Antimicrobial peptides derived from growth factors

Growth factors, comprising diverse protein and peptide families, are involved in a multitude of developmental processes, including embryogenesis, angiogenesis, and wound healing. Here we show that peptides derived from HB-EGF, amphiregulin, hepatocyte growth factor, PDGF-A and PDGF-B, as well as various FGFs are antimicrobial, demonstrating a previously unknown activity of growth factor-derived peptides. The peptides killed the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis, as well as the fungus Candida albicans. Several peptides were also active against the Gram-positive S. aureus. Electron microscopy analysis of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. Furthermore, HB-EGF was antibacterial per se, and its epitope GKRKKKGKGLGKKRDPCLRKYK retained its activity in presence of physiological salt and plasma. No discernible hemolysis was noted for the growth factor-derived peptides. Besides providing novel templates for design of peptide-based antimicrobials, our findings demonstrate a previously undisclosed link between the family of growth factors and antimicrobial peptides, both of which are induced during tissue remodelling and repair.     

Growth factors: Regulation of normal and neoplastic growth

This paper presents an overview of current trends in growth factor research. The first part of the review considers the current classification of growth factors and their receptors. A model of cell proliferation regulation by growth factors is then presented. The final section reviews the latest concepts of the involvement of growth factors in the development of neoplasia.     

Antimicrobial activity of peptides derived from enzymatic hydrolysis of goat milk caseins

In this study, peptide fragments were produced from goat milk casein by proteolytic enzymes trypsin and ficin and combination of both enzymes, and their antimicrobial activity was investigated. After enzymatic treatments, antimicrobial activity against both Gram-positive and Gram-negative bacteria was enhanced. The hydrolysates were passed through ultrafiltration. The obtained hydrolysate by ficin (molecular weight (MW) <3 kDa) showed the highest antimicrobial activity and was selected for further purification by reversed-phase high-performance liquid chromatography (RP-HPLC). Twenty-seven peptide fractions were separated, and their antimicrobial activities were evaluated. The results showed that one of the fraction numbers (14) possessed the highest activity against Escherichia coli and Bacillus cereus. The results suggest that bioactive peptides obtained by digestion of goat milk caseins with ficin could be exploited to as natural antimicrobial agents in pharmaceutical industries     

Role of cytokines and growth factors in radioprotection

Cytokines and growth factors are growing groups of proteins that are responsible for the communication between cells of the immune system, hematopoietic cells, and other cell types. The cloning and large-scale production in a recombinant form of these agents in pharmacological quantities permitted investigations aimed at assessing the benefit they may provide in preserving and restoring functions of tissues compromised by irradiation. We have extensively examined past investigations which suggest that some cytokines and growth factors protect animals from radiation lethality when given prior to or after irradiation, and even in untreated animals, these cytokines serve in innate defenses against external stimuli. In contrast, some cytokines given before irradiation sensitize the animals to radiation lethality. Unfortunately, due to their adverse side effects, these cytokines were not found suitable as radioprotectors. Recent studies suggest that new approaches may bring cytokines and growth factors in clinic for radiation injury. The information and insight gained about therapeutic potential of cytokine manipulation will allow for more rational design of treatment protocols.     

鼻咽癌中生长因子/细胞因子mRNA差异表达分析

目的：考察鼻咽癌中差异表达的生长因子／细胞因子,推测鼻咽癌发生中可能发生的免疫机制。方法：采用ｃＤＮＡ阵列杂交技术,同时对９８个已知与肿瘤相关的生长因子／细胞因子在鼻咽癌、正常因组织及其他几种头颈部鳞癌的ｍＲＮＡ表达水平进行筛查,筛选出在鼻呖癌中差异表达的生长因子／细胞因子基因。结果：在鼻咽癌中上调的基因包括：早期生长应答蛋白１,肝癌来源生长因子,白介素１β,血小板来源生长因子Ａ链,干细胞因子,畸     

Antimicrobial peptides from human platelets

Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin beta-4 (Tbeta-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tbeta-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.     

