Response of Skin-Derived and Metastatic Human Malignant Melanoma Cell Lines to Thymoquinone and Thymoquinone-Loaded Liposomes.

Thymoquinone has been proved to be effective against neoplasms, including skin cancer. Its high lipophilicity, however, may limit its potential use as a drug. Melanoma remains the deadliest of all skin cancers worldwide, due to its high heterogeneity, depending on the stage of the disease. Our goal was to compare the anti-cancer activity of free thymoquinone and thymoquinone-loaded liposomes on two melanoma cell lines that originated from different stages of this cancer: skin-derived A375 and metastatic WM9. We evaluated the proapoptotic effects of free thymoquinone by flow cytometry and Western blot, and its mitotoxicity by means of JC-1 assay. Additionally, we compared the cytotoxicity of free thymoquinone and thymoquinone in liposomes by WST-1 assay. Our results revealed a higher antiproliferative effect of TQ in WM9 cells, whereas its higher proapoptotic activity was observed in the A375 cell line. Moreover, the thymoquinone-loaded liposome was proved to exert stronger cytotoxic effect on both cell lines studied than free thymoquinone. Differences in the response of melanoma cells derived from different stages of the disease to thymoquinone, as well as their different responses to free and carrier-delivered thymoquinone, are essential for the development of new anti-melanoma therapies. However, further research is required to fully understand them.

Plant-Derived Terpenoids: A Promising Tool in the Fight against Melanoma.

Melanoma is responsible for the highest number of skin cancer-caused deaths worldwide. Despite the numerous melanoma-treating options, the fight against it remains challenging, mainly due to its great heterogeneity and plasticity, as well as the high toxicity of standard drugs. Plant-derived terpenoids are a group of plant defense molecules that have been proven effective in killing many different types of cancer cells, both in in vitro experiments and in vivo models. In this review, we focus on recent results in the search for plant terpenoids with anti-melanoma activity. We also report on the synergistic action of combining terpenoids with other plant-derived substances, MAP kinase inhibitors, or radiation. Additionally, we present examples of terpenoid-loaded nanoparticle carriers as anti-melanoma agents that have increased permeation through the cancer tissue.

The Role of Mitochondria Dysfunction in Inflammatory Bowel Diseases and Colorectal Cancer.

Inflammatory bowel disease (IBD) is one of the leading gut chronic inflammation disorders, especially prevalent in Western countries. Recent research suggests that mitochondria play a crucial role in IBD development and progression to the more severe disease-colorectal cancer (CRC). In this review, we focus on the role of mitochondrial mutations and dysfunctions in IBD and CRC. In addition, main mitochondria-related molecular pathways involved in IBD to CRC transition are discussed. Additionally, recent publications dedicated to mitochondria-targeted therapeutic approaches to cure IBD and prevent CRC progression are discussed.

Pentoxifylline as a Potential Adjuvant Therapy for COVID-19: Impeding the Burden of the Cytokine Storm.

The outburst of inflammatory response and hypercoagulability are among the factors contributing to increased mortality in severe COVID-19 cases. Pentoxifylline (PTX), a xanthine-derived drug registered for the treatment of vascular claudication, has been reported to display broad-spectrum anti-inflammatory and immunomodulatory properties via adenosine A2A receptor (A2AR)-related mechanisms, in parallel to its rheological actions. Prior studies have indicated the efficacy of PTX in the treatment of various pulmonary diseases, including the management of acute respiratory distress syndrome of infectious causes. Therefore, PTX has been proposed to have potential benefits in the treatment of SARS-CoV-2 symptoms, as well as its complications. The aim of this review is to discuss available knowledge regarding the role of PTX as a complementary therapeutic in SARS-CoV-2.

The Effectiveness of Supportive Psychotherapy in Weight Loss in a Group of Young Overweight and Obese Women.

