In Silico and In Vitro Studies on an Asymmetrical Porphyrin Derivative with Therapeutic Potential in Skin Disorders.

For developing novel photosensitizers with therapeutic potential in non-malignant and malignant cutaneous disorders, the unsymmetrical porphyrin, 5-(2-hydroxy-3-methoxyphenyl)-10, 15, 20-tris-(4-carboxymethylphenyl) porphyrin, was evaluated in silico and in vitro. The cellular uptake of the investigated porphyrin and its ability to perform photodynamic therapy were investigated in terms of the viability, proliferation, and necrosis of human HaCaT keratinocytes and human Hs27 skin fibroblasts, in correlation with the predictions regarding diffusion through cell membranes, ADMET profile (absorption, distribution, metabolism, elimination, toxicity), and potential pharmacological mechanism. Molecular docking and 250 ns molecular dynamics simulations revealed that P5.2 has the potential to form a relatively stable complex with the carbonic anhydrase IX catalytic site, the lowest predicted free energy of binding (MM/PBSA) being -39.097 kcal/mol. The results of the in vitro study showed that P5.2 is incorporated within 24 h in the investigated cells, especially in HaCaT keratinocytes, indicating its photosensitizing ability. Nevertheless, P5.2 does not exert significant cytotoxicity in "dark" conditions. In turn, PDT induced a decrease in the number of metabolically active HaCaT keratinocytes within 24 h, accompanied by a 4-fold increase in lactate dehydrogenase release, indicating its ability to perform PDT in human skin cells. The experimental results suggest that the asymmetrical porphyrin is a promising candidate theranostics agent for skin disorders.

Modifications in cellular viability, DNA damage and stress responses inflicted in cancer cells by copper-64 ions.

Due to combined therapeutical emissions, a high linear energy transfer Auger-electrons with the longer ranged ß- particles, 64Cu-based radiopharmaceuticals raise particular theragnostic interest in cancer, by joined therapeutic and real-time PET imaging properties. The in vitro study aimed to investigate the biological and molecular background of 64CuCl2 therapy by analyzing the damages and stress responses inflicted in various human normal and tumor cell lines. Colon (HT29 and HCT116) and prostate carcinoma (DU145) cell lines, as well as human normal BJ fibroblasts, were treated up to 72 h with 2-40 MBq/mL 64CuCl2. Radioisotope uptake and retention were assessed, and cell viability/death, DNA damage, oxidative stress, and the expression of 84 stress genes were investigated at various time points after [64Cu]CuCl2 addition. All the investigated cells incorporated 64Cu ions similarly, independent of their tumoral or normal status, but their fate after exposure to [64Cu]CuCl2 was cell-dependent. The most striking cytotoxic effects of the radioisotope were registered in colon carcinoma HCT116 cells, for which a substantial decrease in the number of metabolically active cells, and an increased DNA damage and oxidative stress were registered. The stress gene expression study highlighted the activation of both death and repair mechanisms in these cells, related to extrinsic apoptosis, necrosis/necroptosis or autophagy, and cell cycle arrest, nucleotide excision repair, antioxidant, and hypoxic responses, respectively. The in vitro study indicated that 40 MBq/mL [64Cu]CuCl2 delivers a therapeutic effect in human colon carcinoma, but its use is limited by harmful, yet lower effects on normal fibroblasts. The exposure of tumor cells to 20 MBq/mL [64Cu]CuCl2, might be used for a softer approach aiming for a lower radiotoxicity in normal fibroblasts as compared to tumor cells. This radioactive concentration was able to induce a persistent decrease in the number of metabolically active cells, accompanied by DNA damage and oxidative stress, associated with significant changes in stress gene expression in HCT116 colon cancer cells.

Interaction of Some Asymmetrical Porphyrins with U937 Cell Membranes-In Vitro and In Silico Studies.

