OBJECTIVE: Progelatinase B/proMMP-9 has recently been identified as an indicator of pleural inflammation, presumably originating from granulocytes. The aim of this study was to verify the origin of progelatinase B by simultaneous estimation of specific markers of neutrophil recruitment and activation in pleural effusions following induced pleurisy and pleural injury. MATERIAL AND METHODS: Sixty-three samples of pleural fluid from patients undergoing therapeutic talc pleurodesis (n = 8) and explorative thoracoscopy (n = 3) collected before and at different time intervals after the intervention were analyzed for progelatinase B and neutrophil gelatinase-associated lipocalin (NGAL)-gelatinase complex by substrate electrophoresis, for myeloperoxidase (MPO) and interleukin-8 (IL-8) by immunoadsorbent sandwich assay, as well as for leukocyte count, C-reactive protein (CRP) and total protein (TP). RESULTS: A significant increase in free and NGAL-complexed progelatinase B, MPO and IL-8 was recorded within 48 h following treatment in all subjects. Progelatinase B was strongly correlated with NGAL-gelatinase complex (r = 0.88, p = 0.001), MPO (r = 0.81, p = 0.001), neutrophil count (r = 0.75, p = 0.01) and IL-8 (r = 0.71, p = 0.001), but not with CRP and TP. CONCLUSIONS: The results support the neutrophil origin of the proenzyme, which confirms progelatinase B as an indicator of a local inflammatory reaction. Quantifying the inflammatory reaction may be helpful in the evaluation of both the technical variants of therapeutic pleurodesis and finer discrimination of paraneoplastic effusions.

• MeSH
• biologické markery metabolismus   MeSH
• degranulace buněk fyziologie   MeSH
• financování organizované MeSH
• interleukin-8 metabolismus   MeSH
• kolagenasy biosyntéza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipokaliny MeSH
• matrixová metaloproteinasa 9 MeSH
• metaloendopeptidasy biosyntéza   MeSH
• neutrofily enzymologie   fyziologie   patologie   MeSH
• peroxidasa metabolismus   MeSH
• pleura enzymologie   patologie   zranění   MeSH
• pleurální výpotek enzymologie   patologie   MeSH
• pleuritida enzymologie   patologie   MeSH
• prekurzory enzymů biosyntéza   MeSH
• proteiny akutní fáze metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• senioři MeSH
• želatinasy biosyntéza   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH

Chronická kopř ivka (urtikarie CU) je definována jako přítomnost kopř ivky vyskytující se déle než šest týdnů . Etiologie onemocnění je různorodá, přičemž společ ným znakem většiny urtikarií je aktivace a degranulace žírných buněk a přičemž vyvolávací faktory mohou být jak neimunologického, tak imunologického charakteru. V patogenezi autoimunitní podskupiny je patogenní mechanismus spojován s přítomností protilátek IgG izotypu proti alfa podjednotce vysokoafinitního FcεRI receptoru nebo IgE. Diagnostika chronické kopřivky je obtížná a často založená pouze na klinických a anamnestických údajích. Do dnešního dne byla vyvinuta řada metod k detekci sérových autoprotilátek, je- jich nedostatkem je však značná různorodost v principu stanovení, a tudíž i vysoká metodická a mezilaboratorní variabilita. K nejužívanějším patří intradermální test za užití autologního séra (ASSST), který však nepatří mezi standardizovaná vyšetření. Laboratorní in vitro průkaz anti-FcεRI autoprotilátek je možno provádět funkč ními testy, jako je metoda uvolňování histaminu z basofilů periferní krve nebo imunoana- lytickými metodami jako western blot nebo enzymová imunoanalýza (ELISA). V této práci byla užita modifikace testu aktivace basofilů , kdy basofily zdravého byly stimulovány sérem pacientů s CU. Schopnost aktivoval basofily nealergického dárce vykazovala séra 5 z 11 vyšetřovaných pacientek a lze předpokládat, že aktivace basofilů byla mediována protilátkami proti FcεRI eventuálně anti-IgE protilátkami. Na rozdíl od metod založených na principu imunoanalýzy spočívá pozitivní přínos tohoto testu především v průkazu funkční aktivity séra vázané k FcεRI a odpovědné za aktivaci, resp. degranulaci basofilů a mohl by sloužit jako vhodná metoda pro identifikaci podskupiny pacientů s chronickou kopřivkou autoimunitního původu.

Chronic urticaria (CU) is defined as the presence of urticaria for at least 6 weeks. It is frequently caused by allergic reacti ons; however, there are many nonallergic causes. The majority of chronic urticaria cases have an unknown cause. The autoimmune subgroup is associat ed with the IgG anti-IgE receptor alpha subunit in 35–40% of patients and IgG anti-IgE in an additional 5–10%. These autoantibodies have be en shown to activate blood basophils and cutaneous mast cells in vitro with augmentation of basophil activation by complement and release o f C5a. This autoimmune subgroup can be identified by an autologous skin test or histamine release from human basophils or cutaneous mast ce lls or binding methods as immunoblot and ELISA. However, binding assays do not correlate with these functional assai. Activation of basophils or mast cells causing histamine release is quite specific for chronic urticaria and defines the autoimmune subgroup. To detect autoantibodies to the Fc ε RI in sera of CU patients by a modified serum-induced basophil activation test measured by flow cytometry. Sera of 11 CU patients wer e tested and serum of 5 of these sera upregulated CD63 expression on the surface of basophils. Chronic urticaria serum-induced CD63 expressi on assay seems to be a useful tool for identification of a subset of patiens with autoimmune CU.