Overweight and obesity are among the most widespread health problems worldwide. The primary cause of obesity is an inability to control overeating. Therefore, today, obesity needs to be treated more as an eating disorder, i.e., a mental disorder, and thus, it should be approached as such. Taking the above together, this study aimed to assess the impact of supportive psychotherapy on reducing body weight in young overweight and obese women who attempted slimming therapy and, additionally, the possibility of maintaining the weight-loss effect in the long term. Sixty young women aged 20-30 were randomized into three groups that differed in therapeutic management. With the help of an individually selected diet plan, the highest effectiveness in weight loss was demonstrated in people whose weight reduction was supported by goal-oriented psychotherapy. In this group, a sustained effect of slimming and even further weight loss were observed six months following the discontinuation of the therapy. In conclusion, traditional slimming therapies using an individual diet plan and a dietitian's care are effective; however, supportive psychotherapeutic work provides more beneficial results and maintains the change from a long-term perspective.

Favipiravir in Therapy of Viral Infections.

Favipiravir (FPV) is a novel antiviral drug acting as a competitive inhibitor of RNA-dependent RNA polymerase (RdRp), preventing viral transcription and replication. FPV was approved in Japan in 2014 for therapy of influenza unresponsive to standard antiviral therapies. FPV was also used in the therapy of Ebola virus disease (EVD) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this review, we discuss the mechanisms of action, pharmacokinetic parameters, toxicity, and adverse effects of FPV, as well as clinical studies evaluating the use of FPV in the therapy of influenza virus (IV) infection, EVD, and SARS-CoV-2 infection, along with its effectiveness in treating other human RNA infections.

Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells.

A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPßS) in a 1-1000 µM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100-1000 µM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 µM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.

Electrostatic complex of neurotrophin 4 with dendrimer nanoparticles: controlled release of protein in vitro and in vivo.

Background: NT4 has been regarded as a promising therapeutic protein for treatment of damaged retinal pigment epithelium cells. Purpose: Here, we studied physicochemical parameters of an NT4-polyamidoamine (PAMAM) electrostatic complex, which can provide a sustained concentration of protein in intraocular space over an extended period after delivery. Adsorption/desorption of NT4 molecules to/from positively charged PAMAM dendrimers were precisely determined to control the concentration of bounded/unbounded protein molecules, diffusion coefficient, and size of a protein-laden dendrimer structure. We determined kinetics of NT4 desorption in PBS, vitreous, and damaged retina. Methods: Initially, adsorption of NT4 molecules on PAMAM dendrimers was studied in PBS using dynamic light scattering, electrophoresis, solution depletion, ELISA, and atomic force microscopy. This allowed us precisely to determine desorption of NT4 from nanoparticles under in situ conditions. The maximum coverage of irreversibly adsorbed NT4 determined by ELISA allowed us to devise a robust procedure for preparing stable and well-controlled coverage of NT4 on PAMAM nanoparticles. Thereafter, we studied diffusion of nanospheres containing NT4 molecules by injecting them into vitreous cavities of mice exposed to intravenous injections of sodium iodate and evaluated their intraocular desorption kinetics from drug carriers in vivo. Results: Our measurements revealed NT4-dendrimer nanoparticles can be used for continuous neurotrophic factor delivery, enhancing its distribution into mouse vitreous, as well as damaged retina over 28 days of postinjury observation. Conclusion: Understanding of polyvalent neurotrophin interactions with dendrimer nanoparticles might be useful to obtain well-ordered protein layers, targeting future development of drug-delivery systems, especially for neuroprotection of damaged retinal neurons.

Alteration of Selected Neurotrophic Factors and their Receptor Expression in Mouse Brain Response to Whole-Brain Irradiation.

