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INTRODUCTION This study investigated the role of natural polymers as moisturizers with low toxicity and biodegradability in the cosmetic and pharmaceutical industries. We isolated a polysaccharide extract from Dendrobium candidum (D. candidum) and determined its efficacy in skin hydration when used as an active cosmetic ingredient. METHODS The molecular weight distribution of D. candidum polysaccharides was analyzed via gel permeation chromatography (GPC). We performed real-time reverse transcription PCR (RT-PCR) and western blotting assays to investigate the physiological mechanism of the polysaccharides extracted from D. candidum (PDC). Based on in vitro data, the efficacy of PDC in improving skin condition was tested on the face of 21 volunteers. RESULTS The expression of filaggrin (FLG), caspase-14, and bleomycin hydrolase, which are the major components contributing to skin hydration, was significantly increased in the PDC-treated group. Further, the PDC upregulated the mRNA expression of occludin and claudin-1, which play a key role in epidermal barrier function. In addition, a topical application of PDC markedly increased skin hydration and improved trans-epidermal water loss (TEWL) and skin elasticity after 2 weeks. CONCLUSIONS It is the first study reporting the efficacy of PDC-mediated FLG mechanism associated with positive skin hydration. PDC can be used as an active ingredient in moisturizers. Long-term application of PDC-based moisturizers may result in significant improvement in elasticity and barrier function.

The development of flagellate erythema secondary to bleomycin treatment is a rare adverse effect. The prevalence varies between 8 to 22% of patients and it is becoming rarer. Flagellate erythema presents as a scaly erythematous papule. A lower amount of bleomycin hydrolase in regions with reduced production, such as skin and lungs, decreases bleomycin degradation, which allows its accumulation in tissues, triggering an inflammatory process, especially in patients with lower basal enzyme function. We report a clinical case of a severe presentation of flagellate erythema secondary to a patient ongoing bleomycin, etoposide and platinum (BEP) based chemotherapy of non-seminomatous testis cancer.

Introduction Bleomycin pulmonary toxicity (BPT) is a potentially life-threatening consequence of bleomycin usage in patients. An overproduction of epithelium-derived cytokines, habitually linked to allergic inflammation, has been recently revealed in experimental models of BPT. Methods We assessed retrospectively our cohort of patients with Hodgkin Lymphoma treated with bleomycin between 2014 and 2016 for their demographic, clinical features, including BPT development, atopy status and risk factors for BPT. Then they were invited for allergy testing and blood sample collection. The samples were stimulated with different stimuli (Bleomycin, IL-33, TSLP) for 24 h on cell culture. The culture supernatants were analysed for TGF-β, Galectin3, Arginin, Amphiregulin, Eotaxin, IFNγ, TNFα, IL1β, 4, 5, 6, 10, 13, 17, MIP-1α, and bleomycin hydrolase (BLH) levels. Results The cohort consisted of 51 patients showed that atopy was the only significant risk factor for BPT occurrence (OR: 7.2, p = 0.007). Fourteen subjects were included for blood analysis. The analysis of supernatants at the unstimulated condition revealed that BLH and Amphiregulin were significantly lower in patients who had BPT than controls. The BLH cut-off that best identified a history of BPT was 175.31 (Sensitivity: 62.5%, specificity: 100%). Following the stimulation, BLH reduced compared to the unstimulated condition and the difference between groups remained significant (p Conclusion Our study is the first to report that low levels of bleomycin hydrolase in allergic individuals may be predisposing to a possible pathway of fibrosis.

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C–C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Membrane-derived extracellular vesicles, referred to as microvesicles (MVs), have been proposed to participate in several cancer diseases. In this study, MV fractions were isolated by differential ultracentrifugation from a metastatic breast cancer (BC) cell line MDA-MB-231 and a non-cancerous breast cell line MCF10A, then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 1519 MV proteins were identified from both cell lines. The data obtained were compared to previously analyzed proteins from small extracellular vesicles (sEVs), revealing 1272 proteins present in both MVs and sEVs derived from the MDA-MB-231 cell line. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH) were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells, sEVs, and MVs derived from both cell lines. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in BC and potential in therapeutic applications.

