Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number ... more Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number of cellular systems, but the mechanisms of these activities have still not been clarified unequivocally. Since both drugs are cationic, accumulate in acidic cellular compartments and bind to phospholipids with a consequent increase in lysosomal pH and induce phospholipidosis, we examined the relevance of these common properties to their anti-inflammatory activities. We compared also these effects with effects of concanamycin A, compound which inhibits acidification of lysosomes. All three compounds increased lysosomal pH, accumulation of autophagic vacuoles and ubiquitinated proteins and impaired recycling of TLR4 receptor with consequences in downstream signaling in LPS-stimulated J774A.1 cells. Azithromycin and chloroquine additionally inhibited arachidonic acid release and prostaglandin E2 synthesis. Therefore, impairment of lysosomal functions by azithromycin and chloroquine deregulate TLR4 recycling and signaling and phospholipases activation and lead to anti-inflammatory phenotype in LPS-stimulated J774A.1 cells.
We read with interest the letter by Salvatore Chirumbolo (ref in press) and his precious comments... more We read with interest the letter by Salvatore Chirumbolo (ref in press) and his precious comments on our paper published recently in your journal (1). There are a few points that we would like to discuss. We would like to start with a correction of some statements made in the manuscript of Chirumbolo. Firstly, the so called previous report as mentioned by Chirumbolo (ref in press) is the same publication as in reference 1 but in different phases of publication. Secondly, references 3 and 11 in the paper of Chirumbolo appear to be the same. Thirdly, in the remarks about different mast cell responses between men and women, the referral to reference 11 is incorrect. In this reference (2), no gender differences were discussed at all. Fourthly, the referral to reference 16 to underline the remark about reduction of tryptase levels which may be found (also) in controls throughout a VIT protocol, is also wrong. In this study (from our own group) no VIT protocol was used at all. Furthermore, changes in tryptase levels have not been analyzed in reference 16. Fifthly, the measurement of IgG4 antibodies to wasp venom was only measured to evaluate effects of immunotherapy. Therefore, IgG4 was not measured in patients without wasp venom allergy (and who did not receive immunotherapy). In our opinion, measurement of allergen specific IgG4 antibodies is not recommended for diagnosis of allergy. Other points which are addressed by Chirumbolo might be explained by two main differences in the setup of both studies: Chirumbolo investigated the behavior of isolated basophils (buffy coat) from normal volunteers whereas we explored whole blood basophils in patients with mastocytosis. The main purpose of our study was to compare the BAT in mastocytosis patients with and without wasp venom allergy. Further studies are needed to confirm our data and to elucidate possible pathophysiological mechanisms. Irrespective of the faulty references and inaccurate interpretation for literature data, the main criticism from Chirumbolo being BAT to screen IgE-mediated responses is not relevant because BAT was performed on whole blood in the context of all cells and cytokines with wasp venom containing all constituents. The outcome was measured irrespective of the mechanism. Further studies are needed to elucidate the probable pathways and mechanisms.
Claudins are transmembrane proteins constituting one of three tight junction protein families. In... more Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and fl...
Objectives The patients with hematological malignancies are a vulnerable group to COVID-19, due t... more Objectives The patients with hematological malignancies are a vulnerable group to COVID-19, due to the immunodeficiency resulting from the underlying disease and oncological treatment that significantly impair cellular and humoral immunity. Here we report on a beneficial impact of a passive immunotherapy with convalescent plasma to treat a prolonged, active COVID-19 infection in a patient with a history of nasopharyngeal diffuse large B-cell lymphoma treated with the therapy inducing substantial impairment of particularly humoral arm of immune system. The specific aim was to quantify SARS-CoV2 neutralizing antibodies in a patient plasma during the course of therapy. Materials and methods Besides the standard of care treatment and monitoring, neutralizing antibody titers in patient's serum samples, calibrated according to the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human), were quantified in a time-dependent manner. During the immunotherapy period peripheral blood flow cytometry immunophenotyping was conducted to characterize lymphocyte subpopulations. Results The waves of clinical improvements and worsening coincided with transfused neutralizing antibodies rises and drops in the patient's systemic circulation, proving their contribution in controlling the disease progress. Besides the patient's lack of own humoral immune system, immunophenotyping analysis revealed also the reduced level of helper T-lymphocytes and immune exhaustion of monocytes. Conclusion Therapeutic approach based on convalescent plasma transfusion transformed a prolonged, active COVID-19 infection into a manageable chronic disease.
Abstract: High-throughput prediction of potential drug toxicity is necessary in early stage of dr... more Abstract: High-throughput prediction of potential drug toxicity is necessary in early stage of drug discovery while testing potential hits. This is also a part of the lead optimization process and one of major focus in discovery phase of drug development. In this context, setting-up ...
Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number ... more Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number of cellular systems, but the mechanisms of these activities have still not been clarified unequivocally. Since both drugs are cationic, accumulate in acidic cellular compartments and bind to phospholipids with a consequent increase in lysosomal pH and induce phospholipidosis, we examined the relevance of these common properties to their anti-inflammatory activities. We compared also these effects with effects of concanamycin A, compound which inhibits acidification of lysosomes. All three compounds increased lysosomal pH, accumulation of autophagic vacuoles and ubiquitinated proteins and impaired recycling of TLR4 receptor with consequences in downstream signaling in LPS-stimulated J774A.1 cells. Azithromycin and chloroquine additionally inhibited arachidonic acid release and prostaglandin E2 synthesis. Therefore, impairment of lysosomal functions by azithromycin and chloroquine deregulate TLR4 recycling and signaling and phospholipases activation and lead to anti-inflammatory phenotype in LPS-stimulated J774A.1 cells.