Ionizing radiation can significantly affect brain function in children and young adults, particularly in the hippocampus where neurogenic niches are located. Injury to normal tissue is a major concern when whole-brain irradiation (WBI) is used to treat central nervous system (CNS) tumors, and the pathogenesis of this injury remains poorly understood. We assessed the expression of selected neurotrophins (NTs) and NT receptors (NTRs) in brains of young mice after a single 10 Gy gamma-ray exposure using morphological and molecular analyses [qRT-PCR, Western blot, immunohistochemistry (IHC)] to evaluate WBI-induced injury in its acute phase. Activity of the NT-NTR axes was examined by analysis of ERK and Akt phosphorylation. Using Nissl staining of hippocampus slices to visualize morphological changes, and TUNEL assay and active caspase-3 detection to assess apoptotic cell death, we found evidence of apoptosis and degenerative changes in hippocampal tissue after WBI. Shortly after WBI, we also observed significant overexpression of several NTs (BDNF, NT-3, NGF and GDNF) and NTRs (TrkA, TrkB, TrkC, GFRα-1, and p75NTR) compared to control animals. The upregulated NT and NTR proteins, in part, originated from two analyzed neurogenic areas: the subgranular zone of the hippocampal dentate gyrus and the subventricular zone, as confirmed by IHC. Finally, components of intracellular signaling pathways, including Akt and MAPK, were activated in acute phase after WBI. Given the role of NTs in diverse biological mechanisms, including maintenance and growth of neurons in the adult brain, our findings of altered expression of neurotrophins and their receptors in brain tissue shortly after irradiation suggest that these molecules play a vital role in the pathophysiology of the acute phase of WBI-induced injury.

Growth factors and their receptors derived from human amniotic cells in vitro.

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine.

Endogenous regeneration of damaged retinal pigment epithelium following low dose sodium iodate administration: an insight into the role of glial cells in retinal repair.

The retinal pigment epithelium (RPE) has been reported to demonstrate feasible self-regenerative potential under specific conditions. However, the precise underlying mechanisms involved in this process are still elusive. Here, we performed a sequential morphological, molecular, and functional analysis of retinal injury and subsequent tissue regeneration after intravenous administration of a low dose of sodium iodate (15 mg/kg) in mice over long-term observation, up to 3 months post-injury. To assess the kinetics of the injury/recovery process, the electroretinography (ERG) responses were correlated with ongoing alterations in retinal structure and the global gene expression profile of injured retinas using genome-wide RNA microarray technology, western blotting and immunohistochemical analyses. We observed considerable improvement in the rod cell-mediated ERG response, which was accompanied by the regeneration of RPE within the injury site by the 3rd month post-injury. Our results confirm that the repairing mechanisms within injured retinas involve a significant glial cell reaction marked by glial cell proliferation, migration from their original location toward the injury site, followed by a significant overproduction of NTs such as BDNF, GDNF and NT-3. The global gene expression analysis revealed that initially up-regulated genes associated with cell death, apoptosis, acute response to stress pathways underwent considerable down-regulation in the late post-injury period. Accordingly, the genes implicated in nervous tissue remodeling and neuron development, the regulation of synaptic transmission and the establishment of localization were substantially induced by the 3rd month. Collectively, our observations support the view that Müller glial cells might well play an active role not only in retinal cell reorganization following injury but potentially also in RPE regeneration, which appears to be the key event in retinal reparative process. Furthermore, we provided novel compelling evidence of the crucial role of neurotrophins in the pathophysiology of retinal repair and identified the signaling pathways that are activated during this process.

Circulating hematopoietic stem cell count is a valuable predictor of prematurity complications in preterm newborns.

BACKGROUND: The frequency of preterm labour has risen over the last few years. Hence, there is growing interest in the identification of markers that may facilitate prediction and prevention of premature birth complications. Here, we studied the association of the number of circulating stem cell populations with the incidence of complications typical of prematurity. METHODS: The study groups consisted of 90 preterm (23-36 weeks of gestational age) and 52 full-term (37-41 weeks) infants. Non-hematopoietic stem cells (non-HSCs; CD45-lin-CD184+), enriched in very small embryonic-like stem cells (VSELs), expressing pluripotent (Oct-4, Nanog), early neural (ß-III-tubulin), and oligodendrocyte lineage (Olig-1) genes as well as hematopoietic stem cells (HSCs; CD45+lin-CD184+), and circulating stem/progenitor cells (CSPCs; CD133+CD34+; CD133-CD34+) in association with characteristics of prematurity and preterm morbidity were analyzed in cord blood (CB) and peripheral blood (PB) until the sixth week after delivery. Phenotype analysis was performed using flow cytometry methods. Clonogenic assays suitable for detection of human hematopoietic progenitor cells were also applied. The quantitative parameters were compared between groups by the Mann-Whitney test and between time points by the Friedman test. Fisher's exact test was used for qualitative variables. RESULTS: We found that the number of CB non-HSCs/VSELs is inversely associated with the birth weight of preterm infants. More notably, a high number of CB HSCs is strongly associated with a lower risk of prematurity complications including intraventricular hemorrhage, respiratory distress syndrome, infections, and anemia. The number of HSCs remains stable for the first six weeks of postnatal life. Besides, the number of CSPCs in CB is significantly higher in preterm infants than in full-term neonates (p < 0.0001) and extensively decreases in preterm babies during next six weeks after birth. Finally, the growth of burst-forming unit of erythrocytes (BFU-E) and colony-forming units of granulocyte-macrophage (CFU-GM) obtained from CB of premature neonates is higher than those obtained from CB of full-term infants and strongly correlates with the number of CB-derived CSPCs. CONCLUSION: We conclude that CB HSCs are markedly associated with the development of premature birth complications. Thus, HSCs ought to be considered as the potential target for further research as they may be relevant for predicting and controlling the morbidity of premature infants. Moreover, the observed levels of non-HSCs/VSELs circulating in CB are inversely associated with the birth weight of preterm infants, suggesting non-HSCs/VSELs might be involved in the maturation of fetal organism.

