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To investigate whether the addition of human recombinant epidermal growth factor (h-EGF) to 2% carboxymethyl cellulose gel is more effective in diabetic wound healing than standard treatment, a pilot, double-blind, randomized and controlled clinical trial with therapeutic intervention was performed at a university hospital. The sample consisted of 25 patients (14 in the intervention group that used rh-EGF and 11 in the control group that used 2% carboxymethyl cellulose gel). Data were tabulated in SPSS and analysed by intention to treat, without loss or exclusion of participants. Twenty-five subjects participated with a mean age of 60.6 years, a predominance of males in both groups and 100% prevalence of type-2 diabetes. Within 12 weeks, complete wound healing occurred in three ulcers in the intervention group versus one ulcer in the control group. The percent reduction in the wound area was significantly higher in the intervention group than in the control group (p = 0.049). Concerning the types of tissue, an increase in granulation and epithelial tissue and a reduction in exudate levels were observed in both groups. Decreased slough occurred only in the intervention group. No participant experienced serious or local adverse events during the study period. This study shows that h-EGF is effective, with a statistically significant reduction in wound area, improvement of tissue quality, and safe treatment of chronic wounds. In addition, this study demonstrated that blinding of participants during research using h-EGF is feasible.

An analysis of modern scientific and practical literature leaves no doubt that among the many problems of a dentist, an important place is occupied by the optimization of wound healing of the oral mucosa in case of injuries. Immunodeficiencies of various origins, elderly and senile age, diabetes mellitus, and severe pathology of the cardiovascular system, hypoxic conditions and other somatic diseases reduce the repair potential of the oral mucosa. In addition, all types of metabolism are disrupted, synthesis and humoral transport are reduced, as well as the secretion of growth factors with saliva and their content in the blood and tissue. The healing process of the oral mucosa is slowed down by bacterial colonization of the wound surface, which can increase the area of inflammation. A common method of treating such patients in a dental clinic is the use of agents aimed at regenerative processes in tissues. Along with the positive qualities of modern drugs (good fixation, comfort when used in the oral cavity, wound healing, bactericidal effect on the damaged mucous membrane, reducing pain, accelerating the processes of epithelialization and scar organization), a noticeable weakening of their action in immunodeficient conditions should be noted. Based on this, it is advisable to use recombinant epidermal growth factor (EGFR) in the treatment of traumatic lesions of the oral mucosa, including those caused by the use of orthopedic structures, surgical interventions, especially those accompanied by conditions involving a decrease in the immune status.

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids. Fil: Morgenfeld, Mauro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Vater, Catalina Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Alfano, Edgardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Boccardo, Noelia Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

New high-molecular-weight contrast agents based on polyamidoamine (PAMAM) dendrimers for targeted imaging of malignant tumors characterized by overexpression of human epidermal growth factor receptor (EGFR) and human alpha-fetoprotein receptor (RECAF) were designed. Conjugates of second (G2) and third (G3) generation polyamidoamine dendrimers with 1,4,7,10-tetraazocyclodecane-1,4,7,10-tetraacetic acid (DOTA) were obtained. The quantitative composition of the conjugates was determined by 1HNMR spectroscopy. It was shown that four out of the 16 terminal NH2 groups in G2-DOTA and nine out of the 32 groups in G3-DOTA were modified with DOTA. The morphology, size, and charge of the synthesized macromolecules were characterized by dynamic light scattering and electrophoresis. Gadolinium(III) was loaded into the conjugates and the Gd content was determined by atomic emission spectroscopy. For increasing the selectivity of accumulation in the tumor cells, two recombinant proteins able to bind selectively to EGFR and RECAF, namely, human recombinant epidermal growth factor (rEGF) and human recombinant 3rd domain of alpha-fetoprotein (3dAFPpG), were conjugated with G2 and G3 dendrimers. The conjugates containing vector molecules were mainly accumulated via clathrin-dependent endocytosis, whereas G2-DOTA and G3-DOTA were absorbed via caveolin-dependent endocytosis and macropinocytosis. The dendrimer conjugates with vector molecules were intensely accumulated in A549 cells characterized by high expression of EGFR (Herl) and RECAF, whereas the accumulation of conjugates in the control K562 cells (with low expression of Her1) and in the CD14− population of human unstimulated mononuclear white blood cells was insignificant. The 3dAFPpG-conjugated dendrimers were partly recycled. All synthesized conjugates had a rather low toxicity in the range of 350–450 µmol L−1 (IC50).

Decreased neurogenesis after brain exposure to ionizing radiation is linked to neurocognitive impairments. Using transgenic mouse models, we previously showed that abrogation of radiation-induced senescence, or apoptosis, can partially rescue neurogenesis in the subventricular and hippocampus regions. Here, we evaluate whether the injection of recombinant epidermal growth factor (rEGF) or mesenchymal stromal cells (MSC) engineered to secrete EGF (MSC-EGF) can preserve neurogenesis. Using doublecortin (Dcx) expression and BrdU incorporation assays, we found that the injection of rEGF into the subventricular zone (SVZ) promotes neurogenesis, despite increasing apoptosis, in the brain of irradiated mice. The effect of rEGF was mostly localized, as Dcx expression was not induced in the hippocampus region and limited in the contralateral SVZ. Surprisingly, the injection of bone marrow-derived MSC alone, or secreting EGF, did not result in increased neurogenesis despite the fact that part of the MSC survived a few weeks after injection. Our results suggest that only a supraphysiological concentration of rEGF can promote neurogenesis, likely through a direct mitogenic effect.