The aim of the present study was to assess the effects exerted in vitro by three asymmetrical porphyrins (5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin, 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatozinc(II), and 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatocopper(II)) on the transmembrane potential and the membrane anisotropy of U937 cell lines, using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), respectively, as fluorescent probes for fluorescence spectrophotometry. The results indicate the hyperpolarizing effect of porphyrins in the concentration range of 0.5, 5, and 50 µM on the membrane of human U937 monocytic cells. Moreover, the tested porphyrins were shown to increase membrane anisotropy. Altogether, the results evidence the interaction of asymmetrical porphyrins with the membrane of U937 cells, with potential consequences on cellular homeostasis. Molecular docking simulations, and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy of binding calculations, supported the hypothesis that the investigated porphyrinic compounds could potentially bind to membrane proteins, with a critical role in regulating the transmembrane potential. Thus, both the free base porphyrins and the metalloporphyrins could bind to the SERCA2b (sarco/endoplasmic reticulum ATPase isoform 2b) calcium pump, while the metal complexes may specifically interact and modulate calcium-dependent (large conductance calcium-activated potassium channel, Slo1/KCa1.1), and ATP-sensitive (KATP), potassium channels. Further studies are required to investigate these interactions and their impact on cellular homeostasis and functionality.

Assessment of Some Unsymmetrical Porphyrins as Promising Molecules for Photodynamic Therapy of Cutaneous Disorders.

In order to select for further development novel photosensitizers for photodynamic therapy in cutaneous disorders, three unsymmetrical porphyrins, namely 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.2), 5-(2-hydroxy-5-methoxyphenyl)-10,15,20-tris-(4-carboxymethylphenyl) porphyrin (P3.2), and 5-(2,4-dihydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P4.2), along with their fully symmetrical counterparts 5,10,15,20-tetrakis-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.1) and 5,10,15,20-tetrakis-(4-carboxymethylphenyl) porphyrin (P3.1) were comparatively evaluated. The absorption and fluorescence properties, as well as atomic force microscopy measurements were performed to evaluate the photophysical characteristics as well as morphological and textural properties of the mentioned porphyrins. The cellular uptake of compounds and the effect of photodynamic therapy on the viability, proliferation, and necrosis of human HaCaT keratinocytes, human Hs27 skin fibroblasts, human skin SCL II squamous cell carcinoma, and B16F10 melanoma cells were assessed in vitro, in correlation with the structural and photophysical properties of the investigated porphyrins, and with the predictions regarding diffusion through cell membranes and ADMET properties. All samples were found to be isotropic and self-similar, with slightly different degrees of aggregability, had a relatively low predicted toxicity (class V), and a predicted long half-life after systemic administration. The in vitro study performed on non-malignant and malignant skin-relevant cells highlighted that the asymmetric P2.2 porphyrin qualified among the five investigated porphyrins to be a promising photosensitizer candidate for PDT in skin disorders. P2.2 was shown to accumulate well within cells, and induced by PDT a massive decrease in the number of metabolically active skin cells, partly due to cell death by necrosis. P2.2 had in this respect a better behavior than the symmetric P.2.1 compound and the related asymmetric compound P4.2. The strong action of P2.2-mediated PDT on normal skin cells might be an important drawback for further development of this compound. Meanwhile, the P3.1 and P3.2 compounds were not able to accumulate well in skin cells, and did not elicit significant PDT in vitro. Taken together, our experiments suggest that P2.2 can be a promising candidate for the development of novel photosensitizers for PDT in skin disorders.

Pros and cons of NRF2 activation as adjunctive therapy in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with an important inflammatory component accompanied by deregulated redox-dependent signaling pathways that are feeding back into inflammation. In this context, we bring into focus the transcription factor NRF2, a master redox regulator that exerts exquisite antioxidant and anti-inflammatory effects. The review does not intend to be exhaustive, but to point out arguments sustaining the rationale for applying an NRF2-directed co-treatment in RA as well as its potential limitations. The involvement of NRF2 in RA is emphasized through an analysis of publicly available transcriptomic data on NRF2 target genes and the findings from NRF2-knockout mice. The impact of NRF2 on concurrent pathologic mechanisms in RA is explained by its crosstalk with major redox-sensitive inflammatory and cell death-related pathways, in the context of the increased survival of pathologic cells in RA. The proposed adjunctive therapy targeted to NRF2 is further sustained by the existence of promising NRF2 activators that are in various stages of drug development. The interference of NRF2 with conventional anti-rheumatic therapies is discussed, including the cytoprotective effects of NRF2 for alleviating drug toxicity. From another perspective, the review presents how NRF2 activation would be decreasing the efficacy of synthetic anti-rheumatic drugs by increasing drug efflux. Future perspectives regarding pharmacologic NRF2 activation in RA are finally proposed.

Insight into the Web of Stress Responses Triggered at Gene Expression Level by Porphyrin-PDT in HT29 Human Colon Carcinoma Cells.