Povzetek: Ateroskleroza je bolezen kopičenja maščobnih delcev v žilno steno arterij. Po svoji naravi je progresivna bolezen, ki jo zaznamuje večanje aterosklerotičnega plaka do točke ko le-ta poči in vsebina izpostavi krvnemu obtoku. To na mestu razpoke povzroči nastanek strdka, ki lahko posledično zapre ali zoži arterijo do te mere, da lahko privede do ishemije tkiva katerega taka arterija prehranjuje. Pri srcu to pomeni ishemijo srčne mišičnine – miokardni infarkt. Ob miokardnem infarktu se v sistemsko cirkulacijo zaradi nekroze srčne mišice izločajo vnetni mediatorji, ti pa vplivajo na večje vnetje v aterosklerotičnih lehah na kar kažejo slikovne študije ožilja po ishemičnem dogodku. Klinično to vidimo tudi v povečanem tveganju za ponovni ishemični dogodek, najvišje v roku enega leta po miokardnem infarktu. Na to kažejo podatki iz številnih epidemioloških študij s celega sveta. Modulacija vnetja po miokardnem infarktu lahko zmanjša tveganje za ponovni dogodek, kar so pokazali podatki tako iz predkliničnih kot tudi iz kliničnih študij. V prvem delu doktorske teze predstavim novo pot imunomodulacije po miokardnem infarktu z inhibicijo receptorja za interlevkin 7. V živalski študiji na miših z aterosklerotično boleznijo in pridruženim miokardnim infarktom pokažem, da interlevkin 7 deluje kot monocitni kemotaktični dejavnik, da je količina interlevkina 7 po miokardnem infarktu v žilni steni povišana ter, da se po miokardnem infarktu poveča sinteza mRNA interlevkina 7 v samem ishemično prizadetem miokardu. Imunomodulacija vnetja z inhibitornim monoklonskim protitelesom proti receptorju interlevkina 7 zmanjša kopičenje maščob v žilni steni ter infiltracijo vnetnih celic, predvsem monocitov in makrofagov, kar pomeni zmanjšanje vnetja in stabilizacijo plaka. V prvem delu doktorskega dela tako pokažem doprinos in pomen vnetja in prirojenega imunskega sistema v proces ateroskleroze. Imunski sistem v grobem sestavljata prirojeni imunski sistem, katerega sestavni del so celice kot so monociti in makrofagi, udeleženi v proces ateroskleroze tako v akutni kot kronični fazi bolezni ter pridobljeni imunski sistem. Po začetni, nespecifični fazi vnetja pride do aktivacije specifičnega, pridobljenega dela imunskega sistema, katerega predstavniki so med drugim limfociti T, limfociti B ter antigen predstavitvene celice. Značilnost tega dela imunskega sistema je tarčno, antigensko specifično vnetje, za katerega so značilne interakcije med receptorji na celicah T in MHC molekulami na antigen predstavitvenih celicah. Znano je, da pri procesu ateroskleroze pride do aktivacije avto-reaktivnih limfocitov T in B in specifičnega odziva le-teh proti maščobnim delcem v aterosklerotičnih lehah. En takih delcev je peptid p210, ki je del molekule ApoB100, sestavni del delcev LDL, glavnih krivcev za nastanek vnetja v žilni steni. Raziskave so pokazale obstoj LDL in apoB-100 reaktivnih celic T, vendar do sedaj specifični epitopi pomembni pri aterosklerozi še niso bili potrjeni. V drugem delu doktorskega dela pokažem prisotnost avtoreaktivnih, antigen specifičnih CD8+ limfocitov T, pri katerih stimulacija s peptidom p210 izzove avto-imunski odziv. Avto-imunski odziv temelji na prepoznavi kompleksa antigen-MHC-I molekula s strani celičnega receptorja T. S pomočjo IEDB baze (Immune Epitope Data Base) smo preverili predvideno vezavo peptidnega fragmenta p210 na mišje molekule MHC-I. Na podlagi izbranega peptidnega fragmenta z visoko predvideno vezavno afiniteto na MHC-I smo izdelali fluorescenčno označen pentamer sestavljen iz petih MHC-I molekul, specifičnih za p210 fragment in preverili prisotnost CD8+ celic T, ki se na pentamer vežejo ob specifični prepoznavi epitopa p210. Miši, ki so prejemale aterogeno dieto z visoko vsebnostjo maščob, so izražale večji delež pentamer pozitivnih CD8+ celic T, ki so imele izražen efektorski fenotip. Te celice se ob stimulaciji s peptidom p210 klonalno razmnožijo, kar je značilnost specifične razpoznave antigena s strani T-celičnega receptorja (TCR). Imunomodulacija z imunizacijo s p210 je povečala delež p210-specifičnih pentamer pozitivnih CD8+ limfocitov T in zmanjšala breme ateroskleroze. Interakcija CD8+ limfocitov T z antigen predstavitvenimi celicami temelji na vezavi antigenskega peptida z vezavnim mestom MHC-I molekule na površini antigen predstavitvenih celic in prepoznavi kompleksa antigen/MHC-I s strani T-celičnega receptorja CD8+ limfocitov T. Znano je, da se MHC-I molekule pri bolezenskih stanjih izločajo v krvni obtok. Te topne MHC-I molekule na svoji površini oz. vezavnem mestu nosijo peptidne fragmente, ki predstavljajo potencialne avto-antigene udeležene v boleznska stanja. Z analizo in identifikacijo peptidov, vezanih na topne MHC-I molekule, lahko prepoznamo antigene pomembne pri procesu ateroskleroze in miokardnega infarkta. V tretjem delu teze predstavim novo metodo odkrivanja potencialnih avto-antigenov udeleženih v proces ateroskleroze in po miokardnem infarktu in prikažem translacijski pristop testiranja relevantnosti odkritih peptidov v biološkem modelu ateroskleroze in miokardnega infarkta. Z metodo imunoprecipitacije smo iz krvi pacientov z akutnim koronarnim sindromom pridobljene v AZACS študiji, ki imajo dokazano aterosklerozo, izolirali peptide, ki so bili ob primerjavi z zdravimi kontrolami specifični za paciente. Peptide smo identificirali s pomočjo masne spektrometrije in primerjave aminokislinskega zaporedja z bazo človeških proteinov Uniprot. S to metodo smo odkrili več potencialnih avto-antigenov udeleženih v proces ateroskleroze in miokardnega infarkta. Za nadaljno testiranje biološkega pomena smo izbrali peptide specifične za paciente, ki so imeli na masni spektrometriji najvišjo intenzivnost signala, ali pa so bili skupni največjem številu pacientov (Keratin 8, ARID1A, Bleomicin hidrolaza ter LL-37). Mišje homologe teh peptidov smo testirali na živalskem modelu ateroskleroze ter ateroskleroze s pridruženim miokardnim infarktom. Od izbranih peptidov sta keratin II (mišji analog človeškemu keratinu 8) ter CRAMP (mišji analog človeškemu LL-37) izzvala največji imunski odziv. Keratin II je v mišjem ApoE(-/-) modelu ateroskleroze izzval predvsem efektorski odziv CD8+ limfocitov T, katerega ni bilo videti v mišjem ApoE(-/-) modelu ateroskleroze s pridruženim miokardnim infarktom. To je bilo razvidno tudi ob stimulaciji perifernih mononuklearnih celic izoliranih iz krvi pacientov s stabilno koronarno boleznijo in pacientov z akutnim koronarnim sindromom. Antimikrobni peptid LL-37, fragment proteina hCAP18, ki se nahaja v granulah nevrofilnih granulocitov in igra pomembno vlogo pri nevtrofilnem vnetju, je znan avto-antigen pri avtoimunih boleznih kot je luskavica. Pacienti z luskavico imajo večje breme ateroskleroze kot splošno, zdravo prebivalstvo in pridruženo višje tveganje za srčno-žilne dogodke. Bolniki z dokazano aterosklerozo imajo po drugi strani dvakrat višjo prevalenco luskavice kot zdravo prebivalstvo. Poleg tega je LL-37 moč najti v aterosklerotičnih lehah. Pomanjkanje mišjega ekvivalenta LL-37, imenovanega CRAMP (Cathelicidin Related Antimicrobial Peptide) v mišjem modelu zmanjša aterosklerotično breme. V namen proučevanja vpletenosti CRAMP v proces ateroskleroze pri miših smo sintetizirali skrajšano različico molekule (tCRAMP – truncated CRAMP), ki je vsebovala predvidena vezavna mesta za mišje MHC-I molekule. Stimulacija mišjih imunskih celic je izzvala močan efektorski odziv celic T, ki je bil še posebej poudarjen pri miših z razširjenim aterosklerotičnim bremenom po hranjenju z dieto bogato z maščobami. V odziv sta bili udeleženi tako CD4 kot tudi CD8 T-celična vrsta. Z imunizacijo kot način imunomodulacije smo preverili pomen tCRAMP antigena pri aterosklerozi. Izbrali smo dve imunizacijski dozi, višjo (100ug) in nižjo (20ug), ki sta imeli zelo različen biološki odziv. Nižja imunizacijska doza je preko povečanja deleža CD8+ celic T centralnega spomina ter znižanja deleža vnetnih dendritičnih celic CD11b+ zmanjšala breme aterosklerotične bolezni. Višja imunizacijska doza pa je, ravno obratno, povečala breme aterosklerotične bolezni in povzročila avto-imunsko preobrazbo imunskega sistema. Videti je bilo povišanje avto-reaktivnosti, predvsem z večjim deležem tako CD4+ kot CD8+ efektorskih celic T kot tudi povečanje deleža dendritičnih celic CD11b+, klasično celic vnetnega fenotipa. V doktorski tezi pokažem pomen tako prirojenega kot pridobljenega imunskega sistema v procesu ateroskleroze in vnetja po miokardnem infarktu ter potencialne nove načine imunomodulacije, ki se lahko uporabljajo za zmanjšanje bremena ateroskleroze. Teza predstavi pomen interlevkin 7 vnetne poti in vpletenost le-te v vnetje po miokardnem infarktu. Inhibicija receptorja interlevkina 7 zmanjša posti-ishemično vnetje v aterosklerotičnih lehah. Monoklonska protitelesa proti interlevkinu 7 so bila že prestala varnostno klinično študijo in predstavljajo zanimivo priložnost za klinično uporabo imunomodulacije po miokardnem infarktu. Doktorsko delo je pomembno tudi zaradi predstavitve novega načina raziskovanja avto-antigenov udeleženih v proces ateroskleroze. Določanje imunopeptidoma iz krvi pacientov s koronarno boleznijo je izvedljivo in privede do odkrivanja antigenov biološkega pomena. Predstavim tudi pomembno translacijsko pot raziskovanja in testiranja teh antigenov v pristopu, ki vodi od pacienta do laboratorijskega testiranja hipoteze in nazaj do pacienta in omogoča pridobivanje potencialno pomembnih kliničnih odkritij. V svojem delu tako s translacijskim pristopom pokažem poglobljen pomen avto-antigena LL-37 odkritega v vzorcih krvi pacientov po akutnem koronarnem sindromu v procesu ateroskleroze. Summary: Atherosclerosis is a chronic progressive disease process characterized by lipid accumulation inside the the intimal layer of the arterial wall. The progressive course of the disease can lead to eventual plaque rupture