OBJECTIVE To test whether or not topically administered recombinant human epidermal growth factor (rhEGF) accelerates the early healing phase of oral soft tissue wounds. METHODS One day following the creation of palatal defects (n = 6/animal), 14 dogs were allocated to one of the following five groups: spontaneous healing (SH), vehicle ointment (V), vehicle ointment + rhEGF at concentrations of 1 μg/g (EGF1), 10 μg/g (EGF10) or 50 μg/g (EGF50). Topical administration of ointments was repeated twice per day until sacrifice at days 8 and 16. Wound area was clinically monitored. Keratinocytes proliferation (Ki67-immunolabelling), inflammatory response (IR) and areas of collagen (C) and granulation tissue (GT) were histologically measured. Kruskal-Wallis test with Dunnett correction was used for multiple group statistical comparisons. RESULTS Clinically, in comparison with SH, a significantly smaller wound area was observed in groups EGF1 and EGF10 at day 8 (p

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

OBJECTIVES This study aims to compare the histological healing process and gene expression profile in tendon after the administration of human recombinant epidermal growth factor (hrEGF) and platelet-rich plasma (PRP). MATERIALS AND METHODS A total of 24 mature New Zealand white rabbits (6-month-old; weight 2.5-3.0 kg) were used in the study. Patellar tendons of rabbits were tenotomized and then repaired. Rabbits were separated into three groups and repair areas were injected with 1 mL hrEGF in group 1 (n=9) and 1 mL PRP in group 2 (n=9). No injection was performed in group 3 (controls, n=6). Tissue samples were obtained from the repaired patellar tendons of three rabbits each from groups 1 and 2 and of two rabbits from group 3 at the end of the first, second, and fourth weeks, and these tissues were histologically and genetically assessed. Expression levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, and chemokine receptors (CXCR1, CXCR2) were examined. RESULTS The frequency of neovascularization was detected to be higher in group 1 compared to group 3 at the end of the second and fourth weeks (p=0.018 and p=0.009, respectively). Group 1 was detected to show more increase in terms of the prevalence of tenocytes (p=0.014 and p=0.009, respectively) at the end of the second week, and in terms of collagen intensity at the end of the fourth week (p=0.0018 and p=0.034, respectively) compared to groups 2 and 3. Highest levels of TNF-α, IL-6, and IL-8 were detected in group 1, followed by groups 2 and 3 at all time points. Highest CXCR2 gene expression was detected in group 1. CONCLUSION Compared to PRP, hrEGF caused more increase in healing tissue at neovascularization, tenocyte, fibroblast, collagen, and tissue macrophage levels; and higher levels of TNF-α, IL-6, IL8, and CXCR2. Intralesional hrEGF administration can effectively accelerate tendon healing.

The objective was to evaluate Staphylococcus aureus and Pseudomonas aeruginosa colonisation of wounds treated with recombinant epidermal growth factor (EGF) and platelet-rich plasma (PRP); to analyse the susceptibility profiles of S. aureus and P. aeruginosa isolates from wounds treated with EGF and PRP; and to describe the presence of infection in EGF-treated and PRP-treated wounds. Experimental study was performed using clinical specimens collected with swabs. Patients were treated with PRP and EGF in the outpatient clinic of a university hospital. Forty-three isolates were obtained from 31 patients, 41.9% (13/31) of whom had been treated with EGF and 58.0% (18/31) with PRP. Ten of the 43 isolates were identified as S. aureus, 60.0% (6/10) of which were isolated from PRP-treated wounds. Among the 33 P. aeruginosa isolates, 66.6% (22/33) were isolated from PRP-treated wounds. Regarding antimicrobial susceptibility, only one strain isolated from an EGF-treated wound was identified as methicillin-resistant S. aureus (MRSA). Among the P. aeruginosa isolates, one obtained from a patient treated with EGF was multidrug-resistant. Patients treated with EGF had no infections during the follow-up period, and there was a significant difference between the 1st and 12th week in wound infection improvement in patients treated with PRP (P = .0078).

Anastomotic leaks following intestinal operations may cause devastating effects on patients. Ischemia may also occur at the intestinal walls in the presence of strangulations. In this study, we examined the effects of human recombinant (Hr)-epidermal growth factor (EGF) given at a single intramural dose into the intestinal walls and daily intraperitoneal cavity on ischemia and the healing process of anastomosis.Sixteen male New Zeland white rabbits were randomly divided into four groups (n = 4 in each group). In Group 1, two different segments of ileum were identified and, then, transected and the free ends were sutured each other. In the other groups, ischemia was induced by ligating the mesenteric vascular arcade. After the ischemic induction, Group 2 received intramural injections of %0.9 saline, Group 3 received intramural injections of a single dose of EGF, and Group 4 received intramural and intraperitoneal injections of EGF. Bursting pressures and tissue hydroxyproline levels were analyzed. Necrosis, fibroblastic activity, collagen deposition and neovascularization were also studied.The mean levels of bursting pressures in Group 4 (148.6 ± 25.3 mmHg) were higher than Group 2 (70 ± 21.5 mmHg) (p = 0.001). The mean level of bursting pressures was not statistically significant between Group 1 (170.1 ± 35 mmHg) and Group 4 (p = 0.073). Hydroxyproline levels in Group 2 were lower than Groups 3 and 4. There was a statistically significant difference in the mucosal ischemia, mucosal healing and degree of adhesion, but not in the mural anastomotic healing among the groups.Intramural injection with daily intraperitoneal administration of low-dose EGF enhances the bursting pressure and collagen accumulation in ischemic anastomosis, improving many histological variables associated with ischemic intestinal anastomosis.

BACKGROUND & OBJECTIVES The wide reaching negative social impact of non-healing chronic diabetic ulcers is emphasised by the estimate that 70 million of the world population might be affected by diabetes by 2025 and these people will be physically and socially handicapped if they develop foot problems. A vital cog in the wheel of wound healing is Epidermal Growth Factor. Hence a novel approach for combating longstanding diabetic wounds is external application of growth factors. This study has been done with the view to evaluate the efficacy of Epidermal Growth Factor in accelerating the healing of diabetic foot ulcers and also to assess its tolerability. This study compares the Epidermal growth factor Versus the standard therapy of wound with antiseptic solution and moist saline dressing in healing of diabetic ulcer foot. METHODS Sixty cases of diabetic ulcers (Study group-30 and control group-30) were included in this study. Factors tabulated and analysed. RESULTS & OBSERVATIONS The overall rate of healing of ulcers in EGF group is 86.67% and in the control group is 66.67%. CONCLUSION The conclusion of this study is that recombinant Epidermal Growth Factor is effective in the healing of diabetic ulcers at a significantly higher rate.

