Antitumor activity of bovine lactoferrin and its derived peptides against HepG2 liver cancer cells and Jurkat leukemia cells.

Liver cancer and leukemia are the fourth and first causes, respectively, of cancer death in children and adults worldwide. Moreover, cancer treatments, although beneficial, remain expensive, invasive, toxic, and affect the patient's quality of life. Therefore, new anticancer agents are needed to improve existing agents. Because bovine lactoferrin (bLF) and its derived peptides have antitumor properties, we investigated the anticancer effect of bLF and LF peptides (LFcin17-30, LFampin265-284 and LFchimera) on liver cancer HepG2 cells and leukemia Jurkat cells. HepG2 and Jurkat cells were incubated with bLF and LF peptides. Cell proliferation was quantified by an MTT assay, and cell morphology and damage were visualized by light microscopy or by phalloidin-TRITC/DAPI staining. The discrimination between apoptosis/necrosis was performed by staining with Annexin V-Alexa Fluor 488 and propidium iodide, and the expression of genes related to apoptosis was analyzed in Jurkat cells. Finally, the synergistic interaction of bLF and LF peptides with cisplatin or etoposide was assessed by an MTT assay and the combination index. The present study demonstrated that bLF and LF peptides inhibited the viability of HepG2 and Jurkat cells, inducing damage to the cell monolayer of HepG2 cells and morphological changes in both cell lines. bLF, LFcin17-30, and LFampin265-284 triggered apoptosis in both cell lines, whereas LFchimera induced necrosis. These results suggested that bLF and LF peptides activate apoptosis by increasing the expression of genes of the intrinsic pathway. Additionally, bLF and LF peptides synergistically interacted with cisplatin and etoposide. In conclusion, bLF and LF peptides display anticancer activity against liver cancer and leukemia cells, representing an alternative or improvement in cancer treatment.

Novel Antiamoebic Tyrocidine-Derived Peptide against Brain-Eating Amoebae.

Acanthamoeba castellanii (A. castellanii) can cause Acanthamoeba keratitis, a sight-threatening infection, as well as a fatal brain infection termed granulomatous amoebic encephalitis, mostly in immunocompromised individuals. In contrast, Naegleria fowleri (N. fowleri) causes a deadly infection involving the central nervous system, recognized as primary amoebic encephalitis, mainly in individuals partaking in recreational water activities or those with nasal exposure to contaminated water. Worryingly, mortality rates due to these infections are more than 90%, suggesting the need to find alternative therapies. In this study, antiamoebic activity of a peptide based on the structure of the antibiotic tyrocidine was evaluated against A. castellanii and N. fowleri. The tyrocidine-derived peptide displayed significant amoebicidal efficacy against A. castellanii and N. fowleri. At 250 µg/mL, the peptide drastically reduced amoebae viability up to 13% and 21% after 2 h of incubation against N. fowleri and A. castellanii., whereas, after 24 h of incubation, the peptide showed 86% and 94% amoebicidal activity against A. castellanii and N. fowleri. Furthermore, amoebae pretreated with 100 µg/mL peptide inhibited 35% and 53% A. castellanii and N. fowleri, while, at 250 µg/mL, 84% and 94% A. castellanii and N. fowleri failed to adhere to human cells. Amoeba-mediated cell cytopathogenicity assays revealed 31% and 42% inhibition at 100 µg/mL, while at 250 µg/mL 75% and 86% A. castellanii and N. fowleri were inhibited. Assays revealed inhibition of encystation in both A. castellanii (58% and 93%) and N. fowleri (73% and 97%) at concentrations of 100 and 250 µg/mL respectively. Importantly, tyrocidine-derived peptide depicted minimal cytotoxicity to human cells and, thus, may be a potential candidate in the rational development of a treatment regimen against free-living amoebae infections. Future studies are necessary to elucidate the in vivo effects of tyrocidine-derived peptide against these and other pathogenic amoebae of importance.

Natural and Synthetic Sortase A Substrates Are Processed by Staphylococcus aureus via Different Pathways.

Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.

Lubricating properties of chewing stimulated whole saliva from patients suffering from xerostomia.