Aldehyde dehydrogenase (ALDH) - a promising new candidate for use in preclinical and clinical selection of pluripotent very small embryonic-like stem cells (VSEL SCs) of high long-term repopulating hematopoietic potential.

BACKGROUND: In the field of stem cells (SCs) biology there is great need for a universal marker that can effectively identify the rare populations of SCs in order to characterize and isolate them for research and therapeutic purposes. In line with this idea, the measurement of aldehyde dehydrogenase (ALDH) activity shows promising potential as a common marker for the identification of SCs. Recently discovered very small embryonic-like stem cells (VSEL SCs) have a pluripotent nature and high long-term repopulating hematopoietic potential. The aim of the present study was to determine whether VSEL SCs isolated from umbilical cord blood (CB) and different murine organs express high ALDH activity. MATERIAL/METHODS: To address this issue, 12 CB units were analyzed by employing flow cytometry to detect the following populations: (i) CXCR4(+)/Lin(-)/CD45(-)/ALDH(high), (ii) CD34(+)/Lin(-)/CD45(-)/ALDH(high) and (iii) CD133(+)/Lin(-)/CD45(-)/ALDH(high). Additionally, selected organs from 16 5-week-old female inbred C57BL/6J mice and 16 7-month-old female C57BL/6J mice were analyzed for detection of the following populations: (i) Sca-1(+)/Lin(-)/CD45(-) and (ii) Sca-1(+)/Lin(-)/CD45(+). All these populations were assessed for ALDH activity. RESULTS: We found that CB contains VSEL SCs with high ALDH activity. We also observed that ALDH(high) cells constitute a modest percentage of VSEL SCs present in selected murine organs. CONCLUSIONS: We demonstrated that CB and adult murine organs possess a subpopulation of ALDH(high) VSEL SCs. Above all, the observed high level of ALDH activity can be considered a functional marker of organ-derived pluripotent SCs and allows for simple, efficient isolation of cells with primitive features for their utility in targeted cell therapies.

Neural stem/progenitor cells circulating in peripheral blood of patients with neovascular form of AMD: a novel view on pathophysiology.

BACKGROUND: The neovascular form of age-related macular degeneration (AMD) manifested with choroidal neovascularization (CNV) is one of the leading causes of rapid and irreversible visual loss. Recent reports suggest that bone marrow-derived stem/progenitor cells (SPCs) play a crucial role in the development and progression of the disease. The purpose of this study was to investigate whether or not undifferentiated non-haematopoietic stem cells, including those capable of differentiating into neural phenotypes, play a role in the pathological state of CNV formation. METHODS: Peripheral blood samples were collected from 46 patients diagnosed with CNV and from 46 controls. The CXCR4(+)Lin(-)CD45(-) stem cells were counted and analysed by flow cytometry. Using qRT-PCR and immunocytofluorescence, the expression of early neural and glial cell markers (ß-III-tubulin, nestin, and glial fibrillary acidic protein) in the sorted cells was analysed, and correlated with plasma concentrations of stromal cell-derived factor 1 (SDF-1) (enzyme-linked immunosorbent assay), which is a pivotal chemokine that regulates the trafficking of SPCs. RESULTS: We found that the number of circulating CXCR4(+)Lin(-)CD45(-) cells did not differ in patients with active CNV as compared to the controls. However, we noticed significant intracellular overexpression of ß-III-tubulin in the cells derived from AMD patients. Moreover, we observed significantly lower SDF-1 plasma levels in neovascular AMD patients compared to healthy individuals. CONCLUSIONS: Our findings suggest that neural progenitor cells, together with low SDF-1 concentrations, may play a considerable role in the process of AMD progression. Further investigations aimed at the precise elucidation of these issues may help with the future development of effective prevention against, and the treatment of, this disease.