or erosion, causing exposure of the pro-inflammatory plaque contents to the bloodstream. This is followed by thrombus formation on the rupture site and ischemia of the tissue supplied by the affected artery. If the tissue in question is the myocardium this arterial occlusion causes myocardial infarction. Injury to the myocardium causes a systemic inflammatory reaction with pro-inflammatory cytokine release which impacts atherosclerotic plaque inflammation shown by imaging studies in both preclinical and clinical studies. Epidemiological data show that patient with myocardial infarction are at an increased risk of a recurrent ischemic event in the first year following an index event. Immunomodulation following myocardial infarction has shown to reduce the risk of a recurrent event as evidenced by both preclinical as well as clinical studies. The first part of this thesis is dedicated to a novel pathway of immunomodulation after myocardial infarction by interleukin-7 receptor inhibition. The study was conducted on a mouse ApoE(-/-) model of myocardial infarction and atherosclerosis. We show that interleukin-7 acts as a chemoattractant for monocytes in vivo, its synthesis is increased inside the ishemic myocardium following myocardial infarction and increased levels are found inside the arterial wall following a myocardial infarction as well. Immunomodulation by interleukin-7 receptor blocking by monoclonal antibody reduces the lipid content of atherosclerotic plaque as well as monocyte and macrophage infiltration into the plaque thus reducing inflammation and causing plaque stabilization. The first part of the thesis thus shows the importance of a novel innate inflammatory pathway in atherosclerosis. The human immune system is composed roughly of the innate immune system, represented by cells such as neutrophils, monocytes and macrophages, involved in atherosclerosis in both the acute and chronic stages of disease and the specific, adaptive immune system which is activated after the nonspecific phase of inflammation. The main drivers of this specific part of the immune system are lymphocytes, both T cells and B cells, as well as antigen presenting cells. The MHC/T-cell receptor coupling is a hallmark of the adaptive immune system and is caused by targeted, antigen specific binding of short peptides to both antigen recognition molecules on the cell surfaces of T cells and antigen presenting cells. Data show that activation of auto-reactive T and B cells and antigen specific responses of these cells to lipid particles occur in atherosclerosis. One of the aforementioned particles is p210, part of the apoliprotein ApoB100, a component of LDL, the culprit cholesterol particle involved in plaque inflammation. Research has shown the existence of LDL and ApoB-100 reactive T cells, but specific epitopes involved in atherosclerosis have not yet been discerned. In the second part of this thesis I show the presence of autoreactive, antigen specific CD8+ T lymphocytes, activated by p210 stimulation in an auto-immune manner. This involves recognition and binding of a T-cell receptor to an antigen loaded MHC-I molecule. Potential binding of the p210 peptide to mouse MHC-I was assessed using the Immune Epitope Data Base (IEDB) prediction site and based on binding prediction score a fluorescent pentamer composed of five bound MHC-I molecules was constructed with specificity for p210 epitope recognition. This was used to test for p210 specific CD8+ T cells in a mouse model of atherosclerosis. Mice, fed a high fat, pro-atherogenic diet showed an increase of pentamer positive CD8+ T cells, exhibiting an effector T cell phenotype. Following stimulation, these cells show clonal proliferation, a hallmark of antigen specific recognition by T cell receptor (TCR). Immunomodulation by way of immunization showed a further increase in p210 specific pentamer positive CD8+ T lymphocytes as well as a reduction in atherosclerotic burden in the p210 immunized mice. The CD8+ T cell interaction with antigen presenting cells is based on antigen binding on MHC-I molecules on the surface of antigen presenting cells and recognition of the antigen-MHC-I complex by T cell receptor on the surface of CD8+ T lymphocytes. It's been shown that certain disease states cause MHC-I molecule shedding off the surface of cells. These can be detected in the bloodstream of patients and the peptides bound to these circulating, soluble MHC-I molecules represent possible auto-antigen involved in disease. Analysis of these peptides in patients with coronary artery disease and myocardial infarction can reveal previously unknown antigens important in atherosclerosis and its acute ischemic complication. The third part of this thesis shows a novel way of immunopeptidome profiling in order to reveal new potential antigens involved in atherosclerosis. Presented here is also a translational approach to testing the relevance of these novel peptide antigen in a biological model of atherosclerosis and combined atherosclerosis and myocardial infarction. Immunoprecipitation of soluble MHC-I complexes obtained from blood samples of patients with acute coronary syndrome collected from the AZACS trial revealed peptides specific to patients relative to matched control blood samples. The peptides were identified using mass spectrommetry and the amino acid sequence matched to known peptides from the Uniprot human protein database. The method enabled the identification of potential auto-antigen involved in atherosclerosis and myocardial infarction. In order to further test the biological relevance of the discovery, the peptides, specific to patients with the highest intensity on mass spectrometry were selected as well as peptides common and specific to most of the patient samples. The peptides tested were Keratin 8, ARID1A, Bleomycin Hyrolase and LL-37. Mouse homologues to these peptides were chosen to test auto-reactive responses in an Apo(-/-) mouse model of atherosclerosis as well as a combined ApoE (-/-) mouse model of atherosclerosis and myocardial infarction. Keratin II (murine homolog to human Keratin 8) and CRAMP (murine homolog to LL-37) showed highest immune responses upon stimulation and were chosen for further testing. Keratin II elicited a predominately effector CD8+ T lymphocyte response in the ApoE (-/-) model of atherosclerosis but not in a combined ApoE (-/-) murine model of atherosclerosis and myocardial infarction. This was evident also after testing human peripheral blood mononuclear cells isolated from blood samples taken from patient with either stable coronary artery disease or acute coronary syndrome. The antimicrobial peptide LL-37, a fragment of the larger hCAP-18 and a constituent of neutrophil granules is an important component of neutrophil mediated inflammation. It is a known auto-antigen in psoriasis, a classic auto-immune disease. Patients with psoriasis have increased burden of atherosclerosis compared to the healthy population and a higher risk of cardiovascular complication due to increased risk of plaque rupture. LL-37 has also been shown to be present in atherosclerotic plaque. Deficiency of mouse homolog to LL-37, named CRAMP (Cathelicidin Related Antimicrobial Peptide) in a CRAMP (-/-) murine model with atherosclerosis has shown to reduce atherosclerotic burden. To study the effect of CRAMP in a murine model of atherosclerosis, and to avoid its inherent immune properties, we synthesized a truncated version of CRAMP containing the highest predicted binding sites to mouse MHC-I molecules. Stimulation of murine immune cell populations elicited a strong effector T cell response in both CD4+ and CD8+ T cell populations which was amplified in mice fed a pro-atherogenic high fat diet. Immunization as a way of immunomodulation was used to test the biologic relevance as auto-antigen. Two different immunization doses were used, a higher 100ug dose and a lower 20 ug dose. These two doses exhibited a differential biological effect on atherosclerosis and immune activation. The lower immunization dose elicited a CD8+ Central Memory T cell response and reduced the pro-inflammatory CD11b+ Dendritic Cell populations along with a reduction in the burden of atherosclerotic disease. The higher immunization dose on the other hand increased the level of CD11b+ dendritic cells as well as caused an increase and activation of CD4+ and CD8+ Effector Memory T cells along with a significant increase in atherosclerosis. The higher immunization dose thus caused an increase in auto-reactivity to the auto-antigen and exhibited a seemingly auto-immune transformation of the murine immune system. The thesis outlines the importance of both the innate and adaptive immune systems in atherosclerosis and inflammation following myocardial infarction and potential new ways of immunomodulation effectively reducing the burden of atherosclerotic disease. It shows the importance of the interleukin-7 inflammatory pathway following myocardial infarction. Immunomodulation by inhibition of the IL-7 receptor mitigates the effect of the post-ishemic inflammation on atherosclerotic plaque. Monoclonal antibodies targeting IL-7 have already been tested in a clinical trial and thus represent a promising opportunity for immunomodulation after myocardial infarction in a clinical situation. The thesis outlines an important novel way of self-antigen discovery. Immunopeptidome analysis from patient blood samples is feasible and yields biologically relevant self-peptides involved in atherosclerosis. Furthermore we delineate an important translational pathway to discovery and testing of these antigen in a bedside-bench-bedside approach which produces data relevant to clinical practice and patient health. Our translational pathway revealed the involvement of LL-37, a known auto-antigen in psoriasis as an auto-antigen in atherosclerosis.

