Study of deferiprone-induced agranulocytosis in β-thalassemic patients through a metabolomics approach

ABSTRACT β-Thalassemia is one of the most prevalent forms of congenital blood disorders, characterised by a reduced or absent ability to produce haemoglobin. The mainstay of treatment consists of blood transfusion to maintain the patient’s haemoglobin above 9-10 g/dL. Repeated transfusions result in an excessive accumulation of iron in the body, removal of which is achieved through iron chelating agents. Deferiprone is the first orally bioavailable iron chelator, approved for clinical use in 1997. Considering its potential toxicity, the use of Deferiprone is allowed in Europe only for the treatment of thalassemia major, when Deferoxamine therapy is contraindicated or unappropriated. The main Deferiprone adverse effect is the development of agranulocytosis (in 1–2% of patients). The mechanisms behind this negative effect remain largely unresolved. Currently, only a few metabolomics studies have been performed on neutrophils. Neutrophils are the immune cells forming the major arm of innate immunity. Due to their physiological characteristic, the study of these cells in vitro presents several difficulties meaning that standard protocols for most assays must be optimised. This thesis aimed to establish protocols for the study of the metabolomic profile of human neutrophils in healthy and pathological conditions. In particular, this study aimed to investigate the PMNs (polymorphonuclear leukocytes) metabolic profile of beta thalassemic patients treated with iron chelator Deferiprone, to explore its potential relationship with the onset of agranulocytosis, using a metabolomics approach. Our data demonstrated that analysis of the metabolomic profiles in PMNs with GC-MS, allowed us to identify different metabolites including organic acids, amino acids, fatty acids, and sugars. The results showed a different metabolomic profile between PMNs obtained from patients with Deferiprone-induced agranulocytosis and PMNs of patients without Deferiprone-induced agranulocytosis. Multivariate statistical analysis of GCMS data revealed that the PMNs of patients with Deferiprone-induced agranulocytosis have a metabolic profile characterized by an increase of metabolites directly involved in the metabolic pathways of glutathione synthesis and by a decrease of arachidonic acid stearic acid and inosine. 5 Considering the important physiological roles of these metabolites, our results could shed light on the physiopathological mechanisms of this harmful side effect of DFP treatment. This work is a pilot study that has been only validated in a small independent cohort and, therefore, further confirmation in larger studies is required

Influence of Extraction Techniques on Physicalchemical Characteristics and Volatile Compounds of Extra Virgin Olive Oil

The purpose of this study was to investigate three types of extraction methods of extra virgin olive oil (EVOO) from the same cultivar (Ortice olive cultivar): traditional or pressing (T) system, decanter centrifugation (DC) system and a patented horizontal axis decanter centrifugation (HADC) system. Oil samples were subjected to chemical analyses: free acidity, peroxide value, ultraviolet light absorption K232 and K270, total polyphenols, antioxidant capacity, volatile compounds and olfactory characteristics by electronic nose. The two centrifugation systems showed better free acidity and peroxides value but total polyphenol content was particularly high in extra virgin olive oil produced by patented HADC system. Same volatile substances that positively characterize the oil aroma were found in higher amount in the two centrifugation systems, although some differences have been detected between DC and HADC system, other were found in higher amount in extra virgin olive oil produced by T system. The electronic nose analysis confirmed these results, principal component analysis (PCA) and correlation matrix showed the major differences between EVOO produced by T and HADC system. Taken together the results showed that DC and HADC systems produce EVOO with better characteristics than T system and patented HADC is the best extraction system

Gram variability and prodigiosin production in isolate of Serratia marcescens obtained from blackberries