Photodynamic therapy (PDT), a highly targeted therapy with acceptable side effects, has emerged as a promising therapeutic option in oncologic pathology. One of the issues that needs to be addressed is related to the complex network of cellular responses developed by tumor cells in response to PDT. In this context, this study aims to characterize in vitro the stressors and the corresponding cellular responses triggered by PDT in the human colon carcinoma HT29 cell line, using a new asymmetric porphyrin derivative (P2.2) as a photosensitizer. Besides investigating the ability of P2.2-PDT to reduce the number of viable tumor cells at various P2.2 concentrations and fluences of the activating light, we assessed, using qRT-PCR, the expression levels of 84 genes critically involved in the stress response of PDT-treated cells. Results showed a fluence-dependent decrease of viable tumor cells at 24 h post-PDT, with few cells that seem to escape from PDT. We highlighted following P2.2-PDT the concomitant activation of particular cellular responses to oxidative stress, hypoxia, DNA damage and unfolded protein responses and inflammation. A web of inter-connected stressors was induced by P2.2-PDT, which underlies cell death but also elicits protective mechanisms that may delay tumor cell death or even defend these cells against the deleterious effects of PDT.

Distinctive Under-Expression Profile of Inflammatory and Redox Genes in the Blood of Elderly Patients with Cardiovascular Disease.

PURPOSE: Chronic low-grade inflammation and oxidative stress are present in most of the pathologic mechanisms underlying non-communicable diseases. Inflammation and redox biomarkers might therefore have a value in disease prognosis and therapy response. In this context, we performed a case-control study for assessing in whole blood the expression profile of inflammation and redox-related genes in elderly subjects with various comorbidities. PATIENTS AND METHODS: In the blood of 130 elderly subjects with various pathologies (cardiovascular disease, hypertension, dyslipidemia including hypercholesterolemia, type 2 diabetes mellitus), kept under control by polyvalent disease-specific medication, we investigated by pathway-focused qRT-PCR a panel comprising 84 inflammation-related and 84 redox-related genes. RESULTS: The study highlights a distinctive expression profile of genes critically involved in NF-κB-mediated inflammation and redox signaling in the blood of patients with cardiovascular disease, characterized by significant down-regulation of the genes NFKB2, NFKBIA, RELA, RELB, AKT1, IRF1, STAT1, CD40, LTA, TRAF2, PTGS1, ALOX12, DUOX1, DUOX2, MPO, GSR, TXNRD2, HSPA1A, MSRA, and PDLIM1. This gene expression profile defines the transcriptional status of blood leukocytes in stable disease under medication control, without discriminating between disease- and therapy-related changes. CONCLUSION: The study brings preliminary proof on a minimally invasive strategy for monitoring disease in patients with cardiovascular pathology, from the point of view of inflammation or redox dysregulation in whole blood.

In vitro study of the orthodontic mini-implants influence on the growth of human osteoblasts.

This study aimed to assess the in vitro biocompatibility of titanium (Ti) alloy orthodontic mini-implants by correlating human osteoblasts (HOb) response with chemical composition and surface morphology of mini-implants. HOb were cultivated with or without custom-made and commercial mini-implants, discs and filings. The surface morphology and chemical composition of the implants were assessed under the scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX) microanalysis system. Cell viability, adhesion and proliferation were analyzed by optical microscopy and flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction and lactate dehydrogenase (LDH) release tests were used to assess the cytotoxicity of discs and filings-treated culture medium. Shape, adhesion, and multiplication of HOb were not significantly altered by the presence of mini-implants, discs or filings in culture, even though Ti alloy may exert in vitro a low cytotoxic effect on HOb adhered to discs. Morphology analysis by SEM demonstrated that custom-made mini-implants' surface differs from that of commercial mini-screws in terms of surface finish and roughness, whilst EDX analysis showed largely similar percentages of Ti, aluminum and vanadium for the two types of implants. No major differences were noticed regarding the effect exerted in vitro on HOb by the investigated implants. The new mini-implants have a convenient in vitro cytotoxicity profile on HOb.

Emerging Therapeutic Targets in Oncologic Photodynamic Therapy.