Background: Major complications of diabetes mellitus include cardio vascular disease, chronic kidney disease, diabetic foot ulcers and non-healing wounds. Lack of knowledge and awareness has led to worsening of wounds which can involve deeper tissues and bones also. If treated on time majority of patients can be prevented from undergoing major debilitating surgical procedures such as amputation of toes, foot etc.Methods: In this proposed study, 60 cases of diabetic wounds were studied for healing who were randomly allocated into two groups of 30 patients each. Group A included topical recombinant epidermal growth factor (EGF) application and group B included conventional Betadine dressing for wound healing.Results: Anaemia had a significant role in delaying wound healing in group B patients. Blood sugars had no significant role in delaying wound healing in this study as patients in group A had mean FBS more than group B and still the wound healing was observed better in group A patients. Topical recombinant EGF resulted in faster wound healing than conventional dressings in diabetic wounds. 90% wound healing rates were seen with topical EGF dressings in comparison to 36.67% in conventional betadine dressings. Signs of wound healing i.e., early granulation, decreased discharge, early wound closure were seen faster in group A. Moreover, all patients in group A showed healthy granulation tissue by the end of our study.Conclusions: We concluded that topical recombinant EGF resulted in faster and better wound healing than conventional betadine dressing.

Objective: The aim of this study was to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. Methods: 36 male Sprague Dawley rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: 1- Sham control group where no local augmentation was made, 2- Local Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) augmentation group and 3- Local HA/β-TCP + EGF augmentation group. Rats were euthanized at 8 weeks post-surgery. 6 rats from each group were selected for manual palpation examination, micro-computed tomography analysis and histologic analysis; and the rest was used for biomechanical analysis. Results: Based on manual palpation, there was no fusion in the sham control group. Fusion rate was 33.3% in the HA/β-TCP group and 66.7% in the HA/β-TCP + EGF group (p = 0.085). Micro-CT results revealed that new bone formation was higher in the HA/β-TCP + EGF group (BV/TV: 40% vs. 65%) (p = 0.004). Histologically newly formed bone tissue was more pronounced in the EGF group and compacted and bridging bone spicules were observed. The median maximum bending moment values were 0.51 Nmm (0.42–0.59), 0.73 Nmm (0.49–0.88) and 0.91 Nmm (0.66–1.03) in the sham control, HA/β-TCP and HA/β-TCP + EGF groups, respectively (p = 0.013). The median stiffness values were 1.69 N/mm (1.12–2.18), 1.68 N/mm (1.13–2.74) and 3.10 N/mm (1.66–4.40) as in the previous order (p = 0.087). Conclusion: This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates. Our findings may provide insights to further studies, investigating EGF's clinical usage as an alternative fusion enhancer. Keywords: Epidermal growth factor, Spinal fusion, Bone formation, Posterolateral fusion, Rat model

To analyze compounded recombinant human epidermal growth factor (rhEGF) effectiveness on skin lesions through a case series.Multicentric series of skin lesions treated with topical rhEGF. Site: Patients from 56 different health professionals, three different countries, and two recruitment years.Seventy-seven patients with skin lesions, mean age of 63.15 years (min = 18, max = 95); 53.2% of patients were men and 46.8% were women; 47 of the lesions were ulcers (venous, arterial, and diabetic foot), others were surgical and traumatic wounds, burns, and scars. Most common pathologies were type 2 diabetes mellitus and chronic venous insufficiency. Mean previous evolution time before inclusion was 29.59 months.Cures using compounded topical rhEGF most commonly used rhEGF concentration: 30 μg/g; most used excipient: gel; average time between cures: 36 h (24-48); and mean follow-up: 6.6 weeks (max = 20).Lesions appearance, margins and bed, lesional size evolution, treatment subjective effectiveness, tolerability and comfort through professional oppinion.Qualitative assessment: effectiveness 8.65, tolerability 9.53, and comfort 8.86 (max = 10). Perilesional skin showed improvement in 93.5% of the cases, lesions margins and wound bed appearance improved in 92.2% of the cases, respectively. Wound area decreased a mean average of 66.7%. About 43.3% of included venous ulcers had a greater than 40% cure rate in 4 weeks.Heterogeneity of the included pathologies, limited time follow-up in some cases.Topical rhEGF in individualized formulation (compounding) seems to exhibit effectiveness, comfort, and tolerability. Further larger-size studies with experimental design will allow to establish more precise concentrations, indications, and clinical guidelines.

Introduction: Diabetic foot ulcers are one of the most frequent complications of diabetes; such ulcers cause an increase in the costs of the health care of the diabetic patient and can even cause disability due to amputation in the patient. Although a proportion of patients achieve a spontaneous closure of ulcers, others require medical or surgical treatment. Objective: To determine the cost-effectiveness of the intra- and perilesional application of recombinant human epidermal growth factor (rhEGF), as opposed to conventional therapy for the management of patients diagnosed with Wagner’s 3 or 4 diabetic foot ulcer in Colombia. Methodology: Using a Markov model, the process of care of a diabetic patient with diagnosis of Wagner’s 3 or 4 ulcer receiving conventional treatment, or intra- and perilesional rhEGF, is configured. The evaluation cycles of the treatments are weekly over a 5-year horizon and the outcomes evaluated are quality-adjusted life years (QALYs) and the number of amputations avoided by each treatment scheme, in addition to the total costs for treatments. Results: For the analysed base case, in the outcome of amputations, it was found that the factor presents 39 fewer amputations, in a cohort of 100 patients, compared with conventional treatment. Likewise, QALYs are 0.65 more with the use of rhEGF in an average patient. The estimated cost-utility ratio for the base case would be below the threshold established for Colombia. Conclusions: The intra- and perilesional application of rhEGF is a more effective therapeutic option than conventional therapy in the treatment of patients with Wagner’s 3 or 4 diabetic foot ulcers and is cost-effective, taking as an outcome the QALYs for Colombia from the perspective of the health system.