The bleomycins (BLMs) are widely used in combination therapies for the treatment of various cancers. Dose-dependent and cumulative pulmonary toxicity is the major cause of BLM-associated morbidity, limiting the broad uses of BLMs as anticancer drugs. The organ specificity of BLM-induced toxicity has been correlated with the expression of the hBLMH gene, encoding the human bleomycin hydrolase (hBLMH), which is poorly expressed in the lung. hBLMH hydrolyzes BLMs into the biologically inactive deamido BLMs, thereby protecting organs from BLM-induced toxicity. Here we report (i) expression of hBLMH and production and isolation of recombinant human bleomycin hydrolase (rhBLMH) from E. coli, (ii) structural characterization of deamido BLM A2 and B2 isolated from rhBLMH-catalyzed hydrolysis of BLM A2 and B2, and (iii) kinetic characterization of the rhBLMH-catalyzed hydrolysis of BLM A2 and B2, in comparison with five BLM analogues. rhBLMH from E. coli catalyzes rapid and efficient hydrolysis of all BLMs tested, exhibiting a superior catalytic efficiency for BLM B2. These findings reveal new opportunities to overcome BLM-induced pulmonary toxicity in chemotherapies, potentially by exploring BLM B2 as the preferred congener, engineering designer BLMs with optimized activity for rhBLMH, or co-administrating rhBLMH directly into the lung as a potential protein therapeutic.