BACKGROUND: Reactive oxygen species sustain tumorigenesis and cancer progression through deregulated redox signalling which also sensitizes cancer cells to therapy. Photodynamic therapy (PDT) is a promising anti-cancer therapy based on a provoked singlet oxygen burst, exhibiting a better toxicological profile than chemo- and radiotherapy. Important gaps in the knowledge on underlining molecular mechanisms impede on its translation towards clinical applications. AIMS AND METHODS: The main objective of this review is to critically analyse the knowledge lately gained on therapeutic targets related to redox and inflammatory networks underlining PDT and its outcome in terms of cell death and resistance to therapy. Emerging therapeutic targets and pharmaceutical tools will be documented based on the identified molecular background of PDT. RESULTS: Cellular responses and molecular networks in cancer cells exposed to the PDT-triggered singlet oxygen burst and the associated stresses are analysed using a systems medicine approach, addressing both cell death and repair mechanisms. In the context of immunogenic cell death, therapeutic tools for boosting anti-tumor immunity will be outlined. Finally, the transcription factor NRF2, which is a major coordinator of cytoprotective responses, is presented as a promising pharmacologic target for developing co-therapies designed to increase PDT efficacy. CONCLUSION: There is an urgent need to perform in-depth molecular investigations in the field of PDT and to correlate them with clinical data through a systems medicine approach for highlighting the complex biological signature of PDT. This will definitely guide translation of PDT to clinic and the development of new therapeutic strategies aimed at improving PDT.

Studies on the Synthesis, Photophysical and Biological Evaluation of Some Unsymmetrical Meso-Tetrasubstituted Phenyl Porphyrins.

Abstract: We designed three unsymmetrical meso-tetrasubstituted phenyl porphyrins for further development as theranostic agents for cancer photodynamic therapy (PDT): 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (P2.2), Zn(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Zn(II)2.2) and Cu(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Cu(II)2.2). The porphyrinic compounds were synthesized and their structures were confirmed by elemental analysis, FT-IR, UV-Vis, EPR and NMR. The compounds had a good solubility in polar/nonpolar media. P2.2 and, to a lesser extent, Zn(II)2.2 were fluorescent, albeit with low fluoresence quantum yields. P2.2 and Zn(II)2.2 exhibited PDT-acceptable values of singlet oxygen generation. A "dark" cytotoxicity study was performed using cells that are relevant for the tumor niche (HT-29 colon carcinoma cells and L929 fibroblasts) and for blood (peripheral mononuclear cells). Cellular uptake of fluorescent compounds, cell viability/proliferation and death were evaluated. P2.2 was highlighted as a promising theranostic agent for PDT in solid tumors considering that P2.2 generated PDT-acceptable singlet oxygen yields, accumulated into tumor cells and less in blood cells, exhibited good fluorescence within cells for imagistic detection, and had no significant cytotoxicity in vitro against tumor and normal cells. Complexing of P2.2 with Zn(II) or Cu(II) altered several of its PDT-relevant properties. These are consistent arguments for further developing P2.2 in animal models of solid tumors for in vivo PDT.

In vitro effects of prolonged exposure to quercetin and epigallocatechin gallate of the peripheral blood mononuclear cell membrane.

The study aimed to assess biophysical changes that take place in the peripheral blood mononuclear cell (PBMC) membranes when exposed in vitro to 10 µM quercetin or epigallocatechin gallate (EGCG) for 24 and 48 h. PBMCs isolated from hypercholesterolemia patients were compared to those from normocholesterolemia subjects. The membrane fluidity and transmembrane potential were evaluated and the results were correlated with biochemical parameters relevant to oxidative stress, assessed in the patients' plasma. The baseline value of PBMC membrane anisotropy for the hypercholesterolemia patients was lower than that of the control group. These results correlated with the plasma levels of advanced glycation end products, which were significantly higher in the hypercholesterolemia group, and the total plasma antioxidant status, which was significantly higher in normocholesterolemia subjects. In the case of normocholesterolemia cells in vitro, polyphenols induced a decrease in membrane anisotropy (7.25-11.88% at 24 h, 1.82-2.26% at 48 h) and a hyperpolarizing effect (8.30-8.90% at 24 h and 4.58-13.00% at 48 h). The same effect was induced in hypercholesterolemia cells, but only after 48 h exposure to the polyphenols: the decrease in membrane anisotropy was 5.70% for quercetin and 2.33% for EGCG. After 48 h of in vitro incubation with the polyphenols, PBMCs isolated from hypercholesterolemia patients exhibited the effects that had been registered in cells from normocholesterolemia subjects after 24 h exposure. These results outlined the beneficial action of the studied polyphenols, quercetin and EGCG, as dietary supplements in normocholesterolemia and hypercholesterolemia patients.

