusarium head blight and crown rot on durum wheat caused by Fusarium culmorum

Fusarium culmorum is a soil‐borne fungal pathogen which causes foot and root rot and Fusarium head blight on small‐grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in the contamination of kernels with type B trichothecene mycotoxins. Our knowledge of the pathogenicity factors of this fungus is still limited. A transposon tagging approach based on the mimp1/impala double‐component system has allowed us to select a mutant altered in multiple metabolic and morphological processes, trichothecene production and virulence. The flanking regions of mimp1 were used to seek homologies in the F. culmorum genome, and revealed that mimp1 had reinserted within the last exon of a gene encoding a hypothetical protein of 318 amino acids which contains a ROGDI‐like leucine zipper domain, supposedly playing a protein–protein interaction or regulatory role. By functional complementation and bioinformatic analysis, we characterized the gene as the yeast Rav2 homologue, confirming the high level of divergence in multicellular fungi. Deletion of FcRav2 or its orthologous gene in F. graminearum highlighted its ability to influence a number of functions, including virulence, trichothecene type B biosynthesis, resistance to azoles and resistance to osmotic and oxidative stress. Our results indicate that the FcRav2 protein (and possibly the RAVE complex as a whole) may become a suitable target for new antifungal drug development or the plant‐mediated resistance response in filamentous fungi of agricultural interest.     

Alginate beads as a tool to handle,cryopreserve and culture isolated human primordial/primary follicles

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5–15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME₂SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.     

Signal transduction in T lymphocites under simulated microgravity conditions: involvement of PKC isoforms

Previous data obtained from experiments either in space or in clinostats have shown that: a) human T lymphocytes activation is strongly inhibited; b) the distribution of protein kinase C (PKC) in human leukocytes is altered; c) expression of IL-2 and IL-2-R-alpha is altered. In this study we focus our attention on different isoforms of PKC to determine whether microgravity directly affects the activity and subcellular distribution of PKC. This work was carried out with Con A and anti-CD 28 activated human T cells in simulated microgravity conditions in the Random Positioning Machine (RPM). The cellular fractions (nuclear, cytosolic and membrane) extracted were subjected to Western blotting and RT-PCR analysis.