Stem Cells are mobilized from the bone marrow into the peripheral circulation in response to retinal pigment epithelium damage--a pathophysiological attempt to induce endogenous regeneration.

PURPOSE: Stem cell regeneration of damaged tissue has recently been reported in many different organs. Here, we investigated the mobilization of different stem/progenitor cell (SPC) populations into the peripheral blood (PB), their subsequent homing to the injured retina (IR) and contribution to its regeneration in a retinal pigment epithelium (RPE) damage model induced by sodium iodate (NaIO(3)). METHODS: Mobilization of SPCs was evaluated by flow cytometry. SPCs distribution in IR was assessed using bone marrow (BM)-derived GFP(+)Lin(-) cells transplanted intravenously into NaIO(3)-treated C57Bl/6 mice. The quantity of the chemokine SDF-1 in PB and IR was measured by ELISA and qRT-PCR, respectively. Apoptosis (TUNEL assay), cell proliferation (PCNA analysis) as well as functional retinal activity (electroretinogram) were examined at several time points after NaIO(3) administration. RESULTS: Mobilization of SPCs along with the highest cell proliferation and massive apoptosis within IR were observed on the third day after NaIO(3) administration. Similarly, donor GFP(+)Lin(-) cells were detected in the retina as soon as day 4 after NaIO(3) injection. Plasma levels of SDF-1 did not differ significantly in mice exposed to NaIO(3) compared to healthy controls, however mRNA for SDF-1 was overexpressed locally in IR. Functional retinal recovery was not achieved. CONCLUSION: Our study provides evidence that BM SPCs egress into PB and home to the injured retina, but are not capable of restoring its function. These results indicate that if the range of retinal destruction is profound, endogenous regeneration is ineffective and may ultimately require adjuvant therapeutic transplantation of specific SPCs subpopulations.

Different populations of circulating endothelial cells in patients with age-related macular degeneration: a novel insight into pathogenesis.

PURPOSE: Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) may serve as novel markers of endothelial dysfunction. The presence and clinical implications of CECs and the expression of endothelin (ET)-1, one of the most potent vasoconstrictors, have not been evaluated in patients with the neovascular form of age-related macular degeneration (AMD). This study was conducted to determine the different populations of endothelial cells (ECs) in the peripheral blood of AMD patients and to correlate these findings with the expression of ET-1 and the cytokines and growth factors responsible for EC migration and function. METHODS: Peripheral blood samples were collected from 29 patients with diagnosed neovascular AMD and from 38 healthy control subjects. CD133(-)CD144(+) CECs and CD34(+)CD133(+)CD144(+) EPCs were counted and analyzed by flow cytometry. The intracellular expression of ET-1 in peripheral blood nuclear cells (PBNCs) was studied by using qRT-PCR, Western blot, and immunocytofluorescence assays, and ET-1, IGF-1, VEGF, SDF-1, and HGF plasma concentrations were measured in enzyme-linked immunosorbent assays. RESULTS: Increased CECs and EPCs were found in the AMD patients compared with the counts in healthy individuals. The expression of intracellular ET-1 was significantly elevated in PBNCs from the AMD patients compared with the control subjects. In addition a significantly higher plasma concentration of IGF-1 was observed, but a lower SDF-1 level in the group of AMD patients. CONCLUSIONS: These findings suggest that circulating endothelial cells, together with high ET-1 content, may contribute to the development of AMD. Further prospective investigations on the mechanism involved may be relevant to the potential treatment of this disease.

