Balázs Mayer

Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 –dependent reprogramming of host macrophages to increase their interleukin-10 production

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-α) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups.

Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy‐chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non‐specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.

Reduced inflammatory threshold indicates skin barrier defect in transglutaminase 3 knockout mice

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.

Rapid systemic and local treatments with the antibacterial peptide dimer A3-APO and its monomeric metabolite eliminate bacteria and reduce inflammation in intradermal lesions infected with Propionibacterium acnes and meticillin-resistant Staphylococcus aureus

When administered intramuscularly, the designer antibacterial peptide dimer A3-APO is highly efficacious in mouse models of Acinetobacter baumannii and Staphylococcus aureus burn infections. Here we compared the efficacy of A3-APO and its monomeric metabolite in mouse models of S. aureus and Propionibacterium acnes intradermal infections following administration as intramuscular (i.m.) or topical treatments. In the animal models, either (i) the ears of CD-1 mice were infected with P. acnes or (ii) S. aureus was injected into burn wounds inflicted to the back. A3-APO or the monomer were injected intramuscularly at 5 mg/kg one to three times or were applied three times as 1% local treatment in phosphate-buffered saline or Vaseline(®). Despite being inactive against the strains in vitro, in vivo the skin conditions of the mice were dramatically improved upon peptide treatment regardless of dosing frequency, administration mode or drug valency. In the P. acnes study, A3-APO statistically significantly reduced ear thickness and ear bacterial counts. The amount of ear connective tissue and epithelial macrophages correlated with therapeutic success. Bacterial load in the lesions was more representative of physical improvement than ear dimensions. In the S. aureus model, both peptides eliminated wound bacteria from >10(7) CFU/mg to almost background levels, with monomer treatment being somewhat more successful. In conclusion, A3-APO and its monomeric metabolite very efficiently ameliorate resistant aerobic and anaerobic intradermal infections, but the protection is apparently not due to direct bacterial killing. Immunostimulatory and anti-inflammatory actions are likely involved. Nevertheless, topical and i.m. administrations are equally effective.

Characterization and function of histamine receptors in human bone marrow stromal cells

There are several clinical trials worldwide using bone marrow stromal cells (BMSCs) as a cellular therapy to modulate immune responses in patients suffering from various inflammatory conditions. A deeper understanding of the molecular mechanisms involved in this modulatory effect could help us design better, more effective protocols to treat immune mediated diseases. In this study, we demonstrated that human BMSCs express H1, H2, and H4 histamine receptors and they respond to histamine stimulation with an increased interleukin 6 (IL‐6) production both in vitro and in vivo. Using different receptor antagonists, we pinpointed the importance of the H1 histamine receptor, while Western blot analysis and application of various mitogen‐activated protein kinase inhibitors highlighted the role of p38, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase kinases in the observed effect. When BMSCs were pretreated with either histamine or degranulated human mast cells, they exhibited an enhanced IL‐6‐dependent antiapoptotic effect on neutrophil granulocytes. Based on these observations, it is likely that introduction of BMSCs into a histamine‐rich environment (such as any allergic setting) or pretreatment of these cells with synthetic histamine could have a significant modulatory effect on the therapeutic potential of BMSCs. STEM CELLS 2012; 30:222–231.

NFκB induces overexpression of bovine FcRn: a novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of FcRn.

Among the many functions of the neonatal Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen processing occurs. We previously reported that transgenic (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response. NFκB induces human FcRn expression and our previous in silico analysis suggested NFκB binding sites in the promoter region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we report the identification of three NFκB transcription binding sites in the promoter region of this gene using luciferase reporter gene technology, electromobility shift assay and supershift analysis. Stimulation of primary bovine endothelial cells with the Toll-like receptor-4 ligand lipopolysaccharide (LPS), which mediates its effect via NFκB, resulted in rapid upregulation of the bFcRn expression and a control gene, bovine E-selectin. This rapid bFcRn gene induction was also observed in the spleen of bFcRn Tg mice treated with intraperitoneally injected LPS, analyzed by northern blot analysis. Finally, NFκB-mediated bFcRn upregulation was confirmed at the protein level in macrophages isolated from the bFcRn Tg mice using flow cytometry with a newly developed FcRn specific monoclonal antibody that does not cross-react with the mouse FcRn. We conclude that NFκB regulates bFcRn expression and thus optimizes its functions, e.g., in the professional antigen presenting cells, and contributes to the much augmented humoral immune response in the bFcRn Tg mice.

Transglutaminases in autoimmune and inherited skin diseases: The phenomena of epitope spreading and functional compensation

Transglutaminases (TGs) are structurally and functionally related enzymes that modify the post‐translational structure and activity of proteins or peptides, and thus are able to turn on or switch off their function. Depending on location and activities, TGs are able to modify the signalling, the function and the fate of cells and extracellular connective tissues. Besides mouse models, human diseases enable us to appreciate the function of various TGs. In this study, skin diseases induced by genetic damages or autoimmune targeting of these enzymes will be discussed. TG1, TG3 and TG5 contribute to the cutaneous barrier and thus to the integrity and function of epidermis. TGM1 mutations related to autosomal recessive ichthyosis subtypes, TGM5 mutations to a mild epidermolysis bullosa phenotype and as novelty TGM3 mutation to uncombable hair syndrome will be discussed. Autoimmunity to TG2, TG3 and TG6 may develop in a few of those genetically determined individuals who lost tolerance to gluten, and manifest as coeliac disease, dermatitis herpetiformis or gluten‐dependent neurological symptoms, respectively. These gluten responder diseases commonly occur in combination. In autoimmune diseases, the epitope spreading is remarkable, while in some inherited pathologies, a unique compensation of the lost enzyme function is noted.