We read with interest the letter by Salvatore Chirumbolo (ref in press) and his precious comments... more We read with interest the letter by Salvatore Chirumbolo (ref in press) and his precious comments on our paper published recently in your journal (1). There are a few points that we would like to discuss. We would like to start with a correction of some statements made in the manuscript of Chirumbolo. Firstly, the so called previous report as mentioned by Chirumbolo (ref in press) is the same publication as in reference 1 but in different phases of publication. Secondly, references 3 and 11 in the paper of Chirumbolo appear to be the same. Thirdly, in the remarks about different mast cell responses between men and women, the referral to reference 11 is incorrect. In this reference (2), no gender differences were discussed at all. Fourthly, the referral to reference 16 to underline the remark about reduction of tryptase levels which may be found (also) in controls throughout a VIT protocol, is also wrong. In this study (from our own group) no VIT protocol was used at all. Furthermore, changes in tryptase levels have not been analyzed in reference 16. Fifthly, the measurement of IgG4 antibodies to wasp venom was only measured to evaluate effects of immunotherapy. Therefore, IgG4 was not measured in patients without wasp venom allergy (and who did not receive immunotherapy). In our opinion, measurement of allergen specific IgG4 antibodies is not recommended for diagnosis of allergy. Other points which are addressed by Chirumbolo might be explained by two main differences in the setup of both studies: Chirumbolo investigated the behavior of isolated basophils (buffy coat) from normal volunteers whereas we explored whole blood basophils in patients with mastocytosis. The main purpose of our study was to compare the BAT in mastocytosis patients with and without wasp venom allergy. Further studies are needed to confirm our data and to elucidate possible pathophysiological mechanisms. Irrespective of the faulty references and inaccurate interpretation for literature data, the main criticism from Chirumbolo being BAT to screen IgE-mediated responses is not relevant because BAT was performed on whole blood in the context of all cells and cytokines with wasp venom containing all constituents. The outcome was measured irrespective of the mechanism. Further studies are needed to elucidate the probable pathways and mechanisms.
Claudins are transmembrane proteins constituting one of three tight junction protein families. In... more Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and fl...
Objectives The patients with hematological malignancies are a vulnerable group to COVID-19, due t... more Objectives The patients with hematological malignancies are a vulnerable group to COVID-19, due to the immunodeficiency resulting from the underlying disease and oncological treatment that significantly impair cellular and humoral immunity. Here we report on a beneficial impact of a passive immunotherapy with convalescent plasma to treat a prolonged, active COVID-19 infection in a patient with a history of nasopharyngeal diffuse large B-cell lymphoma treated with the therapy inducing substantial impairment of particularly humoral arm of immune system. The specific aim was to quantify SARS-CoV2 neutralizing antibodies in a patient plasma during the course of therapy. Materials and methods Besides the standard of care treatment and monitoring, neutralizing antibody titers in patient's serum samples, calibrated according to the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human), were quantified in a time-dependent manner. During the immunotherapy period peripheral blood flow cytometry immunophenotyping was conducted to characterize lymphocyte subpopulations. Results The waves of clinical improvements and worsening coincided with transfused neutralizing antibodies rises and drops in the patient's systemic circulation, proving their contribution in controlling the disease progress. Besides the patient's lack of own humoral immune system, immunophenotyping analysis revealed also the reduced level of helper T-lymphocytes and immune exhaustion of monocytes. Conclusion Therapeutic approach based on convalescent plasma transfusion transformed a prolonged, active COVID-19 infection into a manageable chronic disease.
Abstract: High-throughput prediction of potential drug toxicity is necessary in early stage of dr... more Abstract: High-throughput prediction of potential drug toxicity is necessary in early stage of drug discovery while testing potential hits. This is also a part of the lead optimization process and one of major focus in discovery phase of drug development. In this context, setting-up ...
We are pleased to offer you an intensive, five-day, hands-on flow cytometry course. Lectures and ... more We are pleased to offer you an intensive, five-day, hands-on flow cytometry course. Lectures and lab work will be teached by two main tutors, two flow cytometry experts from University of Zürich and Children's Hospital Srebrnjak. In addition, guest specialists organised by sponsors will give 45 minutes talks, twice per day during commercial scientific tutorials, on their specialised techniques and various uses of flow cytometry.
No experience in flow cytometry is required to attend the course. Any scientist (technicians, graduate students, postgraduate students, postdocs, researchers) with biology / biochemistry / biotechnology / veterinary medicine / medicine related field are welcome to register, from the beginners level up to intermediate level of flow cytometry knowledge. The goal of the course is to allow participants to gain experience of what a flow cytometer is capable, how to plan, execute and perform polychromatic flow cytometry experiments, how to succesfuly analyse data obtained and create and present final results.
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No experience in flow cytometry is required to attend the course. Any scientist (technicians, graduate students, postgraduate students, postdocs, researchers) with biology / biochemistry / biotechnology / veterinary medicine / medicine related field are welcome to register, from the beginners level up to intermediate level of flow cytometry knowledge. The goal of the course is to allow participants to gain experience of what a flow cytometer is capable, how to plan, execute and perform polychromatic flow cytometry experiments, how to succesfuly analyse data obtained and create and present final results.