Bleomycin hydrolase (BLMH) is a well-conserved cysteine protease widely expressed in several mammalian tissues. In skin, which contains high levels of BLMH, this protease is involved in the degradation of citrullinated filaggrin monomers into free amino acids important for skin hydration. Interestingly, the expression and activity of BLMH is reduced in patients with atopic dermatitis (AD) and psoriasis, and BLMH knockout mice acquire tail dermatitis. Apart from its already known function, we have discovered a novel role of BLMH in the regulation of inflammatory chemokines and wound healing. We show that lowered BLMH levels in keratinocytes result in increased release of the pro-inflammatory chemokines CXCL8 and GROα, which are upregulated in skin from AD patients compared to healthy individuals. Conditioned media from keratinocytes expressing low levels of BLMH increased chemotaxis by neutrophils and caused a delayed wound healing in the presence of low-level TNFα. This defective wound healing was improved by blocking the shared receptor of CXCL8 and GROα, namely CXCR2, using a specific receptor antagonist. Collectively, our results present a novel function of BLMH in regulating the secretion of chemokines involved in inflammation and wound healing in human keratinocytes.

Bleomycin (BLM) is a broadly used antibiotic to treat different types of cancer. It can be hydrolyzed by bleomycin hydrolase (BLMH), which eventually influences the anti-tumor efficacy of BLM. Therefore, it is particularly important to detect BLM and BLMH. Herein, we demonstrated highly sensitive detection of BLM and BLMH by a simple and convenient liquid crystal (LC)-based sensing platform for the first time. 5CB (a nematic LC) doped with the cationic surfactant OTAB was working as the sensing platform. When the OTAB-laden 5CB interface was in contact with an aqueous solution of ssDNA, LCs displayed a bright image due to disruption of the arrangement of OTAB monolayers by ssDNA, indicating the planar orientation of LCs at the aqueous/LC interface. When BLM·Fe(II) and ssDNA were both present in the aqueous solution, ssDNA underwent irreversible cleavage, which prevented disruption of the arrangement of OTAB monolayers. Accordingly, LCs showed a dark image, suggesting the homeotropic orientation of LCs at the aqueous/LC interface. However, when BLM·Fe(II) was enzymatically hydrolyzed by BLMH, LCs remained the bright image. This approach showed high sensitivity for the detection of BLM and BLMH with the limits of detection of 0.2 nM and 0.3 ng/mL, respectively. Besides, the detection of BLM and BLMH was successfully achieved in human serum. This method has the advantages of high sensitivity, robust stability, simple operation, low cost, and easy detection through naked eyes, which makes it a potential candidate for applications in clinical analysis.