WOS: 000414976900026, PubMed ID: 29151322, Background/aim: Intralesional recombinant epidermal growth factor (EGF) is a new treatment approach for diabetic foot ulcer, approved in 2006. EGF therapy is given as an adjunct to the standard treatment regimen of antibiotics, surgery, and hyperbaric oxygen. EGF accelerates the healing of diabetic foot ulcers and reduces healing time. This single-center study was conducted to evaluate the outcomes of intralesional EGF therapy in patients with diabetic foot ulcers. Materials and methods: We present the data of the follow-up patients treated in our clinics. Fifteen patients with diabetic foot ulcers or infections, who had been followed up and treated in our clinics, were included in this retrospective study. All patients were administered intralesional injections of 75 mu g of EGF after treatment for infection on their diabetic foot ulcers, three times a week on alternate days. The patients were monitored with respect to treatment response and side effects of EGF. Results: Thirteen patients (86.7%) developed new granulation tissue, 10 patients (66.7%) had complete wound closure, and three patients (20%) showed partial wound closure. No serious side effects requiring discontinuation of EGF therapy were observed. A total of twenty-one bacterial agents were isolated in thirteen patients, and no bacterial growth was observed in the tissue cultures of two patients. Pseudomonas aeruginosa was the most common isolated infectious agent in the tissue cultures (n: 6, 28%). Conclusion: Intralesional injection of EGF on top of the standard treatment regimen appears to be a useful adjuvant therapy option in selected patients.

Apocrine sweat glands in bovine skin are involved in thermoregulation. Human, horse, and sheep sweat gland epithelial cells have been isolated and grown in vitro. The present study was conducted to identify a method to isolate bovine sweat glands and culture apocrine bovine sweat gland epithelial cells in vitro. Mechanical shearing, collagenase digestion, centrifugation, and neutral red staining were used to identify and isolate the apocrine glands from skin. Bovine sweat glands in situ and after isolation comprised 2 major cell types consisting of a single layer of cuboidal epithelial cells resting on a layer of myoepithelial cells. In situ, the glands were embedded in a collagen matrix primarily comprising fibroblasts, and some of these cells were also present in the isolated material. The isolated material was transferred to complete medium (keratinocyte serum-free medium, bovine pituitary extract, and human recombinant epidermal growth factor + 2.5% fetal bovine serum) in a T 25 flask (Falcon, Franklin Lakes, NJ) with media film and then incubated at 37°C for 24 h. After sweat glands adhered to the bottom of the flask, an additional 2 mL of complete medium was added and the medium was changed every 3 d. Isolated apocrine sweat glands and bovine sweat gland epithelial cells were immunostained for cytokeratin and fibroblast specific protein, indicating fibroblast-free cultures.

Heng Liu,1,2 Xiao Chen,1 Wei Xue,1 Chengchao Chu,2 Yu Liu,2 Haipeng Tong,1 Xuesong Du,1 Tian Xie,1 Gang Liu,2 Weiguo Zhang1,3 1Department of Radiology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 2State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, Fujian, 3Chongqing Clinical Research Center for Imaging and Nuclear Medicine, Chongqing, People’s Republic of China Abstract: The highly infiltrative and invasive nature of glioma cells often leads to blurred tumor margins, resulting in incomplete tumor resection and tumor recurrence. Accurate detection and precise delineation of glioma help in preoperative delineation, surgical planning and survival prediction. In this study, recombinant epidermal growth factor-like domain-1, derived from human coagulation factor VII, was conjugated to iron oxide nanoparticles (IONPs) for targeted glioma magnetic resonance (MR) imaging. The synthesized EGF1-EGFP-IONPs exhibited excellent targeting ability toward tissue factor (TF)-positive U87MG cells and human umbilical vein endothelial cells in vitro, and demonstrated persistent and efficient MR contrast enhancement up to 12 h for preclinical glioma models with high targeting specificity in vivo. They hold great potential for clinical translation and developing targeted theranostics against brain glioma. Keywords: epidermal growth factor-like domain-1, tissue factor, iron oxide nanoparticles, MRI, glioma

Background: Human recombinant epidermal growth factor has been shown to be effective in corneal healing when applied topically. The purpose of this preliminary study was to observe whether re-epithelization occurred in patients with non-healing corneal defects treated with a bandage contact lenses impregnated with epidermal growth factor. Design: Prospective non-comparative interventional case series study. Epidermal growth factor-impregnated bandage contact lenses (created through passive transfer of epidermal growth factor into hydrogel contact lenses of high water content) were used to passively release epidermal growth factor to the corneal surface of the damaged eye. Participants: Nine clinical patients who presented for tertiary care at the University of British Columbia Eye Care Centre at Vancouver General Hospital. Methods: All patients had clinically significant delayed corneal re-epithelization that had not healed despite standard treatments including conventional bandage contact lenses and topical medications. Causes of delayed re-epithelization varied from corneal injuries (e.g. alkali burns, recurrent corneal erosions) to recent corneal surgery (photorefractive keratectomy, phototherapeutic keratectomy, penetrating keratoplasty). Main Outcome Measures: Closure of wounds. Results: Re-epithelialization was seen in the corneas of seven of the nine patients within 8 days after insertion of the epidermal growth factor-treated bandage contact lens into the damaged eye. The drug delivery system appeared to be most effective in non-inflamed corneas. Conclusions: Preliminary results indicate that bandage contact lenses impregnated with epidermal growth factor may be helpful in promoting re-epithelization in corneas with delayed healing. Efficacy appears to be reduced for vascularized and significantly inflamed corneas.

Agonist and antagonist drugs acting on epidermal growth factor receptor (EGFR) signaling are emerging as a new possibility for pharmaceutical study and clinical manipulation of some skin and corneal disorders. EGFR activation appears to be effective in reducing the time of reepithelialization after corneal wound healing, with potential uses in penetrating keratoplasty, refractive surgery, alkali burns, diabetic keratopathy, keratopathy following chemotherapy, cornea transplantation, and dry eye. Most of the studies show therapeutic advantages of human recombinant epidermal growth factor (hrEGF) eye drops without showing adverse effects. In contrast, EGFR inhibition delays epithelial cell proliferation and stratification during corneal regeneration.The aim of this review is to summarize the most seminal discoveries and recent advances so as to clarify the role of the EGFR system in corneal physiology and pharmacology. Epidermal growth factor eye drops could be a first-choice treatment for promoting regeneration in numerous epithelial defects in the medium to long term.