Effects of zearalenone on oxidative stress and inflammation in weanling piglets.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of Fusarium genera. Piglets were fed for 18 days with a control or a ZEN (316 ppb) contaminated diet. At the end of the experiment tissue samples were taken for assessment of: lymphocyte proliferation, monocytes and granulocytes respiratory burst, inflammatory cytokine synthesis in blood and liver, expression of genes involved in oxidative stress or in inflammation, plasma biochemical parameters, total antioxidant status and nitric oxide synthesis. In blood, ZEN increases the respiratory burst of monocytes and the inflammatory cytokine (TNF alpha, IL-1 beta, IFN gamma) synthesis, while in liver, ZEN decreases the synthesis of all inflammatory cytokines investigated. In liver and spleen, different effect on the expression of genes involved in oxidative stress and inflammation was observed. While in liver, ZEN decrease the expression of cyclooxigenase gene, but increase the expression of glutathione peroxydase and catalase genes; in spleen, ZEN induces a decrease of the superoxide dismutase gene expression together with an increase of the cyclooxigenase. In conclusion, our results showed that liver, spleen and blood may also be target tissues in weanling piglets fed ZEN contaminated diet, with different effects on oxidative stress and inflammation.

Membranar effects exerted in vitro by polyphenols - quercetin, epigallocatechin gallate and curcumin - on HUVEC and Jurkat cells, relevant for diabetes mellitus.

Polyphenols are largely studied for their beneficial action in various pathologies, but the correlation with their effects on cell membranes is still elusive. In the present study we assessed the effects exerted in vitro by quercetin, epigallocatechin gallate and curcumin on membrane fluidity and transmembrane potential of human umbilical vein endothelial cells and Jurkat T lymphoblasts, in experimental conditions mimicking diabetes mellitus, i.e. high glucose conditions or increased concentration of advanced glycation end products. Results showed that the investigated polyphenols had beneficial effects on cell membranes altered in diabetic conditions, by restoring transmembrane potential and by membrane "stiffening". Moreover, they limited the release of pro-inflammatory factors, like monocyte chemotactic protein-1. These effects were more obvious for cells exposed to advanced glycation end products specific for the late stages of diabetes. Apparently, the inhibitory action of polyphenols on lipid peroxidation was associated with a decrease of membrane fluidity. Concluding, our in vitro study highlighted the potential beneficial action of polyphenols mainly in the late stages of diabetes, exerted at the level of membrane fluidity and transmembrane potential, accompanied by an anti-inflammatory effect on endothelial and immune cells.

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential.

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.

Effects of zearalenone and its derivatives on porcine immune response.

Zearalenone (ZEN), a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. In this in vitro study, we compared the effects of zearalenone (ZEN) and some of its derivatives: α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL), and zearalanone (ZAN) on several peripheral blood mononuclear cell (PBMC) parameters: cytotoxicity, proliferation, as well as antibody and cytokine synthesis. The amounts of toxins necessary to inhibit viability, in a dehydrogenase enzyme activity assay (MTT test), by 50% were: 22.7 µM for ZEN, 29.1 µM for α-ZOL, 17.3 µM for ß-ZOL and 26.3 µM for ZAN. The administration of 10 µM toxin induced a decrease in the ConA stimulated proliferation of PBMC by 19.6% for ZAN, 45.4% for ZEN, 43.6% for α-ZOL and 85.2% for ß-ZOL, when compared to the control stimulated cells. Also, ZEN and its metabolites at concentrations higher than 5 µM induced a significant decrease of the IgG, IgA or IgM levels. Concentrations of 5 and 10 µM of ZEN and ZAN significantly decreased the TNF-α synthesis in the supernatant of the stimulated cells; 10 µM of ZAN also decreased IL-8 synthesis. In conclusion, our results show that ZEN and ZEN derivatives altered several parameters of the humoral and cellular immune response. Therefore, our results are clinically relevant as ZEN and its metabolites are frequent contaminants of animal feed and we have shown that intoxicated animals are incapable of inducing an adequate immune response.

Assessment of the potential of a boron-fructose additive in counteracting the toxic effect of Fusarium mycotoxins.