The article description and significance to dermatologists: bleomycin flagellate dermatitis is a rare cutaneous manifestation, believed to be due to the lack of bleomycin hydrolase enzyme in the skin, which inactivates bleomycin, resulting in its accumulation. This is thought to be a dose-dependent reaction, and doses over 200 U and higher may increase risk. This case describes a male developing a pruritic, erythematous linear flagellated dermatitis to the lower back after his third cycle of bleomycin, etoposide and cisplatin for a stage 3 seminoma. Pruritis resolved and erythema improved with the treatment of bilastine and desoximetasone cream. It is important to recognize this condition because untreated pruritis may lead to increased impairment of the skin barrier in already immunocompromised patient populations. This may also give further evidence to having ongoing and continuing collaboration between Dermatology and Medical Oncology for any patients presenting with a new rash undergoing chemotherapy treatments.

Ubiquitin-conjugating protein 9 (Ubc9), the sole enzyme for sumoylation, plays critical roles in many physiological functions, such as DNA damage repair and genome integrity. Its overexpression led to poor prognosis and drug resistance in tumor chemotherapy. However, the underlying mechanism by which Ubc9 promotes tumor progress and influences the susceptibility to antitumor agents remains elusive. In this study, we used nine antitumor agents with distinct actions to explore Ubc9-mediated resistance in human breast carcinoma MCF-7 cells. Increase of susceptibility, respectively, to boningmycin, hydroxycamptothecine, cis-dichlorodiamineplatinum, 5-fluorouracil, vepeside and gemcitabine, but not for doxorubicin, vincristine and norcantharidin, was observed after the knockdown of Ubc9 protein level with RNA interference. Reduction of bleomycin hydrolase and poly(ADP-ribose) polymerase-1 levels after knockdown of Ubc9 suggests their contribution to Ubc9-mediated drug resistance. This is the first report on the sensitivity to hydroxycamptothecine, cis-dichlorodiamineplatinum and gemcitabine that increased after knockdown of bleomycin hydrolase at protein level. In conclusion, Ubc9 plays different roles of action in antitumor agents in chemotherapy. The process requires bleomycin hydrolase and poly(ADP-ribose) polymerase-1. The results are beneficial to deeply understanding of Ubc9 functions and for precise prediction of chemotherapy outcomes in tumors.

BACKGROUND: Knowledge of the ethnic differences and effects of photodamage on the relative amounts of natural moisturizing factor (NMF) together with filaggrin processing enzymes in facial stratum corneum is limited. Our aim was to characterize the activities of calpain-1 (C-1), bleomycin hydrolase (BH) and the levels of pyrrolidone carboxylic acid (PCA) as a marker for total NMF levels and to relate them to plasmin activities and corneocyte maturation. METHODS: Enzyme activities, PCA levels and corneocyte maturation were determined from facial tape strippings of photoexposed cheek and photoprotected post-auricular areas (PA) of healthy Caucasian (C), Black African (BA) and albino African (AA) female subjects living in South Africa. RESULTS: PCA concentration levels were of the order AA > BA > C subjects, and the highest activities of BH were present in the AA subjects. BH activities were greater on the photoexposed sites for the BA and C subjects, but they were only numerically elevated in the AA subjects. Photoprotected sites had an increase in C-1 activity in pigmented groups (C and BA), whereas in the AA subjects, the opposite was measured. Plasmin activities were greater on the cheek compared with the PA site for the AA and C subjects, but the activity was low in the BA subjects. In both test sites, the AA, but not the BA and C subjects, had smaller, parakeratotic and less mature corneocytes. CONCLUSION: Variation in PCA levels has been found for different ethnic groups in this study (AA > BA > C subjects). The values in the AA subjects are surprising as one might expect that the lack of pigmentation, and thereby increased photodamage, might lead to lower levels. Increased BH, but not C-1 activity, was observed in the AA subjects indicating that BH is associated with PCA production to a greater extent. Surprisingly, corneocyte maturation is still impaired with elevated PCA levels in AA subjects. The higher levels of plasmin and BH activities on the cheeks, especially for AA and C subjects, suggest that they can be used as markers for epidermal photodamage.