OBJECTIVES: The study aimed to quantify the lubricating properties of chewing stimulated whole saliva from healthy controls (n = 22), from patients suffering from primary Sjögren's syndrome (n = 37) and from patients undergoing head-and-neck radiotherapy (n = 34). MATERIALS AND METHODS: All participants had to complete the Xerostomia Inventory questionnaire to score dry mouth sensation. Lubrication was measured using an ex vivo tongue-enamel friction system in terms of Relief and Relief period. MUC5b and total protein concentrations of the saliva samples were measured by an enzyme-linked immunosorbent assay and a bicinchoninic acid assay, respectively. RESULTS: Relief of Sjögren's patients' saliva and post-irradiation patients' saliva was similar compared with healthy controls, but saliva from post-irradiation patients lubricated significantly better than saliva from Sjögren's patients. The Relief period was similar between the three groups. The Relief and Relief period were higher for saliva samples post-irradiation compared to pre-irradiation. MUC5b and total protein concentrations were comparable in all groups. MUC5b and total protein output were significantly lower in patients subjected to radiotherapy compared to saliva from healthy controls and pre-irradiation patients. MUC5b concentrations positively correlated with lubricating properties of post-irradiation patient saliva. CONCLUSIONS: The lubricating properties of patient saliva were not any worse than healthy controls. Lower flow rate leads to lower availability of saliva in the oral cavity and decreases the overall output of protein and MUC5b, which might result in an insufficient replenishing of the mucosal salivary film. CLINICAL RELEVANCE: An insufficient replenishing might underlie the sensation of a dry mouth and loss of oral function.

Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation.

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of ß-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.

Bovine lactoferrin and lactoferrin peptides affect endometrial and cervical cancer cell lines.

Cervical, uterine, and ovarian cancers are the most common malignancies of the female genital tract worldwide. Despite advances in prevention, early diagnosis, effective screening, and treatment programs, mortality remains high. Consequently, it is important to search for new treatments. The activity of bovine lactoferrin (bLF) and LF peptides against several types of cancer has been studied; however, only a few studies report the effect of bLF and LF peptides against cervical and endometrial cancers. In this study, we explored the effect of bLF as well as LF chimera and its constituent peptides LFcin17-30 and LFampin265-284 on the viability of cervical (HeLa, SiHa) and endometrial (KLE, HEC-1A) cancer cell lines. Cell proliferation was quantified with an MTT assay, cell morphological changes and damage were determined by Giemsa and phalloidin-TRITC and DAPI staining, and apoptotic and necrotic cells were identified by Alexa Fluor® 488 Annexin V and propidium iodide staining. Additionally, the effect of combinations of bLF and LF peptides with cisplatin was assessed. bLF and LF peptides inhibited the proliferation of uterine cancer cells and caused cellular morphological changes and damage to cell monolayers. bLF induced apoptosis, LFcin17-30 and LFampin265-284 induced apoptosis and necrosis, and LF chimera induced necrosis. Additionally, bLF and LF chimera showed an additive interaction with cisplatin against uterine cancer cells.

D-LL-31 enhances biofilm-eradicating effect of currently used antibiotics for chronic rhinosinusitis and its immunomodulatory activity on human lung epithelial cells.

Chronic rhinosinusitis (CRS) is a chronic disease that involves long-term inflammation of the nasal cavity and paranasal sinuses. Bacterial biofilms present on the sinus mucosa of certain patients reportedly exhibit resistance against traditional antibiotics, as evidenced by relapse, resulting in severe disease. The aim of this study was to determine the killing activity of human cathelicidin antimicrobial peptides (LL-37, LL-31) and their D-enantiomers (D-LL-37, D-LL-31), alone and in combination with conventional antibiotics (amoxicillin; AMX and tobramycin; TOB), against bacteria grown as biofilm, and to investigate the biological activities of the peptides on human lung epithelial cells. D-LL-31 was the most effective peptide against bacteria under biofilm-stimulating conditions based on IC50 values. The synergistic effect of D-LL-31 with AMX and TOB decreased the IC50 values of antibiotics by 16-fold and could eliminate the biofilm matrix in all tested bacterial strains. D-LL-31 did not cause cytotoxic effects in A549 cells at 25 µM after 24 h of incubation. Moreover, a cytokine array indicated that there was no significant induction of the cytokines involving in immunopathogenesis of CRS in the presence of D-LL-31. However, a tissue-remodeling-associated protein was observed that may prevent the progression of nasal polyposis in CRS patients. Therefore, a combination of D-LL-31 with AMX or TOB may improve the efficacy of currently used antibiotics to kill biofilm-embedded bacteria and eliminate the biofilm matrix. This combination might be clinically applicable for treatment of patients with biofilm-associated CRS.

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes.

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus.

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.

Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells.

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control-scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

DMBT1 inhibition of Pseudomonas aeruginosa twitching motility involves its N-glycosylation and cannot be conferred by the Scavenger Receptor Cysteine-Rich bacteria-binding peptide domain.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

A rapid, non-invasive tool for periodontitis screening in a medical care setting.