Fernández‐Montequín JI, Valenzuela‐Silva CM, González Díaz O, Savigne W, Sancho‐Soutelo N, Rivero‐Fernández F, Sánchez‐Penton P, Morejón‐Vega L, Artaza‐Sanz H, García‐Herrera A, González‐Benavides C, Hernández‐Cañete CM, Vázquez‐Proenza A, Berlanga‐Acosta J, López‐Saura PA, for the Cuban Diabetic Foot Study Group. Intra‐lesional injections of recombinant human epidermal growth factor promote granulation and healing in advanced diabetic foot ulcers: multicenter, randomised, placebo‐controlled, double‐blind study. A multicenter, double‐blind, placebo‐controlled trial was carried out to evaluate the intra‐lesional infiltration of recombinant epidermal growth factor (EGF) in Wagner's grade 3 or 4 diabetic foot ulcers (DFUs). Subjects (149) were randomised to receive EGF (75 or 25 µg) or placebo, three times per week for 8 weeks and standard good wound care. The main endpoint was granulation tissue covering ≥ 50% of the ulcer at 2 weeks. It was achieved by 19/48 controls versus 44/53 in the 75 µg group [odds ratio (OR): 7·5; 95% confidence interval (CI): 2·9–18·9] and 34/48 in the 25 µg group (OR: 3·7; 1·6–8·7). Secondary outcome variables such as end‐of‐treatment complete granulation response (28/48 controls, 46/53 with 75 µg and 34/48 with 25 µg EGF), time‐to‐complete response (controls: 5 weeks; both EGF dose groups: 3 weeks), and wound closure after follow‐up (25/48 controls, 40/53 with 75 µg and 25/48 with 25 µg EGF) were also treatment dependent. Multivariate analyses yielded that they were significantly enhanced by 75 µg EGF treatment and neuropathic versus ischemic ulcers. Most adverse events were mild and no drug‐related severe adverse reactions were reported. It was concluded that recombinant human EGF (rhEGF) local injections offer a favourable risk–benefit balance in patients with advanced DFU.

An optimized process for protein encapsulation was applied to formulate epidermal growth factor (rhEGF)-poly-epsilon-caprolactone microspheres. Microparticles mean size was 3.8 microm +/- 0.2 and the encapsulation efficiency was 41.9% +/- 2.6. rhEGF recovery after the encapsulation process was approximately 70% (41.9% inside the microspheres and 30% still active in the external phase). In vitro release experiments in McIlvaine buffered solution showed a rhEGF sustained release over 4 days. Skin absorption studies conducted on full-thickness human skin using the Franz cell method showed that 20% rhEGF was released from the microspheres after 24 h exposure. Microspheres accumulated in the stratum corneum where they may act as a rhEGF reservoir. Therefore, rhEGF-PCL microparticles seemed to be promising systems due to their ability to provide locally a sustained release of rhEGF in skin layers.

Background : Intralesional recombinant epidermal growth factor (EGF) was produced in the Centre for Genetic Engineering and Biotechnology (CIGB), Cuba, in 1988 and licensed in 2006. Because it may accelerate wound healing, it is a potential new treatment option in patients with a diabetic foot wound (whether infected or not) as an adjunct to standard treatment (i.e. debridement, antibiotics). We conducted the initial evaluation of EGF for diabetic foot wounds in Turkey. Methods : We enrolled 17 patients who were hospitalized in various medical centers for a foot ulcer and/or infection and for whom below the knee amputation was suggested to all except one. All patients received 75 μg intralesional EGF three times per week on alternate days. Results : The appearance of new granulation tissue on the wound site (≥75%) was observed in 13 patients (76%), and complete wound closure was observed in 3 patients (18%), yielding a ‘complete recovery’ rate of 94%. The most common side effects were tremor ( n= 10, 59%) and nausea ( n= 6, 35%). In only one case,a serious side effect requiring cessation of EGF treatment was noted. That patient experienced severe hypotension at the 16th application session, and treatment was discontinued. At baseline, a total of 21 causative bacteria were isolated from 15 patients, whereascultures were sterile in two patients. The most frequently isolated species was Pseudomonas aeruginosa . Conclusion : Thus, this preliminary study suggests that EGF seems to be a potential adjunctive treatment option in patients with limb-threatening diabetic foot wounds. Keywords: diabetic foot; intralesional epidermal growth factor; treatment (Published: 11 August 2015) Citation: Diabetic Foot & Ankle 2015, 6: 28419 - http://dx.doi.org/10.3402/dfa.v6.28419

Background aims Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. Methods Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers ( ABCG2 and P63α ) and differentiation markers ( CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43 ). Fluorescence-activated cell sorter analysis was performed for ABCG2. Results The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3 , CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies. Conclusions Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.

This research addresses the development and in vitro evaluation of lipid nanoparticle (NP)-based dressings to optimize the delivery of human recombinant epidermal growth factor (rhEGF) for the topical treatment of chronic wounds. The systems investigated were rhEGF-loaded solid lipid nanoparticles (rhEGF-SLN) and rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) formulated in wound dressings comprising either semi-solid hydrogels or fibrin-based solid scaffolds. Following detailed characterisation of the NP, in vitro diffusion cell experiments (coupled with dermatopharmacokinetic measurements), together with confocal microscopic imaging, conducted on both intact skin samples, and those from which the barrier (the stratum corneum) had been removed, revealed that (a) the particles remained essentially superficially located for at least up to 48 h post-application, (b) rhEGF released on the surface of intact skin was unable to penetrate to the deeper, viable layers, and (c) sustained release of growth factor from the NP “drug reservoirs” into barrier-compromised skin was observed. There were no significant differences between the in vitro performance of rhEGF-SLN and rhEGF-NLC, irrespective of the formulation employed. It is concluded that, because of their potentially longer-term stability, the fibrin-based scaffolds may be the most suitable approach to formulate rhEGF-loaded lipid nanoparticles.

Objectives This study was conducted to examine the tissue distribution of human recombinant epidermal growth factor (EGF) after multiple intravenous and subcutaneous injections in mice. Methods Male BALB/c mice were divided into (1) EGF 1 mg/kg intravenous dose, (2) EGF 5 mg/kg intravenous dose, (3) drug-free intravenous control, (4) EGF 1 mg/kg subcutaneous dose, (5) EGF 5 mg/kg subcutaneous dose and (6) drug-free subcutaneous control groups. EGF and drug-free dosing solutions were injected by intravenous and subcutaneous injections once a day for 3 days. EGF concentrations in serum and tissues of kidney, liver, lung, small intestine and tongue were determined by ELISA. Key findings As the intravenous and subcutaneous doses were increased from 1 to 5 mg/kg, serum Cmax and area under the concentration–time curve (AUC) values were increased dose-proportionally. In lung, tongue and small intestine, increases in AUC were dose-proportional after intravenous injections, but greater than dose-proportional after subcutaneous injections. The fold-increases in Cmax and AUC values were lowest in liver and highest in kidney. Conclusion Based on Cmax and AUC data, the systemic exposure achieved by subcutaneous injections was comparable with that achieved by intravenous injections.