Plasma biomarkers that identify abdominal aortic aneurysm (AAA) rupture risk would greatly assist in stratifying patients with small aneurysms. Identification of such biomarkers has hitherto been unsuccessful over a range of studies using different methods. The present study used an alternative proteomic approach to find new, potential plasma AAA biomarker candidates. Pre-fractionated plasma samples from twelve patients with AAA and eight matched controls without aneurysm were analyzed by mass spectrometry applying a tandem mass tag (TMT) technique. Eight proteins were differentially regulated in patients compared to controls, including decreased levels of the enzyme bleomycin hydrolase. The down-regulation of this enzyme was confirmed in an extended validation study using an enzyme-linked immunosorbent assay (ELISA). The TMT-based proteomic approach thus identified novel potential plasma biomarkers for AAA.

Summary Background Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels of filaggrin degradation products [natural moisturizing factors (NMFs)], activities of filaggrin‐processing enzymes [bleomycin hydrolase (BH) and calpain‐1 (C‐1)] and plasmin, and corneocyte envelope (CE) maturity in early life are largely unknown. Objectives We conducted a cross‐sectional, observational study investigating regional and age‐dependent variations in NMF levels, activity of proteases and CE maturity in stratum corneum (SC) from infants to determine whether these factors could explain the observed predilection sites for AD in early life. Methods We measured NMF using a tape‐stripping method at seven sites in the SC of 129 children (aged < 12 months to 72 months) and in three sites in 56 neonates and infants (< 48 h to 3 months). In 37 of these neonates and infants, corneocyte size, maturity, BH, C‐1 and plasmin activities were determined. Results NMF levels are low at birth and increase with age. Cheek SC, compared with elbow flexure and nasal tip, has the lowest NMF in the first year of life and is the slowest to reach stable levels. Cheek corneocytes remain immature. Plasmin, BH and C‐1 activities are all elevated by 1 month of age in exposed cheek skin, but not in elbow skin. Conclusions Regional and temporal differences in NMF levels, CE maturity and protease activities may explain the predilection for AD to affect the cheeks initially and are supportive of this site as key for allergen priming in early childhood. These observations will help design early intervention and treatment strategies for AD., What's already known about this topic? Atopic dermatitis (AD) frequently starts in early infancy, and the first eczematous lesions emerge on the cheeks.Filaggrin is a major structural protein in the stratum corneum (SC).Filaggrin deficiency is associated with the development of AD and, in the context of AD, food allergies and asthma.Filaggrin is metabolized into natural moisturizing factors (NMFs), which can be measured in the SC. What does this study add? Regional differences in NMF levels, corneocyte envelope immaturity and protease activities may help explain why infantile AD most often initially affects the cheeks.Filaggrin processing, corneocyte maturity, and protease activities show regional and temporal differences in infant skin.These findings may explain disease patterns in early‐life AD. What is the translational message? Cheek skin may be highly relevant for allergen priming.Emollient therapy at the vulnerable cheek site might help to prevent AD and/or food sensitization. Linked Editorial: https://doi.org/10.1111/bjd.16806. https://doi.org/10.1111/bjd.16959 available online https://goo.gl/Uqv3dl

The combination of a negative D-dimer and a Wells score can rule out, but not confirm, a diagnosis of deep venous thrombosis (DVT). We aimed to identify new diagnostic biomarkers for DVT and to investigate their relationship with hypercoagulability markers [D-dimer and activated protein C-protein C inhibitor (APC-PCI) complex]. We screened 92 cardiovascular-specific proteins in plasma samples from 45 confirmed DVT patients and 45 age- and sex-matched non-DVT patients selected from a prospective multicentre diagnostic management study (SCORE) by Proseek Multiplex CVDIII96×96. Plasma levels of 30 proteins were significantly different between DVT and non-DVT patients. After Bonferroni correction, plasma levels of seven proteins: P-selectin, transferrin receptor protein 1, von Willebrand factor, tissue factor pathway inhibitor, osteopontin (OPN), bleomycin hydrolase and ST2 protein remained significantly different. The area under curve (AUC) for these proteins ranged from 0·70 to 0·84. Furthermore, all seven identified proteins were significantly associated with markers of hypercoagulability. A combination of OPN and APC-PCI had the best ability to discriminate DVT from non-DVT patients (AUC = 0·94; sensitivity = 89% and specificity = s84%). In conclusion, we identified multiple proteins associated with markers of hypercoagulability and with a potential to become novel diagnostic biomarkers for DVT. (Less)

Summary Background Maintenance of water balance in the stratum corneum (SC) is determined by the content of intercellular lipids and natural moisturizing factors (NMFs) in corneocytes. Aim To investigate the association between the NMFs and (pro)filaggrin and the proteases responsible for the processing of (pro)filaggrin to NMFs in the SC of hydrated and dry skin areas of healthy human subjects. Methods The SC hydration state and the transepidermal water loss (TEWL) were measured using a Corneometer and a Tewameter, respectively. Proteases, (pro)filaggrin and NMFs were extracted from SC samples obtained by tape-stripping of the tested skin. Expression levels of (pro)filaggrin were determined by dot blotting and western blotting, and total NMFs by ultra-high performance liquid chromatography. Expression of the proteases caspase-14, calpain-1 and bleomycin hydrolase was measured by western blotting. Results The levels of (pro)filaggrin were not significantly different between hydrated and dry skin, whereas the level of total NMFs was significantly reduced in dry skin. A negative correlation between (pro)filaggrin and NMFs was found in dry skin (Pearson correlation coefficient r = − 0.57, *P