BACKGROUND: Since periodontitis is bi-directionally associated with several systemic diseases, such as diabetes mellitus and cardiovascular diseases, it is important for medical professionals in a non-dental setting to be able examine their patients for symptoms of periodontitis, and urge them to visit a dentist if necessary. However, they often lack the time, knowledge and resources to do so. We aim to develop and assess "quick and easy" screening tools for periodontitis, based on self-reported oral health (SROH), demographics and/or salivary biomarkers, intended for use by medical professionals in a non-dental setting. METHODS: Consecutive, new patients from our outpatient clinic were recruited. A SROH questionnaire (8 questions) was conducted, followed by a 30 s oral rinse sampling protocol. A complete clinical periodontal examination provided the golden standard periodontitis classification: no/mild, moderate or severe periodontitis. Total periodontitis was defined as having either moderate or severe. Albumin and matrix metalloproteinase-8 concentrations, and chitinase and protease activities were measured in the oral rinses. Binary logistic regression analyses with backward elimination were used to create prediction models for both total and severe periodontitis. Model 1 included SROH, demographics and biomarkers. The biomarkers were omitted in the analysis for model 2, while model 3 only included the SROH questionnaire. The area under the receiver operating characteristic curves (AUROCC) provided the accuracy of each model. The regression equations were used to create scoring algorithms, composed of the remaining predictors, each with its own weight. RESULTS: Of the 156 patients participating in this study, 67% were classified with total periodontitis and 33% had severe periodontitis. The models for total periodontitis achieved an AUROCC of 0.91 for model 1, 0.88 for model 2 and 0.81 for model 3. For severe periodontitis, this was 0.89 for model 1, 0.82 for model 2 and 0.78 for model 3. The algorithm for total periodontitis (model 2), which we consider valid for the Dutch population, was applied to create a freely accessible, web-based screening tool. CONCLUSIONS: The prediction models for total and severe periodontitis proved to be feasible and accurate, resulting in easily applicable screening tools, intended for a non-dental setting.

EphA2 Targeted Doxorubicin-Nanoliposomes for Osteosarcoma Treatment.

PURPOSE: To employ Doxorubicin-loaded liposomes, modified with YSA-peptide to target EphA2, to reduce adverse effects against primary bone cells and maximize toxicity against Saos-2 osteosarcoma cells. METHODS: PEGylated liposomes were prepared by thin film method using Dipalmitoylphosphatidylcholine (DPPC), cholesterol and distearylphosphatidylethanolamine-polyethyleneglycol conjugate (DSPE-mPEG) in 67.9:29.1:3 M ratios, and loaded with DOX (L-DOX) by pH-gradient method. Targeted liposomes (YSA-L-DOX), were prepared by conjugating YSA-peptide to DSPE-mPEG. Liposomes were physicochemically characterized and tested in cellular toxicity assays. RESULTS: YSA conjugation efficiency was >98%. Size and polydispersity index of both L-DOX and YSA-L-DOX were around 88 nm and 0.188, respectively. Both had similar zeta potential, and 85% DOX loading efficiencies. DOX release kinetics followed the Korsmeyer-Peppa model, and showed comparable release for both formulations from 1-8 h, and a plateau of 29% after 48 h. Both formulations could be stably stored for ≥6 months at 4°C in the dark. Toxicity assays showed a significant 1.91-fold higher cytotoxicity compared to free DOX in the Saos-2 cells, and 2-fold lesser toxicity in primary bone cells compared to the Saos-2 cells. Cellular uptake studies showed higher and more nuclear uptake in YSA-L-DOX compared to L-DOX treated cells. CONCLUSIONS: YSA-L-DOX vesicles might be effective for targeted treatment of osteosarcoma.

Saliva-Derived Host Defense Peptides Histatin1 and LL-37 Increase Secretion of Antimicrobial Skin and Oral Mucosa Chemokine CCL20 in an IL-1α-Independent Manner.

Even though skin and oral mucosae are continuously in contact with commensal and opportunistic microorganisms, they generally remain healthy and uninflamed. Host defense peptides (HDPs) make up the body's first line of defense against many invading pathogens and are involved in the orchestration of innate immunity and the inflammatory response. In this study, we investigated the effect of two salivary HDPs, LL-37 and Hst1, on the inflammatory and antimicrobial response by skin and oral mucosa (gingiva) keratinocytes and fibroblasts. The potent antimicrobial chemokine CCL20 was investigated and compared with chemokines CCL2, CXCL1, CXCL8, and CCL27 and proinflammatory cytokines IL-1α and IL-6. Keratinocyte-fibroblast cocultures showed a synergistic increase in CCL20 secretion upon Hst1 and LL-37 exposure compared to monocultures. These cocultures also showed increased IL-6, CXCL1, CXCL8, and CCL2 secretion, which was IL-1α dependent. Secretion of the antimicrobial chemokine CCL20 was clearly IL-1α independent. These results indicate that salivary peptides can stimulate skin as well as gingiva cells to secrete antimicrobial chemokines as part of the hosts' defense to counteract infection.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

The scavenging capacity of DMBT1 is impaired by germline deletions.