Sodium iodate selectively injuries the posterior pole of the retina in a dose-dependent manner: morphological and electrophysiological study.

Sequential morphological and functional features of retinal damage in mice exposed to different doses (40 vs. 20 mg/kg) of sodium iodate (NaIO(3)) were analyzed. Retinal morphology, apoptosis (TUNEL assay), and function (electroretinography; ERG) were examined at several time points after NaIO(3) administration. The higher dose of NaIO(3) caused progressive degeneration of the whole retinal area and total suppression of scotopic and photopic ERG. In contrast, the lower dose induced much less severe degeneration in peripheral part of retina along with a moderate decline of b- and a-wave amplitudes in ERG, corroborating the presence of regions within retina that retain their function. The peak of photoreceptor apoptosis was found on the 3rd day, but the lower dose induced more intense reaction within the central retina than in its peripheral region. In conclusion, these results indicate that peripheral area of the retina reveals better resistance to NaIO(3) injury than its central part.

Identification of a specific IgE-binding protein from narrow-leafed lupin (L. Angustifolius) seeds.

UNLABELLED: Since lupin has been introduced as a food ingredient on the market there are more and more reports concerning its allergenic properties. However, only few narrow-leafed lupin proteins have yet been characterized as specific IgE-binding molecules and identified. The aim of the study has been to find and identify the main narrow-leafed lupin globulins that bind to specific IgEs from the sera of lupin-allergic people. Isolated lupin globulins were subjected to immunoblotting with the sera from people who suffered from lupin allergy. Incubation with alpha-methyl-D-galactopyranoside was performed to eliminate possible binding of unspecific human IgEs. The proteins binding specific IgEs from lupin-allergic patients' sera were identified by means of mass spectrometry. Western blot analysis revealed 2 signals corresponding to lupin globulins that bound to specific IgEs from the sera of people allergic to lupin. The globulins were identified as conglutin-gamma and its smaller subunit. The results suggested that individuals that displayed lupin allergy symptoms reacted to conglutin-gamma. PRACTICAL APPLICATION: The results of the study can contribute to identification of yet undetected allergens of narrow-leafed lupin. This, in turn, can make lupin-fortified products safer for the consumers.

Clinical relevance of thyroid dysfunction in human haematopoiesis: biochemical and molecular studies.

OBJECTIVE: Abnormalities in haematological parameters have been noted in patients with thyroid diseases. Nevertheless, the exact mechanism of thyroid hormones' (THs) action on human haematopoiesis is still not entirely clear. DESIGN: The influence of THs through TH receptors (TRalpha-1 and TRbeta-1) on haematopoiesis in patients with hypo- and hyperthyroidism was analysed. METHODS: TR gene expression at the mRNA and protein levels in human CD34(+)-enriched haematopoietic progenitor cells (HPCs) obtained from the peripheral blood of patients with thyroid disorders and healthy volunteers was analysed. The cell populations were also investigated for clonogenic growth of granulocyte macrophage-colony forming units and erythrocyte-burst forming units (BFU-E). The level of apoptosis was determined by annexin V/propidium iodide staining and quantitative RT-PCR. RESULTS: The studies revealed that hypo- and hyperthyroidism modify TR gene expression in HPCs in vivo. TH deficiency resulted in a decrease in total blood counts and clonogenic potential of BFU-E. In contrast, hyperthyroid patients presented increased clonogenic growth and BFU-E number and significantly higher expressions of cell cycle-regulating genes such as those for PCNA and cyclin D1. Finally, an increase in the frequency of apoptotic CD34(+)-enriched HPCs in hypo- and hyperthyroidism with a modulation of apoptosis-related genes was detected. CONCLUSIONS: The following conclusions were derived: i) TR expression in human haematopoietic cells depends on TH status, ii) both hypo- and hyperthyroidism significantly influence clonogenicity and induce apoptosis in CD34(+)-enriched HPCs and iii) the molecular mechanism by which THs influence haematopoiesis might provide a basis for designing novel therapeutic interventions in thyroid diseases.