Epidermal growth factor (EGF) is a potent stimulant of epithelialisation. However, topical application of EGF to achieve facilitated re-epithelialisation in partial thickness wounds has been controversial. A total of 10 pigs, each with eight 4 x 4 cm partial thickness wounds, were treated twice a day for 10 days to observe the effect of human recombinant EGF in concentrations of 0.1, 1, 5, 10, 25 ug/g, vehicle only and two controls. The control and the vehicle-only wounds each demonstrated 100% healing time (HT100) of 9.31 +/- 1.34 and 8.5 +/- 1.12 while the wounds treated with EGF ointment with concentrations of 0.1 (HT100 = 6.4 +/- 0.71), 1 (HT100 = 5.2 +/- 0.63), 5 (HT100 = 5.8 +/- 0.85), 10 (HT100 = 7.1 +/- 1.45) and 25 ug/g (HT100 = 7.4 + 0.57) demonstrated significant reduction in time to achieve re-epithelialisation. Among the EGF-treated wounds, the wounds treated with EGF concentrations of 1 and 5 ug/g achieved the fastest re-epithelialisation with evidence of substantial increase in basal keratinocyte activity observed through Ki-67 activity. In conclusion, this article demonstrates the efficacy of human recombinant EGF in facilitating re-epithelialisation of partial thickness wounds with the most efficient healing found in EGF concentrations of 1 and 5 ug/g.

Incubation of human dermal fibroblasts in keratinocyte-conditioned culture medium led to a 5.7-fold increase in the level of matrix metalloproteinase-1. Virtually all of the matrix metalloproteinase-1 – inducing activity could be related to agonists acting through members of the epidermal growth factor receptor family or to agonists acting through the interleukin-1 receptor. The same keratinocyte-conditioned medium also induced a modest increase in fibroblast proliferation (approximately 1.8-fold). Growth-stimulating activity could be attributed to epidermal growth factor receptor (but not interleukin-1 receptor) function. In fibroblasts exposed to keratinocyte-conditioned medium, mitogen-activated protein kinase signalling through both the extracellular signal-related kinase pathway and p38 pathway occurred. When recombinant epidermal growth factor or recombinant interleukin-1β were used as a control, they induced mitogen-activated protein kinase signalling consistent with the combined effects of epidermal growth factor receptor – specific and interleukin-1 receptor – specific agonists in keratinocyte-conditioned medium. Recombinant epidermal growth factor stimulated both matrix metalloproteinase-1 induction and proliferation while recombinant interleukin-1β stimulated matrix metalloproteinase-1 elaboration but not fibroblast growth. An inhibitor of extracellular signal-related kinase pathway signalling (U0126) blocked induction of matrix metalloproteinase-1 production induced by keratinocyte-conditioned medium (as well as by epidermal growth factor or interleukin-1β), and also inhibited proliferation. A p38 signalling inhibitor (SB203580) blocked matrix metalloproteinase-1 elaboration induced by keratinocyte-conditioned medium or interleukin-1β, but did not inhibit matrix metalloproteinase-1 elaboration or cell growth induced by epidermal growth factor. These data indicate that keratinocyte-fibroblast interactions are mediated by multiple stimulating agents acting on specific receptors to induce signalling through different mitogen-activated protein kinase pathways leading to altered expression of key biological functions. British Journal of Cancer (2002) 87, 457–464. doi:10.1038/sj.bjc.6600478 www.bjcancer.com © 2002 Cancer Research UK

Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

To assess effects of supraphysiologic doses of human recombinant epidermal growth factor(1-48) (rhEGF(1-48)) on neonatal rats, 10 litters of Wistar rats/treatment group were given 0 (formulated vehicle), 10, 100, or 1000 microg/kg daily by subcutaneous injection on postnatal days (PND) 1 through 6. Clinical signs, body weight, acquisition of developmental landmarks and reflexes, and behavior were monitored during treatment and for 5 weeks thereafter (to PND 42). A subset of animals was euthanized weekly from PND 7-28 and necropsied. Selected tissues were examined microscopically. Body weight gain at 1000 microg/kg during treatment was significantly less than control. Precocious incisor eruption, eye opening, vaginal opening, and preputial separation occurred at 100 and/or 1000 microg/kg. Acquisition of reflexes (negative geotaxis, wire maneuver, acoustic startle reflex, and visual placing) was delayed at 1000 microg/kg. Acquisition of adult locomotion was also delayed at 1000 microg/kg. These effects were transient, as locomotor activity at PND 28 and 42 did not differ from control. Effects on acoustic-startle responding persisted in females to final assessment on PND 42. Habituation to repeated acoustic stimuli was impaired, as well as response inhibition following a prepulse acoustic stimulus. rhEGF(1-48) induced structural changes in the skin, retina, kidney, oral and nasal mucosa, lung, and liver. Many of these changes were consistent with the expected mitogenic activity of rhEGF(1-48) and were transient in nature, as severity and incidence diminished with time. An exception was changes observed in the retina at 1000 microg/kg (rosettes/folds and focal defects in the outer nuclear/photoreceptor layers) that were still present 3 weeks after termination of treatment. Acceleration of developmental landmarks; suppression of reflexes, behavior, and somatic growth; and mitogenic responses in epidermal tissues have been reported in rodents treated with epidermal growth factor (EGF) derived from various mammalian species. These results demonstrate that a 48-amino acid fragment of human EGF produced by recombinant technology also induces such effects.