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

Incorporation of a Valine-Leucine-Lysine-Containing Substrate in the Bacterial Cell Wall.

The emergence of antibiotic-resistant bacteria is a major public health threat, and therefore novel antimicrobial targets and strategies are urgently needed. In this regard, cell-wall-associated proteases are envisaged as interesting antimicrobial targets due to their role in cell wall remodeling. Here, we describe the discovery and characteristics of a protease substrate that is processed by a bacterial cell-wall-associated protease. Stationary-phase grown Gram-positive bacteria were incubated with fluorogenic protease substrates, and their cleavage and covalent incorporation into the cell wall was analyzed. Of all of the substrates used, only one substrate, containing a valine-leucine-lysine (VLK) motif, was covalently incorporated into the bacterial cell wall. Linkage of the VLK-peptide substrate appeared unrelated to sortase A and B activity, as both wild-type and sortase A and B knock out Staphylococcus aureus strains incorporated this substrate into their cell wall with comparable efficiency. Additionally, the VLK-peptide substrate showed significantly higher incorporation in the cell wall of VanA-positive Enterococcus faecium strains than in VanB- and vancomycin-susceptible isolates. In conclusion, the VLK-peptide substrate identified in this study shows promise as a vehicle for targeting antimicrobial compounds and diagnostic contrast agents to the bacterial cell wall.

Salivary agglutinin is the major component in human saliva that modulates the lectin pathway of the complement system.

Saliva interacts with blood after mucosal damage or leakage of gingival crevicular fluid. Surface-adsorbed salivary agglutinin (SAG) activates the lectin pathway (LP) of the complement system via mannose-binding lectin, while SAG in solution inhibits complement activation. In the present study we investigated if, next to SAG, whole and glandular saliva itself and other salivary glycoproteins activate or inhibit the LP. Complement activation was measured by detecting C4 deposition on microtiter plates coated with saliva or purified proteins. Complement inhibition was measured after incubating serum with saliva or proteins in microtiter plates coated with mannan, an LP activator. Adsorbed whole, sublingual and submandibular saliva showed LP-dependent complement activation. Blood group secretors, but not non-secretors, activated the LP. Saliva of both secretors and non-secretors inhibited C4 deposition on mannan. After depletion of SAG, saliva no longer inhibited the LP. Other salivary proteins, including amylase, MUC5B and histatin 2, did not activate or inhibit the LP. Surface-adsorbed whole saliva and glandular saliva samples activate the LP of complement, depending on the presence of SAG and the secretor status of the donor. In solution, saliva inhibits the LP, depending on the presence of SAG, but independent of the secretor status.

The Influence of Chronic Wound Extracts on Inflammatory Cytokine and Histatin Stability.

Chronic ulcers represent a major health burden in our society. Despite many available therapies, a large number of ulcers do not heal. Protein based therapies fail in part due to proteolytic activity in the chronic wound bed. The aim of this in vitro study was to determine whether typical inflammatory cytokines and human salivary histatins remain stable when incubated with chronic wound extracts. Furthermore we determined whether a short exposure of histatins or cytokines was sufficient to exert long term effects on fibroblast migration. Stability of human recombinant cytokines IL-6 and CXCL8, and histatin variants (Hst1, Hst2, cyclic Hst1, minimal active domain of Hst1) in the presence of chronic wound extracts isolated from non-healing ulcers, was monitored by capillary zone electrophoresis. Migration-stimulating activity was assessed using a dermal fibroblast wound healing scratch assay. Histatins and cytokines stayed stable in saline for > 24 h at 37°C, making them ideal as an off-the-shelf product. However, incubation with chronic wound extracts resulted in serious breakdown of Hst1 and Hst2 (~50% in 8 h) and to lesser extent cyclic Hst1 and the minimal active domain of Hst1 (~20% in 8 h). The cytokines IL-6 and CXCL8 were more stable in chronic wound extracts (~40% degradation in 96 h). An initial 8-hour pulse of histatins or cytokines during a 96-hour study period was sufficient to stimulate fibroblast migration equally well as a continuous 96-hour exposure, indicating that they may possibly be used as novel bioactive therapeutics, exerting their activity for up to four days after a single exposure.