Spatial gradients of chemotropic factors from immobilized patterns to guide axonal growth and regeneration

In this study, we investigated the effect of varying localized concentration gradients of NGF and Sema3A on the axonal outgrowth of embryonic chick DRG explants and primary neurons in vitro. Immobilized 2D NGF or Sema3A micropatterns were produced using photolithography on tissue culture cover slips. Two distinct regions were identified: slow, with little or no change in concentration of chemotropic factor; and steep, with a transition from low to high. The direction of axonal outgrowth was defined as proximal or distal, with proximal growing towards the higher concentration of immobilized NGF/Sema3A and vice versa for distal. Axons grew preferentially in the proximal direction when explants were seeded onto steep NGF, and distally in response to steep Sema3A. On slow NGF, or on slow Sema3A there was no difference in the directional specificity of axonal outgrowth. DRG primary neurons seeded onto steep NGF migrated proximally, whereas neurons seeded onto slow NGF migrated in all directions. Conversely, neurons seeded onto steep or slow Sema3A did not extend any axons. Our 2D immobilized micropatterns of chemotropic factors show promise for further development of in vitro nerve tissue engineering studies.     

Micro-structured,spontaneously eroding hydrogels accelerate endothelialization through presentation of conjugated growth factors

Growth factors represent highly potent and highly efficacious means of communication to cells. At the same time, these proteins are fragile and relatively small sized – rendering their immobilization and controlled release from biomaterials challenging. In this work, we establish a method to incorporate growth factors into the physical hydrogels based on poly(vinyl alcohol), PVA. The latter have a long and successful history of biomedical applications and approval for diverse use in human patients, but are also characterized with scant opportunities for bioconjugation and functionalization. Herein, we develop the conjugation of growth factors to the micro-structured, spontaneously eroding physical hydrogels based on PVA. Protein conjugation was elaborated using model substrates, albumin and lysozyme, which aided to reveal specificity of chemical reactions and benign, non-harmful nature of the established protocols. Surface-adhered format of hydrogel analyses allowed to quantify bioconjugation reactions and enzymatic activity of the immobilized proteins and to visualize the hydrogels with immobilized cargo. In cell culture, immobilized growth factors were effective in communicating to adhering cells and specifically enhanced proliferation rates of the cells containing the corresponding receptors. At the same time, proliferation of the cells devoid of these receptors was un-altered.     

Role of growth factors on periodontal repair

Regeneration of periodontal structures lost during periodontal diseases constitutes a complex biological process regulated among others by interactions between cells and growth factors. Growth factors are biologically active polypeptides affecting the proliferation, chemotaxis and differentiation of cells from epithelium, bone and connective tissue. They express their action by binding to specific cell-surface receptors present on various target cells including osteoblasts, cementoblasts and periodontal ligament fibroblasts. The observation that growth factors participate in all cell functions led to exogenous application during periodontal tissue repair aiming to their use as an alternative therapeutic approach to periodontal therapy. Cell types and cultures conditions, dose, carrier materials, application requirements are of critical importance in the outcome of periodontal repair. The purpose of this article is to review the literature with respect to the biological actions of PDGF, TGF, FGF, IGF and EGF on periodontal cells and tissues, which are involved in periodontal regeneration.     

Pancreatic cancer: the potential clinical relevance of alterations in growth factors and their receptors

Molecular alterations play a key role in the pathogenesis of gastrointestinal cancers. In the present paper we describe relevant molecular alterations in human pancreatic adenocarcinomas. Overexpression of growth factor receptors (EGF receptor, c-erbB2, c-erbB3, TGF receptor I–III), growth factors (EGF, TGF, TGF-1-3, aFGF, bFGF), adhesion molecules (ICAM-1, ELAM-1) and gene mutations (p53, K-ras, DCC, APC) are present in a significant number of these tumors. These changes stimulate tumor growth and enhance the metastatic behavior of pancreatic cancer cells and thereby may contribute to shorter postoperative survival following tumor resection.Abbreviations EGF Epidermal growth factor - ELAM Endothelial leukocyte adhesion molecule - aFGF Acidic fibroblast growth factor - bFGF Basic fibroblast growth factor - HER Human EGF-receptor - ICAM Intercellular adhesion molecule - TGF Transforming growth factor     