Scope Hyperhomocysteinemia (HHcy) induced by dietary or genetic factors is linked to kidney disease. Bleomycin hydrolase (Blmh) metabolizes Hcy-thiolactone to Hcy. We aimed to explain the role of dietary HHcy in kidney disease. Methods and results We examined kidney proteome in dietary HHcy and Blmh-knockout mouse models using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We found that the kidney proteome was altered by dietary HHcy and the Blmh −/− genotype. Proteins involved in metabolism of lipoprotein (ApoA1), amino acid and protein (Acy1, Hspd1), carbohydrate (Pdhb, Fbp1-isoform 1, Eno1), and energy metabolism (Ndufs8, Ldhd) were down-regulated. Proteins involved in carbohydrate metabolism (Fbp1-isoform 2), oxidative stress response (Prdx2), and detoxification (Glod4) were up-regulated. The Blmh −/− genotype down-regulated Glod4 isoform 3 mRNA but did not affect isoform 1 mRNA expression in mouse kidneys, suggesting post-transcriptional regulation of the Glod4 protein by the Blmh +/+ genotype. Responses of ApoA1, Acy1, Hspd1, Ndufs8, Fbp1, Eno1, and Prdx2 to HHcy and/or Blmh deficiency mimic their responses to renal disease. Conclusion Our findings indicate that Blmh interacts with diverse cellular processes – lipoprotein, amino acid and protein, carbohydrate, and energy metabolisms, detoxification, antioxidant defenses – that are essential for normal kidney homeostasis and that deregulation of these processes can account for the involvement of HHcy in kidney disease.

Detoxification of the glycopeptide bleomycin is mediated by bleomycin hydrolase, a cysteine aminopeptidase identified in a variety of organisms. The opportunistic fungal pathogen Candida albicans is known to exhibit increased resistance to bleomycin when compared to other yeast. Presented here is the cloning of the C. albicans Lap3p aminopeptidase, predicted by sequence identity to be the Candida form of bleomycin hydrolase. C. albicans Lap3p is functionally capable to replace the Saccharomyces cerevisiae Lap3p in vivo. Furthermore, the Candida enzyme was found to function as a cysteine aminopeptidase in vivo. It is shown here that upon introduction into a lap3 deletion strain of S. cerevisiae, the C. albicans Lap3p aminopeptidase does not significantly alter the response of Saccharomyces to bleomycin. These results suggest that C. albicans Lap3p does not function as the sole factor involved in bleomycin detoxification, and may require an accessory protein or co-factor in order to efficiently mediate this process in Candida. This study provides the first evidence of a functional description of the C. albicans Lap3p cysteine aminopeptidase, and provides the foundation for further mechanistic studies of the role of this protein in the cellular processes of Candida. Key words: Candida albicans, bleomycin detoxification, Lap3p aminopeptidase, cysteine protease.

Bleomycin is a chemotherapeutic agent commonly used to treat curable diseases such as germinative tumors and Hodgkin’s lymphoma. The major limitation of bleomycin therapy is pulmonary toxicity, which can be life threatening in up to 10% of patients receiving the drug. The mechanism of bleomycin-induced pneumonitis (BIP) involves oxidative damage, relative deficiency of the deactivating enzyme bleomycin hydrolase, genetic susceptibility, and the elaboration of inflammatory cytokines. Ultimately, BIP can progress to lung fibrosis. The diagnosis of BIP is established by the combination of systemic symptoms, radiological and histological findings, and respiratory function tests abnormalities, while other disorders should be excluded. Although the diagnosis and pathophysiology of this disease have been better characterized over the past few years, there is no effective therapy for the disease. In general, the clinical picture is extremely complex. A greater understanding of the BIP pathogenesis may lead to the development of new agents capable of preventing or even treating the injury already present. Physicians who prescribe bleomycin must be aware of the potential pulmonary toxicity, especially in the presence of risk factors. This review will focus on BIP, mainly regarding recent advances and perspectives in diagnosis and treatment.

Alzheimer disease (AD) is a progressive and irreversible neurodegenerative disorder that is characterized by cognitive decline, memory loss and confusion. The E4 allele of the apolipoprotein E gene (APOE) is associated with AD and it is the main genetic risk factor for disease. Although the exact physiological function is unknown, bleomycin hydrolase (BLMH) may also be associated with AD development, although previous immunohistochemical findings havebeen inconsistent. Therefore, the purpose ofthis study was to evaluate the genotypic and allele frequencies of theAPOEgene andBLMH1450 G> A polymorphism and assessBLMHexpression using PCR-RFLP and RT-qPCR analyses ofblood samples from patients with Alzheimer disease (AD), healthy elderly adults (EC) andhealthyyoung subjects(YC). BLMHexpression wassignificantly different among groups (p= 0.015) and there was substantial reduction with age and with AD. TheAPOEandBLMHgenotype frequency did not diverge from the Hardy-Weinberg equilibrium. There was a higher frequency of genotype 3/3 inall subjects (61.1%) and the AD group demonstrated a higher frequency of allele 4; however, differences ingenotype and allele distributions were statistically different among groups.