Targeting bacterial infections by using immunomodulatory host defence peptides and proteins

Bacterial infections and sepsis are amongst the leading causes of death worldwide. Sepsis is caused by an uncontrolled systemic host response towards an invading pathogen. Due to the engagement of various systems including cellmediated responses, the coagulation and complement cascades, treatment of sepsis remains challenging. This is reflected in mortality rates of about 50 % for patients with septic shock, despite improved health care and antibiotic usage. Endogenous host defence peptides (HDPs) are essential components of the innate defence against invading pathogens. They exert multiple biological functions, and apart from their antimicrobial effects they may modulate inflammation, coagulation, and chemotaxis. Due to increasing bacterial resistance and due to the complex nature of severe bacterial infections, HDPs are today considered valuable candidates in the development of novel treatments for infections. In papers I-III the ability of HDPs, derived either from the C-terminus of human thrombin (HVF18, GKY25) or human tissue factor pathway inhibitor 2 (EDC34), to modulate innate immune responses caused by bacteria or lipopolysaccharide (LPS), were investigated. Thrombin-derived peptides significantly blocked LPS-induced responses including tissue-factor driven coagulation in vitro and in vivo and thereby improved survival in experimental animal models of LPS-induced shock and Pseudomonas aeruginosa sepsis (Paper I). The data in Paper II, demonstrate that the inhibition of these pro-inflammatory responses by GKY25, is not solely dependent on extracellular LPS-scavenging, but involves interactions with macrophages and monocytes. The TFPI-2 peptide (EDC34) also significantly modulated the coagulation cascade in vitro and in vivo and efficiently killed bacteria by enhancing complementmediated killing. The combination of these functions lead to improved survival in experimental models of E. coli or Pseudomonas sepsis (Paper III). In conclusion, the discovered immunomodulatory properties of these HDPs clearly indicate their potential for the development of new treatments for bacterial infections. Finally, a novel role of the abundant plasma protein heparin cofactor II (HCII) in host defence was discovered. HCII belongs to the class of serine proteinase inhibitors (serpins) and specifically inhibits human thrombin. However, its precise physiological role remained enigmatic. Paper IV shows that cleavage of HCII by human neutrophil elastase induces a conformational change in the HCII molecule, thereby uncovering a previously unknown antibacterial function of HCII

Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides

Effects of linear amphiphilicity on membrane interactions of antimicrobial peptides were investigated by ellipsometry, dual polarization interferometry, fluorescence spectroscopy, light scattering, and circular dichroism. In doing so, the thrombin-derived GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) was compared to WFF25 (WFFFYYLIIGGGVVTHQQRKKKKDE) of identical composition, but with amino acids sorted according to hydrophobicity, the latter peptide thus displaying pronounced linear amphiphilicity. In addition, GKY25d (GKYG(f) YTH(v) FRL(k) KWI(q) KVI(d) QFGE; with an identical sequence but with selected D-amino acid substitutions) was included as a control peptide, for which conformationally induced (helix-related) amphiphilicity was suppressed. Through its pronounced linear amphiphilicity, WFF25, but not the less amphiphilic GKY25 and GKY25d, forms aggregates in solution. Through its terminal W/F stretch, WFF25 also displays pronounced selectivity, with higher membrane binding and liposome rupture than GKY25 and GKY25d for anionic membranes, but suppressed peptide insertion and lytic effects for zwitterionic ones. In addition, WFF25 binds extensively to anionic polyelectrolyte components in bacterial membranes, i.e., lipopolysaccharide and lipoteichoic acid, resulting in reduced antimicrobial effects through peptide scavenging, not seen for the less amphiphilic GKY25 and GKY25d peptides. Taken together, the results thus demonstrate a series of striking effects for highly amphiphilic peptides, which need to be recognized in the development of such compounds as potential peptide therapeutics

An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo.

Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock

Structure-Activity Studies and Therapeutic Potential of Host Defense Peptides of Human Thrombin

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length-and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (> 12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest

Hepatocyte-Targeted Expression by Integrase-Defective Lentiviral Vectors Induces Antigen-Specific Tolerance in Mice with Low Genotoxic Risk

Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity. Conclusion: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis. (hepatology 2011;

Teledermatology: Comparison of Store-and-Forward Versus Live Interactive Video Conferencing

A decreasing number of dermatologists and an increasing number of patients in Western countries have led to a relative lack of clinicians providing expert dermatologic care. This, in turn, has prolonged wait times for patients to be examined, putting them at risk. Store-and-forward teledermatology improves patient access to dermatologists through asynchronous consultations, reducing wait times to obtain a consultation. However, live video conferencing as a synchronous service is also frequently used by practitioners because it allows immediate interaction between patient and physician. This raises the question of which of the two approaches is superior in terms of quality of care and convenience. There are pros and cons for each in terms of technical requirements and features. This viewpoint compares the two techniques based on a literature review and a clinical perspective to help dermatologists assess the value of teledermatology and determine which techniques would be valuable in their practice

Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy

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A Face-Aging App for Smoking Cessation in a Waiting Room Setting: Pilot Study in an HIV Outpatient Clinic

Background: There is strong evidence for the effectiveness of addressing tobacco use in health care settings. However, few smokers receive cessation advice when visiting a hospital. Implementing smoking cessation technology in outpatient waiting rooms could be an effective strategy for change, with the potential to expose almost all patients visiting a health care provider without preluding physician action needed. Objective: The objective of this study was to develop an intervention for smoking cessation that would make use of the time patients spend in a waiting room by passively exposing them to a face-aging, public morphing, tablet-based app, to pilot the intervention in a waiting room of an HIV outpatient clinic, and to measure the perceptions of this intervention among smoking and nonsmoking HIV patients. Methods: We developed a kiosk version of our 3-dimensional face-aging app Smokerface, which shows the user how their face would look with or without cigarette smoking 1 to 15 years in the future. We placed a tablet with the app running on a table in the middle of the waiting room of our HIV outpatient clinic, connected to a large monitor attached to the opposite wall. A researcher noted all the patients who were using the waiting room. If a patient did not initiate app use within 30 seconds of waiting time, the researcher encouraged him or her to do so. Those using the app were asked to complete a questionnaire. Results: During a 19-day period, 464 patients visited the waiting room, of whom 187 (40.3%) tried the app and 179 (38.6%) completed the questionnaire. Of those who completed the questionnaire, 139 of 176 (79.0%) were men and 84 of 179 (46.9%) were smokers. Of the smokers, 55 of 81 (68%) said the intervention motivated them to quit (men: 45, 68%;women: 10, 67%);41 (51%) said that it motivated them to discuss quitting with their doctor (men: 32, 49%;women: 9, 60%);and 72 (91%) perceived the intervention as fun (men: 57, 90%;women: 15, 94%). Of the nonsmokers, 92 (98%) said that it motivated them never to take up smoking (men: 72, 99%;women: 20, 95%). Among all patients, 102 (22.0%) watched another patient try the app without trying it themselves;thus, a total of 289 (62.3%) of the 464 patients were exposed to the intervention (average waiting time 21 minutes). Conclusions: A face-aging app implemented in a waiting room provides a novel opportunity to motivate patients visiting a health care provider to quit smoking, to address quitting at their subsequent appointment and thereby encourage physician-delivered smoking cessation, or not to take up smoking

Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A–producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis

A skin cancer prevention facial-aging mobile app for secondary schools in Brazil : appearance-focused interventional study.

Background: The incidence of melanoma is increasing faster than any other major cancer both in Brazil and worldwide. Southeast Brazil has especially high incidences of melanoma, and early detection is low. Exposure to ultraviolet (UV) radiation is a primary risk factor for developing melanoma. Increasing attractiveness is a major motivation among adolescents for tanning. A medical student-delivered intervention that takes advantage of the broad availability of mobile phones and adolescents? interest in their appearance indicated effectiveness in a recent study from Germany. However, the effect in a high-UV index country with a high melanoma prevalence and the capability of medical students to implement such an intervention remain unknown. Objective: In this pilot study, our objective was to investigate the preliminary success and implementability of a photoaging intervention to prevent skin cancer in Brazilian adolescents. Methods: We implemented a free photoaging mobile phone app (Sunface) in 15 secondary school classes in southeast Brazil. Medical students ?mirrored? the pupils? altered 3-dimensional (3D) selfies reacting to touch on tablets via a projector in front of their whole grade accompanied by a brief discussion of means of UV protection. An anonymous questionnaire capturing sociodemographic data and risk factors for melanoma measured the perceptions of the intervention on 5-point Likert scales among 356 pupils of both sexes (13-19 years old; median age 16 years) in grades 8 to 12 of 2 secondary schools in Brazil. Results: We measured more than 90% agreement in both items that measured motivation to reduce UV exposure and only 5.6% disagreement: 322 (90.5%) agreed or strongly agreed that their 3D selfie motivated them to avoid using a tanning bed, and 321 (90.2%) that it motivated them to improve their sun protection; 20 pupils (5.6%) disagreed with both items. The perceived effect on motivation was higher in female pupils in both tanning bed avoidance (n=198, 92.6% agreement in females vs n=123, 87.2% agreement in males) and increased use of sun protection (n=197, 92.1% agreement in females vs n=123, 87.2% agreement in males) and independent of age or skin type. All medical students involved filled in a process evaluation revealing that they all perceived the intervention as effective and unproblematic, and that all pupils tried the app in their presence. Conclusions: The photoaging intervention was effective in changing behavioral predictors for UV protection in Brazilian adolescents. The predictors measured indicated an even higher prospective effectiveness in southeast Brazil than in Germany (>90% agreement in Brazil vs >60% agreement in Germany to both items that measured motivation to reduce UV exposure) in accordance with the theory of planned behavior. Medical students are capable of complete implementation. A randomized controlled trial measuring prospective effects in Brazil is planned as a result of this study