Therapeutic foam scaffolds incorporating biopolymer-shelled mesoporous nanospheres with growth factors

A novel therapeutic scaffolding system of engineered nanocarriers within a foam matrix for the long-term and sequential delivery of growth factors is reported. Mesoporous silica nanospheres were first functionalized to have an enlarged mesopore size (12.2 nm) and aminated surface, which was then shelled by a biopolymer, poly(lactic acid) (PLA) or poly(ethylene glycol) (PEG), via electrospraying. The hybrid nanocarrier was subsequently combined with collagen to produce foam scaffolds. Bovine serum albumin (BSA), used as a model protein, was effectively loaded within the enlarged nanospheres. The biopolymer shell substantially prolonged the release period of BSA (2–3 weeks from shelled nanospheres vs. within 1 week from bare nanospheres), and the release rate was highly dependent on the shell composition (PEG > PLA). Collagen foam scaffolding of the shelled nanocarrier further slowed down the protein release, while enabling the incorporation of a rapidly releasing protein, which is effective for sequential protein delivery. Acidic fibroblast growth factor (aFGF), loaded onto the shelled-nanocarrier scaffolds, was released over a month at a highly sustainable rate, profiling a release pattern similar to that of BSA. The biological activity of the aFGF was evidenced by the significant proliferation of osteoblastic precursor cells in the aFGF-releasing scaffolds. Furthermore, the aFGF-delivering scaffolds implanted in rat subcutaneous tissue for 2 weeks showed a substantially enhanced invasion of fibroblasts with a homogeneous population. Taken together, it is concluded that the biopolymer encapsulation of mesoporous nanospheres effectively prolongs the release of growth factors over weeks to a month, providing a nanocarrier platform for a long-term growth factor delivery. Moreover, the foam scaffolding of the nanocarrier system is a potential therapeutic three-dimensional matrix for cell culture and tissue engineering.     

Glycosaminoglycan synthesis and shedding induced by growth factors are cell and compound specific

The interactions between growth factors and sulphated glycosaminoglycans (GAG) have been extensively studied. The aim of this study is to investigate if growth factors would show specificity of action on the synthesis and shedding of sulphated GAG, using two different cell lines: endothelial and smooth muscle cells. The cells were grown in the presence or absence of growth factors: EGF, FGF2, VEGF121, VEGF165. Transfection assays were also performed using recombinant pcDNA3.1, containing VEGF165 cDNA. In order to analyse the different types of GAG the cells were metabolically labelled with [³⁵S]-sulphate. At low doses, VEGF121 was the only growth factor able to increase both the synthesis and secretion of heparan sulphate (HS) in endothelial cells. Over expression of VEGF165 stimulated HS synthesis in both cells. The combined results showed that growth factors affect GAG synthesis in a cell specific and dose dependent manner.     

Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)

The beta-chain of the respiratory protein hemoglobin (Hbbeta), has recently been identified in novel sites, including mammalian macrophages and alveolar epithelium, as well as in gill microsomes of fish. However, the functional significance of extra-erythrocytically expressed hemoglobin has been unclear. Here we show inducible expression and upregulation of antimicrobial peptides (AMPs) homologous to Hbbeta in the gill epithelium of channel catfish (Ictalurus punctatus) in response to parasitic (Ichthyophthirius multifiliis, ich) infection. One peptide (HbbetaP-1), while having activity against some fish bacterial pathogens (e.g., Aeromonas hydrophila), had especially potent antiparasitic activity that was specifically lethal (lytic) to the feeding (trophont) stage of ich and also appeared to accelerate the differentiation of trophonts. However, it had no apparent effect on either the disseminative (theront) or reproductive (tomont) stages, nor was it lytic to channel catfish erythrocytes. Fish experimentally challenged with ich confirmed that the HbbetaP-1 sequence was both transcribed and translated in skin and gill epithelium, the target tissues for ich. The Hb AMP concentration expressed in vivo appeared to be well within the antiparasitic concentrations measured in vitro. Our findings suggest that hemoglobin-derived AMPs might play a significant role in the non-specific immune response.     