Prodigiosin is a secondary metabolite produced by Gram negative Serratia marcescens (S. marcescens) bacteria and it is characterized by a pyrrolyl pyrromethene skeleton and different alkyl substituents. In its production are involved different enzymes and it is a pigment capable of inducing the apoptosis of several cancer cell lines, so its antioxidant, antifungal, antitumoral and antibiotic potential is very important. Prodigiosin production needs dissolved oxygen, an incubation time of ≈ 60 hours, a solution pH of ≈ 8, a growth temperature of 28°C, and presence of dissolved phosphates. Its production is controlled by a complex regulatory network of both N-acyl-L-homoserine lactone-quorum-sensing-dependent and independent pathways. Prodigiosin can exist in two forms, depending on the hydrogen ion concentration of the solution: in an acid medium, the red pigment exhibits a sharp spectral peak at 535 nm, while in the alkaline medium the pigment is coloured orange-yellow and has a broader spectral curve at 470 nm. In my work, three isolates of Serratia marcescens from blackberry fruit (Rubus fruticosus, cultivar "Polar"), later named D2, D4 and D6, were used, while the isolate Serratia marcescens var kiliensis PCM 550 (designated as PJ), from the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław was used as reference microorganism. The aims of the work were, firstly, to characterize the pigment prodigiosin and, secondly, to verify if the Gram variability of S. marcescens, noticed in previous works, was correlated to prodigiosin production. To understand the spectrophotometric characteristics of the pigment, extraction and purification methods were performed. On the other hand, the Gram variability was studied through growth curve analysis in LB and LB + 1% glucose, which inhibits the prodigiosin production: in this way, slide samples at different timepoints were collected for Gram staining. Further understanding of the phenomenon was proved by HPLC analysis for lipopolysaccharides’ (LPS) carbohydrates, isolated from bacteria grown in shaking conditions, where no prodigiosin production was seen, and stable conditions, where prodigiosin was observed. All these analyses are supported by previous information found in the literature to understand the growing conditions of S. marcescens (e.g., oxygen, glucose, and nutrients influence) and the localization of prodigiosin.Prodigiosin is a secondary metabolite produced by Gram negative Serratia marcescens (S. marcescens) bacteria and it is characterized by a pyrrolyl pyrromethene skeleton and different alkyl substituents. In its production are involved different enzymes and it is a pigment capable of inducing the apoptosis of several cancer cell lines, so its antioxidant, antifungal, antitumoral and antibiotic potential is very important. Prodigiosin production needs dissolved oxygen, an incubation time of ≈ 60 hours, a solution pH of ≈ 8, a growth temperature of 28°C, and presence of dissolved phosphates. Its production is controlled by a complex regulatory network of both N-acyl-L-homoserine lactone-quorum-sensing-dependent and independent pathways. Prodigiosin can exist in two forms, depending on the hydrogen ion concentration of the solution: in an acid medium, the red pigment exhibits a sharp spectral peak at 535 nm, while in the alkaline medium the pigment is coloured orange-yellow and has a broader spectral curve at 470 nm. In my work, three isolates of Serratia marcescens from blackberry fruit (Rubus fruticosus, cultivar "Polar"), later named D2, D4 and D6, were used, while the isolate Serratia marcescens var kiliensis PCM 550 (designated as PJ), from the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław was used as reference microorganism. The aims of the work were, firstly, to characterize the pigment prodigiosin and, secondly, to verify if the Gram variability of S. marcescens, noticed in previous works, was correlated to prodigiosin production. To understand the spectrophotometric characteristics of the pigment, extraction and purification methods were performed. On the other hand, the Gram variability was studied through growth curve analysis in LB and LB + 1% glucose, which inhibits the prodigiosin production: in this way, slide samples at different timepoints were collected for Gram staining. Further understanding of the phenomenon was proved by HPLC analysis for lipopolysaccharides’ (LPS) carbohydrates, isolated from bacteria grown in shaking conditions, where no prodigiosin production was seen, and stable conditions, where prodigiosin was observed. All these analyses are supported by previous information found in the literature to understand the growing conditions of S. marcescens (e.g., oxygen, glucose, and nutrients influence) and the localization of prodigiosin

Oxidative stress protection by newly synthesized nitrogen compounds with pharmacological potential

In this study we used new nitrogen compounds obtained by organic synthesis whose structure predicted an antioxidant potential and then an eventual development as molecules of pharmacological interest in diseases involving oxidative stress. The compounds, identified as FMA4, FMA5, FMA7 and FMA8 differ in the presence of hydroxyl groups located in the C-3 and/or C-4 position of a phenolic unit, which is possibly responsible for their free radicals buffering capacity. Data from the DPPH discoloration method confirm the high antiradical efficiency of the compounds. The results obtained with cellular models (L929 and PC12) show that they are not toxic and really protect from membrane lipid peroxidation induced by the ascorbate-iron oxidant pair. The level of protection correlates with the drugs lipophilic profile and is sometimes superior to trolox and equivalent to that observed for a-tocopherol. The compounds FMA4 and FMA7 presented also a high protection from cell death evaluated in the presence of a staurosporine apoptotic stimulus. That protection results in a significant reduction of caspase-3 activity induced by staurosporine which by its turn seems to result from a protection observed in the membrane receptor pathway (caspase-8) together with a protection observed in the mitochondrial pathway (caspase-9). Taken together the results obtained with the new compounds, with linear chains, open up perspectives for their use as therapeutical agents, namely as antioxidants and protectors of apoptotic pathways. On the other hand the slight pro-oxidant profile obtained with the cyclic structures suggests a different therapeutic potential that is under current investigation.Fundação para a Ciência e a Tecnologia (FCT) - POCTI and/or FEDER programmes, SFRH/BD/17174/2004, SFRH/BD/3185/2000