Background/Aim: Both epidermal growth factor and insulin-like growth factor I play a role in connection with the liver. In the present study, the possible interaction of these two growth factor systems was studied by investigating the effect of epidermal growth factor or insulin-like growth factor I treatment on the expression of the epidermal growth factor receptor, and its activating ligands, transforming growth factor-α and epidermal growth factor. Methods: Fifty-five male rats received no treatment, human recombinant epidermal growth factor or human recombinant insulin-like growth factor I for either 3 or 7 days. The amount of epidermal growth factor receptor, transforming growth factor-α, and epidermal growth factor mRNA was quantitated by a calibrated user-friendly RT-PCR assay (CURT-PCR), and the expression of transforming growth factor-α and epidermal growth factor peptides was quantitated by ELISA. Results: Control liver ( n =16) contained a mean (±SD) value of 12.7±7.4×10 −18 mol epidermal growth factor receptor mRNA, 3.8±2.0×10 −18 mol transforming growth factor-α mRNA and 0.8±0.4×10 −18 mol epidermal growth factor mRNA per μg total RNA and 9.8±1.6 fmol/mg protein epidermal growth factor and 144±22 fmol/mg protein transforming growth factor-α. Both epidermal growth factor and insulin-like growth factor I treatment increased the expression of mRNA for transforming growth factor-α and epidermal growth factor receptor, as well as the expression of transforming growth factor-α peptide. The level of epidermal growth factor receptor and transforming growth factor-α mRNA expression was found to correlate both in control and growth factor-treated animals, whereas the expression of epidermal growth factor receptor and epidermal growth factor showed no correlation. Marked differences were seen upon activation of the two growth factor systems, as epidermal growth factor, but not insulin-like growth factor I treatment, increased the plasma concentration of urea and decreased the concentration of insulin-like growth factor I and the liver enzymes, alanine aminotransferase and alkaline phosphatase. Conclusion: Our results show that epidermal growth factor and insulin-like growth factor I, which belong to two different growth factor systems, both induce a correlated upregulation of transforming growth factor-α and epidermal growth factor receptor mRNA in rat liver. Although marked differences were observed after treatment with either epidermal growth factor or insulin-like growth factor I on the liver as reflected in the plasma concentrations of e.g. liver enzymes, a common motif in their action involves an upregulation of the expression of the epidermal growth factor system.

The topical anti-inflammatory activity of epidermal growth factor (EGF) was evaluated in inflammation models induced by 12-Otetradecanoylphorbol-13-acetate, croton oil and arachidonic acid. When EGF (1.5, 3 or 6 μg/ear) was coapplied with each inflammatory agent, there was a dose-related decrease in inflammation as assessed by ear punch weights, myeloperoxidase activity as well as by histopathological studies. The precise anti-inflammatory action of EGF is yet unclear, but we believe that interference with arachidonic acid metabolism may play an important role.

Epidermal growth factor (EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6×His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], anEscherichia coli host strain, in amount of 30–40% of total proteins present inE. coli extract by the addition of isopropylthio-β-galactopyranoside (IPTG). The recombinant EGF purified using a Ni2+-NTA affinity column chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NIH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.

The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [ 3 H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [ 35 S]sulfate incorporation, and collagen synthesis by [ 3 H]proline incorporation. The presence of specific receptors was confirmed by [ 125 I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5–2.5 ng/ml) stimulated [ 3 H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (≤1%) and was lost at high (≥7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-β and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites ( Kd 0.18 n m ). There were about 4.5 × 10 9 binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions.

The intralesional injection of recombinant human epidermal growth factor (rhEGF) has been recently approved and introduced in several countries for the treatment of advanced diabetic foot ulcers (DFU), based on the results of five exploratory and one confirmatory, phase III clinical trials in 344 subjects. A significant stimulatory effect of this product on the healing process, given by development of granulation tissue and re-epithelization was shown in these trials, as well as a reduction in lesion recurrences during follow-up, and a tendency to a reduction of the risk of amputations, with an acceptable safety profile. However, products not always perform the same way in current medical practice. The present review summarizes the clinical information available from the intralesional use of rhEGF for advanced DFU and shows that in this case the postmarketing experiences in more than 2000 subjects confirm the results of the clinical trials, with 75% probability of complete granulation response, 61% healing, and a 16% absolute and 71% relative reduction of the risk of amputation. The benefit includes ischemic patients. The safety profile in current practice was satisfactory. Serious adverse events are not attributable to the treatment but to the underlying conditions of the patients. No evidence of neoplasia promotion by the growth factor has been found. The benefit-risk ratio of the procedure is favorable.

1. Epidermal growth factor is secreted into the gut lumen as a 53 amino acid peptide in vivo. Several preliminary studies have examined the clinical benefit of recombinant epidermal growth factor in intestinal disease, but there is inconsistency in the form of epidermal growth factor used (some using epidermal growth factor 1–48 and some epidermal growth factor 1–53), making comparisons difficult to evaluate. 2. We therefore examined the relative potency of recombinant epidermal growth factor 1–48 and epidermal growth factor 1–53 in stimulating [3H]thymidine uptake into primary rat hepatocytes at various doses in vitro and on various parameters of proliferation within the gastrointestinal tract when infused intravenously at 50 nmol day−1 kg−1 into rats receiving total parenteral nutrition. 3. Epidermal growth factor 1–53 was about twice as potent as epidermal growth factor 1–48 in its ability to stimulate [3H]thymidine incorporation into rat hepatocytes in vitro. In vivo studies supported this finding as, in both the small and large intestine, epidermal growth factor 1–53 caused about a 30% greater stimulation of intestinal growth than epidermal growth factor 1–48 (P < 0.001). 4. The 1–48 truncated form of epidermal growth factor is less active than the full-length molecule in vitro and in vivo. This must be borne in mind when comparing clinical trials that use different forms of recombinant epidermal growth factor.

The retinal pigment epithelial (RPE) cell is a potent regulatory cell within the retina. It helps to maintain normal retinal activity, and following gamma interferon (IFN-gamma) exposure, it may express major histocompatibility complex class II molecules and function as an antigen-presenting cell. Since interleukin-1 (IL-1) and IL-6 are potent cytokines observed in ocular inflammatory processes, we initiated studies to evaluate conditions which enable RPE cells to produce these cytokines. Cultures of human RPE cells from two eye donors were established and characterized, and enzyme immunoassays were employed to screen for IL-1 and IL-6 production. Treatment of RPE cells with lipopolysaccharide (LPS) or recombinant tumor necrosis factor alpha, IL-1, or IFN-gamma resulted in a significant level of secretion of IL-6. In contrast, treatment with recombinant epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, or transforming growth factor alpha, or LPS can dramatically augment the secretion of IL-6 by RPE cells. Thus, these inflammatory mediators can act alone or synergistically with IFN-gamma to activate RPE cells and dramatically increase the expression and secretion of IL-6. In contrast, IL-1 was not detected following stimulation with any of the above-mentioned cytokines or LPS. Characterization of IL-6 protein production by RPE cells revealed that 98% of the protein is promptly secreted by the cell, its induction is dependent upon the time and concentration of the stimulant, and the continuous presence of the stimulant is required for IL-6 production. Moreover, Western blot (immunoblot) analysis of secreted proteins revealed that IL-6 was produced in multiple molecular forms.(ABSTRACT TRUNCATED AT 250 WORDS)