深圳市7家医院常见病原菌耐药性分析

目的了解深圳市区级、街道人民医院常见病原菌的耐药情况,为临床合理使用抗菌药物提供依据。方法对深圳市7家区级、街道二级甲等医院2009年1～9月分离的1765株病原菌进行药敏试验,用WHONET5.5软件对数据进行统计分析。结果大肠埃希菌对广谱青霉素、头孢呋辛、复方新诺明的耐药率均〉60%,肺炎克雷伯菌对广谱青霉素、第一和第二代头孢菌素的耐药率〉40%,两种菌对碳青酶烯类的敏感率为95%～100%,对氨基糖苷类阿米卡星敏感率达90%～95%,产ESBLs率分别为44.6%和29.0%。铜绿假单胞菌对氨苄西林、氨苄西林/舒巴坦、第一和第二代头孢菌素的耐药率〉90%,对氨基糖苷类阿米卡星的敏感率为91.4%,对碳青酶烯类和氟喹诺酮类药物敏感率为70%～80%。未发现耐万古霉素的葡萄球菌。结论深圳市区级、街道人民医院常见病原菌对抗菌药物的耐药性普遍存在,应长期开展对病原菌的耐药性监测。     

Hyperplasia of epithelium adjacent to transitional cell carcinoma can be induced by growth factors through paracrine pathways

Hyperplasia of transitional cell epithelium adjacent to human transitional cell carcinomas (TCC) is a common finding in pathology. This hyperplasia may be a precancerous aberration. Alternatively, it has been suggested that the hyperplasia is due to paracrine action of tumour-derived growth factors. In this study we tested the latter hypothesis using the mouse tumorigenic TCC cell line NUC-1. Transplantation of NUC-1 tumour cells into the urinary bladder submucosa of syngeneic mice in vivo induced hyperplasia of normal adjacent urothelium in all tested mice. Implantation of normal mouse bladder mucosa did not induce urothelial hyperplasia. In vitro, conditioned medium of NUC-1 cells induced the proliferation of the mouse urothelial cell line g/G, which closely resembles normal urothelial cells. This induction was inhibited by transforming growth factor ₁ (TGF₁). Similarly, TGF₁ inhibited the fibroblast growth factor-1 (FGF-1) and FGF-2 induced proliferation of g/G cells. Chemico-physical examination, bioassays with conditioned media, and RNA analysis of NUC-1 cells revealed that these cells secreted a growth factor with FGF-like properties. These results indicate that epithelial hyperplasia surrounding carcinomas is not necessarily a precancerous aberration, but may result from direct paracrine action of tumour-derived growth factors.     

The development of a serum-free medium utilizing the interaction between growth factors and biomaterials

To promote clinical application of cartilage tissue engineering, we should establish a serum-free chondrocyte growth medium. The serum-free medium would increase the cell numbers by more than 20-fold within one week, which proliferation ability almost matches that of serum-based one. For that, we examined the combinations of growth factors and the methods to enhance their effects by making use of the interaction with biomaterials. From various growth factors that are contained within the serum, we made the cocktail of FGF-2 (100 ng/mL), insulin (5 μg/mL), EGF (10 pg/mL), PDGF (625 pg/mL) and TGF-β (5 pg/mL), which increased the chondrocyte numbers by approximately 3-fold for 7 days. Moreover, we used the biomaterials including albumin and hyaluronan as the carrier of those factors. By direct mixing of those factors with biomaterials before the administration to the medium, the medium containing those mixture showed the chondrocyte growth of approximately a 25-fold increase by day 10. In this medium, the FGF-2 or insulin concentration hardly decreased, and rather enhanced the activation of ERK. Due to the optimal usage of biomaterials, this serum-free medium will realize a constant harvest of chondrocytes and could contribute to the safety and quality in regenerative medicine.