• Klíčová slova
• chronická kopřivka, CD63,
• MeSH
• autoprotilátky krev   škodlivé účinky   MeSH
• bazofily imunologie   MeSH
• kožní testy MeSH
• lidé MeSH
• receptory IgE antagonisté a inhibitory   fyziologie   MeSH
• test degranulace bazofilů MeSH
• urtikarie * diagnóza   imunologie   krev   patologie   MeSH
• Check Tag
• lidé MeSH

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides possess strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5-40 microM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt alpha-helical conformation in such anisotropic environments.

• MeSH
• antibakteriální látky farmakologie   chemická syntéza   chemie   MeSH
• Bacillus subtilis účinky léků   MeSH
• degranulace buněk MeSH
• erytrocyty účinky léků   MeSH
• Escherichia coli účinky léků   MeSH
• financování organizované MeSH
• hemolýza fyziologie   účinky léků   MeSH
• hydrofobní a hydrofilní interakce MeSH
• inhibiční koncentrace 50 MeSH
• kationické antimikrobiální peptidy farmakologie   chemická syntéza   chemie   MeSH
• krysa rodu rattus MeSH
• mastocyty fyziologie   účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární sekvence - údaje MeSH
• peptidy farmakologie   chemická syntéza   chemie   izolace a purifikace   účinky léků   MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• sekvence aminokyselin MeSH
• sršňovití chemie   MeSH
• Staphylococcus aureus účinky léků   MeSH
• substituce aminokyselin MeSH
• vosí jedy farmakologie   genetika   chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

• MeSH
• aktiny metabolismus   MeSH
• buněčná adheze MeSH
• chemotaxe MeSH
• cytokiny genetika   metabolismus   MeSH
• fosforylace MeSH
• galektin 3 genetika   metabolismus   MeSH
• lyzozomy metabolismus   MeSH
• malá interferující RNA MeSH
• mastocyty cytologie   fyziologie   MeSH
• myši inbrední BALB C MeSH
• prostaglandin D2 metabolismus   MeSH
• receptory IgE genetika   metabolismus   MeSH
• signální transdukce MeSH
• ubikvitinace MeSH
• vápník metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.

• MeSH
• antiinfekční látky farmakologie   chemická syntéza   chemie   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   MeSH
• hemolýza účinky léků   MeSH
• kationické antimikrobiální peptidy farmakologie   chemie   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• mastocyty metabolismus   účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• peptidy farmakologie   chemická syntéza   chemie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky farmakologie   chemická syntéza   chemie   MeSH
• screeningové testy protinádorových léčiv MeSH
• včelí jedy chemie   MeSH
• včely chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH(2) by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content alpha-helices, which indicates that melectin can adopt an amphipathic alpha-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the alpha-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.

• MeSH
• antibakteriální látky farmakologie   chemická syntéza   izolace a purifikace   MeSH
• cirkulární dichroismus MeSH
• degranulace buněk účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   MeSH
• hemolýza účinky léků   MeSH
• hydrofobní a hydrofilní interakce MeSH
• inhibiční koncentrace 50 MeSH
• kationické antimikrobiální peptidy farmakologie   chemická syntéza   izolace a purifikace   MeSH
• krysa rodu rattus MeSH
• mastocyty fyziologie   účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární sekvence - údaje MeSH
• potkani Wistar MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• včelí jedy chemie   MeSH
• včely MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Mast cells play an effector role in innate immunity, allergy, and inflammation. Antigen-mediated activation of mast cells initiates signaling events leading to Ca2+ response and the release of inflammatory and allergic mediators from granules. Diseases associated with deregulated mast cell functions are hard to treat and there is an increasing demand for new therapeutic strategies. Miltefosine (hexadecylphosphocholine) is a new candidate for treatment of mast cell-driven diseases as it inhibits activation of mast cells. It has been proposed that miltefosine acts as a lipid raft modulator through its interference with the structural organization of surface receptors in the cell membrane. However, molecular mechanisms of its action are not fully understood. Here, we report that in antigen-activated bone marrow-derived mast cells (BMMCs), miltefosine inhibits degranulation, reorganization of microtubules, as well as antigen-induced chemotaxis. While aggregation and tyrosine phosphorylation of IgE receptors were suppressed in activated cells pre-treated with miltefosine, overall tyrosine phosphorylation levels of Lyn and Syk kinases, and Ca2+ influx were not inhibited. In contrast, lipid raft disruptor methyl-β-cyclodextrin attenuated the Ca2+ influx. Tagged-miltefosine rapidly localized into the cell interior, and live-cell imaging of BMMCs with labeled intracellular granules disclosed that miltefosine inhibited movement of some granules. Immunoprecipitation and in vitro kinase assays revealed that miltefosine inhibited Ca2+- and diacylglycerol-regulated conventional protein kinase C (cPKC) isoforms that are important for mast cell degranulation. Inhibition of cPKCs by specific inhibitor Ly333531 affected activation of BMMCs in the same way as miltefosine. Collectively, our data suggest that miltefosine modulates mast cells both at the plasma membrane and in the cytosol by inhibition of cPKCs. This alters intracellular signaling pathway(s) directed to microtubules, degranulation, and migration.

• Publikační typ
• časopisecké články MeSH