Trichotecenes are mycotoxins produced by Fusarium sp., which may contaminate animal feeds and human food. A feeding trial was conducted to evaluate the effect of a fusarotoxin-contaminated diet, and to explore the counteracting potential of a calcium fructoborate (CFrB) additive on performance, typical health biochemistry parameters and immune response in weaned pigs. A naturally contaminated maize, containing low doses of deoxynivalenol, zearalenone, fumonisins and T-2/HT-2 toxins (1790, 20, 0·6 and 90 parts per billion), was included in a maize-soyabean meal diet, and given ad libitum to eight weaned piglets (two groups: four pigs/group) for a period of 24 d. CFrB was administered to one of the contaminated groups and to another four piglets as a daily supplement, following the manufacturer's recommendation. A decrease in performance was observed in contaminated animals at this concentration of feed toxins, which was ameliorated by the dietary CFrB supplementation. Fusarium toxins also altered the pig immune response by increasing (P < 0·05) the ex vivo peripheral blood mononuclear cell proliferation (111·7 % in comparison with control), the respiratory burst of porcine granulocytes (15·4 % for responsive cells v. 5·1 % for unstimulated cells and 70·95 v. 22·65 % for stimulated cells, respectively), the percentage of peripheral T, CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) subsets and the synthesis of IL-1ß, TNF-α and IL-8 (123·8, 217·1 and 255·1 %, respectively). The diet containing the CFrB additive reduced these exacerbated cellular immune responses induced by Fusarium toxins. However, consumption of CFrB did not counteract the effect of mycotoxins on biochemistry parameters, and increased plasma IgM and IgG of contaminated pigs.

Preliminary study on the immunologic background of good clinical outcome in rheumatoid arthritis patients after one month therapy with leflunomide.

This preliminary study focuses on early peripheral cellular immune changes after 1 month therapy with leflunomide, in 18 patients with severe rheumatoid arthritis, previously treated with methotrexate. A good clinical outcome of disease was documented and we showed that a particular target of short-time leflunomide therapy in rheumatoid arthritis was the peripheral innate immune system (NK cells and the population of granulocytes developing phagocytosis and superoxide anion production when challenged ex vivo with zymosan particles). Meanwhile, the high inter-individual variability of adaptive immunity required data analysis in subgroups of patients. We showed that the abnormal increase of peripheral leukocytes counts, or the decrease towards normal values of the CD4:CD8 lymphocytes ratio, or the inhibition of uridine uptake by ex vivo activated lymphocytes were consistent with a positive clinical evolution, proved by the reduction of tender/swollen joints, morning stiffness duration or acute phase response. We emphasized that significant benefits of short-term leflunomide therapy were associated with functional suppression of peripheral B lymphocytes. Hence, the positive evolution of rheumatoid arthritis patients seemed to be specifically linked to early drug-induced changes of trafficking or uridine metabolism of mononuclear cells.

Laboratory diagnosis of infectious diarrhoea syndrome; a three years study in two hospitals of infectious diseases.

Infectious diarrhoea is a syndrome caused by a variety of bacterial, viral and parasitic organisms which represents a major cause of morbidity and mortality all over the world. The wide diversity of etiological agents impairs the surveillance and the diagnosis and affects the correct treatment applied to reduce the long-term complications. Besides well known enteric pathogens such as Salmonella, Shigella and Yersinia, a high number of emergent and re-emergent aetiologies are now recognised to be at the origin of diarrhoea. The lack of a correct diagnostic algorithm and adequate methods of analyses leads to under-evaluation and incertitude in an important number of clinical cases. Our study was designed as a complex analysis of the stool specimens collected from the patients, in the purpose to improve the laboratory diagnostic and to enhance the number of confirmed cases of infectious diarrhoea. A number of 756 samples from inpatients with diarrhoea were tested targeting pathogenic and opportunistic bacteria, viruses and parasites by classical and molecular methods. We documented that, in case of non-Salmonella, non-Shigella, non-Yersinia diarrhoea, the quality of diagnostic was improved by increasing the percentage of positive specimens to 22.49% compared to 11.12% when only bacteria, 5.56% when only viruses and 4.10% when only parasites were investigated. The laboratory data are of great value in evaluating the diarrhoea syndrome offering the documentation for an accurate epidemiological response and an adequate treatment.