Regulation of polymeric immunoglobulin receptor by reovirus in intestinal epithelial cells

Most pathogens invade the human body through mucosal surfaces. Enteric viruses are one such group of pathogens that are of great public health significance because they cause a number of serious diseases including gastroenteritis, hepatitis and poliomyelitis. Gastrointestinal disease is the second most common cause of morbidity worldwide, with diarrhea ranking first among infectious diseases in the categories of frequency and mortality in children. The mucosal epithelium provides the first line of defense against invading pathogens by serving as the first sensors of microbial infection and launching an innate immune response that leads to the development of adaptive immunity. An immunologic function of the mucosal epithelium is to mediate transcytosis of secretory immunoglobulin A (sIgA) from the lamina propria into the intestinal lumen. Transcytosis of IgA is dependent on the polymeric immunoglobulin receptor (pIgR) expressed by mucosal and glandular epithelial cells because one molecule of pIgR must be synthesized for each molecule of transported IgA. Thus, pIgR plays a role in mucosal host defense, and factors that influence expression of pIgR could affect mucosal immunity. This dissertation describes efforts to understand the role of intestinal epithelial cells (IECs) as an active participant of mucosal immunity during enteric virus infection. We used reovirus to demonstrate that a newly appreciated role of the IECs in responding to infection is upregulation of pIgR expression, which appears to serve as an innate host defense mechanism. Our studies demonstrate that reovirus upregulates pIgR expression in the intestinal epithelial cell line HT-29 in a replication independent fashion, and that binding of virus to cellular receptors and partial disassembly of virus inside acidified endosomes are required steps for this innate immune response by IECs. In addition, we demonstrate that activation of calpain and NFkappaB signaling and reovirus-induced PIGR gene transcription in IECs upregulate pIgR expression during infection. Signaling induced by virus-host interaction might serve to augment pIgR-mediated transcytosis of IgA in vivo, thereby linking the innate and acquired immune responses to enteric viruses. Our studies will contribute to improving human health by advancing understanding of IEC biology and function, viral pathogenesis, and induction of innate and adaptive immunity to viruses, particularly in the intestine

Microbial characterization of discarded breads

Producción CientíficaBread is one of the most wasted products in distribution systems and in households. To consider reusing it, the presence of microorganisms must be taken into account. In this study, the microbial load of crumb and crust of four kinds of bread, sold in two different types of establishments, has been compared separately. Counts of Bacillus spp, sulphite reducing bacteria, coliforms, E. coli, Salmonella spp, lactic acid bacteria, Aerobic plate count, molds, and yeasts were determined. The microorganisms found were identified. The microbial load of discarded breads was similar to that of cereal flours found in other studies. In general, a greater presence of bacteria in breads from small stores than in those from supermarkets has been found. Some of the least sold breads in small bakeries, such as wholemeal or candeal, presented a higher microbial load. Although their quantity or type are not a concern for people's health, good practices must be implemented to reduce health risks in products made from discarded bread.LWT Volume 173, 2023, 114291Junta de Castilla y León (project VA177P20

Phenolic compounds in extra virgin olive oil stimulate human osteoblastic cell proliferation

In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11–16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18–22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9–13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly

Surface expression of secretory component and HLA class II DR antigen on glandular epithelial cells from human endometrium and two endometrial adenocarcinoma cell lines

The expression of secretory component (SC) by human glandular endometrial cells cultured in vitro was significantly increased by estradiol in the medium. Interferon-gamma and interleukin-4 stimulated the expression of SC only in the presence of estrogen. Tumor necrosis factor-alpha plus estrogen also caused a significant increase in the number of cells expressing SC. HLA class II antigen DR was detected on few glandular epithelial cells of human endometrium cultured in control medium, whereas interferon-gamma and interleukin-4, but not tumor necrosis factor-alpha, caused significant increases in the expression of DR. Estrogen in the culture medium did not significantly affect DR expression. The human endometrial adenocarcinoma cell lines, HEC and RL-95, expressed SC in approximately 50 and 20% of the cells. Also, approximately 20% of the RL-95 cells stained for DR antigen. Interferon-gamma did not influence the degree of expression of either surface marker of the two cell lines. Cells of both lines bound polymeric IgA and IgM but showed little to no binding of monomeric IgA, IgG, or an IgM previously shown not to bind SC.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44844/1/10875_2004_Article_BF00919384.pd