Decreased fibrinolytic potential and morphological changes of fibrin structure in dermatitis herpetiformis

BACKGROUND Recently, high prevalence of cryofibrinogenaemia has been observed in plasma of untreated dermatitis herpetiformis (DH) patients, and the pathological IgA and TG3 deposits in the papillary dermis were found to co-localize with fibrin and fibrinogen. OBJECTIVE To study the fibrinolytic potential in plasma of untreated, dapsone and or/gluten-free diet treated DH patients as well as the in vitro effect of dapsone on the fibrinolytic profile. METHOD Plasma samples of 23 DH patients, 19 healthy subjects and 5 pemphigus vulgaris patients were investigated by a turbidimetric-clot lysis assay. Out of them 5 DH plasma samples representing different fibrinolytic parameters, and 3 healthy controls were selected for parallel fibrin clot preparation. The clot fibrin structure was examined by scanning electron microscopy (SEM), and the diameters of 900 fibrin fibres were determined in each clot. RESULTS A significantly prolonged clot lysis time was detected in untreated DH patients. The turbidity values of DH plasma clots indicated an altered fibrin structure that was also confirmed by SEM: significantly thicker fibrin fibers were observed in untreated, TG3 antibody positive DH patients compared to healthy controls, whereas the fiber diameters of dapsone-treated patients were similar or thinner than the control values. In line with the structural changes of fibrin, the fibrinolytic profile of 5 DH patients under dapsone treatment approached the control values. CONCLUSION This study revealed that the fibrinolytic potential was impaired in the plasma of untreated DH patients, whereas dapsone corrected the fibrinolytic defect. These data suggest a pathogenic role for plasma-derived factors in the development of skin symptoms and add a new aspect to the long-known beneficial, symptomatic effect of dapsone in active DH.

Ex vivo nonlinear microscopy imaging of Ehlers–Danlos syndrome-affected skin

Ehlers–Danlos syndrome (EDS) is the name for a heterogenous group of rare genetic connective tissue disorders with an overall incidence of 1 in 5000. The histological characteristics of EDS have been previously described in detail in the late 1970s and early 1980s. Since that time, the classification of EDS has undergone significant changes, yet the description of the histological features of collagen morphology in different EDS subtypes has endured the test of time. Nonlinear microscopy techniques can be utilized for non-invasive in vivo label-free imaging of the skin. Among these techniques, two-photon absorption fluorescence (TPF) microscopy can visualize endogenous fluorophores, such as elastin, while the morphology of collagen fibers can be assessed by second-harmonic generation (SHG) microscopy. In our present work, we performed TPF and SHG microscopy imaging on ex vivo skin samples of one patient with classical EDS and two patients with vascular EDS and two healthy controls. We detected irregular, loosely dispersed collagen fibers in a non-parallel arrangement in the dermis of the EDS patients, while as expected, there was no noticeable impairment in the elastin content. Based on further studies on a larger number of patients, in vivo nonlinear microscopic imaging could be utilized for the assessment of the skin status of EDS patients in the future.

Tumorőssejtek szerepe a melanoma progressziójában és heterogenitásában

Over the past decade a rare cell population called cancer stem cells has been identified in both solid tumors and hematologic cancers. These cells are reminiscent of somatic and embryonic stem cells and play a critical role in the initiation and progression of malignancies. As all stem cells, they are able to undergo asymmetric cell division and hence renew themselves and create various other progenies with heterogenous phenotypes. A growing body of literature suggested that stem cell subpopulations contribute significantly to the growth and metastatic properties of melanoma. This review gives a comprehensive overview of the current literature on melanoma stem cells, with a special emphasis on the signaling pathways responsible for the homeostatic growth of melanocytes and the uncontrolled proliferation of melanoma cells. The importance of the local microenvironment are demonstrated through summarizing the role of various cell types, soluble factors and cell adhesion molecules in the progression of melanoma and the creation of treatment resistant cancer cell clones. Last but not least, the models of melanoma progression will be introduced and a variety of cellular markers will be presented that may be used to identify and therapeutically target melanoma. Orv. Hetil., 2016, 157(34), 1339-1348.Absztrakt A szolid es hematologiai tumorok legtobbjeben mara olyan sejtpopulaciokat azonositottak, amelyek a daganatok kis szazalekat alkotjak, megis kiemelkedő szerepet toltenek be a daganat terjedesenek előmozditasaban. Ezek az ugynevezett tumorőssejtek a szomatikus es embrionalis őssejtekhez hasonlo viselkedest mutatnak, aszimmetrikus osztodassal onmegujitasra kepesek es heterogen sejtpopulaciokat is letrehoznak. Egyre tobb kutatas alatamasztja, hogy a malignus melanomak progresszioja mogott is tumoros őssejtek allnak. Nem tisztazott kerdes azonban, hogy a tumorigenicitasert vajon kizarolag melanomaőssejtek szubpopulacioi felelősek vagy pluripotens őssejtte barmely melanomasejt dedifferencialodhat. Jelen kozlemeny a pluripotens melanomaőssejtekről kivan atfogo kepet nyujtani, kulonos tekintettel azokra a mechanizmusokra, amelyek a melanocyta-őssejtek differencialodasat szabalyozzak, ugyanakkor a melanomaőssejtekben szabalyozatlanul műkodnek. Bemutatasra kerul a mikrokornyezet sejtjeinek, sejtadhezios m...