An enhanced formation of bone, dentin, and collagen fibers in periodontal wounds after application of polypeptide growth factors has recently been reported. However, the complex environment in vivo makes it impossible to determine the specific effects of growth factors on various cells involved in the wound-healing process. We have therefore investigated the mitogenic and morphogenic effects of recombinant epidermal growth factor (rEGF), natural platelet-derived growth factor (nPDGF), and natural fibroblast growth factor (nFGF) on periodontal ligament fibroblast-like cells. A cell line was established from rat PDL tissue. The cell line was characterized according to morphology, growth pattern, cytoskeletal proteins, and growth kinetics. The mitogenic effect of growth factors was assessed by incorporation of [3H]thymidine in the cellular DNA for 4 hours. Differences between groups of observations were assessed by the Student t-test. The morphogenic effects of growth factors were described with respect to growth pattern, cell orientation, and cell and nucleus form after a random photographic recording. The fibroblast-like cell type and the non-transformed phenotype of the cell line have been identified by the presence of parameters considered to be characteristic of a normal fibroblast-like cell line. The morphogenic analysis of both experimental and control cultures showed a monolayer of adherent cells with spindle or stellate morphology, a random alignment and round or elongated nuclei. Incorporation of [3H]-thymidine was increased in a dose-dependent manner by all growth factors. Maximal effect on the DNA synthesis was: rEGF, 131%; nPDGF, 274%; and nFGF, 182%.(ABSTRACT TRUNCATED AT 250 WORDS)

CIMAvax-EGF consists of a human recombinant epidermal growth factor (EGF), coupled to P64k, a recombinant carrier protein from N. meningitis, and Montanide ISA 51 as adjuvant. The vaccine immunization induces a specific antibody production, inhibiting the EGF/EGF-R interaction through EGF deprivation. The objective of this study was to assess the CIMAvax-EGF toxicity in Sprague Dawley rats after intramuscular administration of repeated doses (6 months) and at the same time to determine if rat is a relevant species for studying CIMAvax-EGF vaccine. Rats were randomly distributed into four groups: control, Montanide ISA 51, treated with 1× and 15× of human total dose of the antigen. Animals were immunized weekly during 9 weeks, plus 9 immunizations every 14 days. Rats were inspected daily for clinical signs. Body weight, food consumption, and rectal temperature were measured during the administration of doses. Blood samples were collected for hematological, serum biochemical determinations and EGF titles at the beginning, three months and at the end of experimentation. Gross necropsy and histological examination of tissues were performed on animals at the end of the assay. Vaccine provoked the apparition of antibodies against EGF in the rats, demonstrating rat species relevance in these studies. Body weight gain, food and water consumption were not affected. CIMAvax-EGF and Montanide ISA 51 produced local damage at the administration site, showing multiple cysts and granulomas. Both vaccine-treated groups showed neutrophil elevation, besides an AST increase probably related to the damage at the administration site. Rectal temperature was found to be significantly higher in 15× treated group after immunizations, probably induced by the inflammatory process at the injection site. In summary, the clinical pathology findings together with the body temperature results, appear to be caused by the inflammatory reaction at the administration site of the vaccine, mainly mediated by the oil-based adjuvant Montanide ISA 51, probably enhanced by the immunological properties of the antigen. This study showed evidences that intramuscular administration during 26 weeks of CIMAvax-EGF at doses up to 15× human total dose is well tolerated in rats and it has a clinical importance since this long lasting study in relevant species allows to treat cancer patients with tumors during long periods with relative weight safety margin.

In order to achieve the most satisfactory results in tissue expansion, a cream containing human recombinant epidermal growth factor (EGF) was applied daily to the expanding skin. The capacity of EGF to stimulate tissue repair has been evaluated on expanded skin flaps, comparing surgical procedures carried out with and without EGF. The present clinical, morphological, immunohistochemical and morphometric results demonstrate that EGF used together with the tissue expander may improve skin repair after mechanical stretching. In EGF-expanded skin flaps, clinical results show a reduction in teleangectasiae and histological evidence of a more regular texture in the dermal tissue, together with a significant thickening of the epidermal malpighian layer. Without EGF some signs of immaturity, such as abundance of fibronectin, persist for longer. EGF seems to be a helpful support to improve the tissue expansion process.

Stress and incomplete gastrointestinal development in early-weaned piglets represent significant challenges in commercial swine farming. Orally ingested recombinant epidermal growth factor (EGF) has been shown to remain biologically active in the gastrointestinal tract as well as stimulate intestinal development, reducing the incidence of pathogen infection and diarrhea. We have previously shown that the food-grade bacterium Lactococcus lactis can be genetically altered to express biologically active EGF when fed to early-weaned mice. In this study, we assigned 8 pigs to each of 4 groups that were given EGF-expressing L. lactis (EGF-LL), empty vector-expressing L. lactis (EV-LL), recombinant human EGF, or unsupplemented bacterial media, all of which were delivered as 50-mL i.g. doses twice per day. All pigs were killed after 14 d to examine intestinal morphology. Pigs in the EGF-LL group had greater jejunal and duodenal villus heights (P < 0.0001) and intestinal length (P = 0.049) than pigs in the control group. Immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA) revealed that the proliferation of intestinal cells was significantly greater in the EGF-LL group than in the control group. PCNA expression and intestinal length also were greater in the EV-LL group, which received L. lactis that did not express EGF, than in the control group (P = 0.049), further supporting the use of naturally occurring intestinal microbes as desirable vectors for recombinant protein delivery. Our data demonstrates the feasibility of delivering a growth factor using common probiotic bacteria to farm animals for commercial practice.

The effects of human recombinant epidermal growth factor (EGF) on the growth of MKN-28 human gastric carcinoma, transplanted into nude mice were studied. Modulation of tissue cAMP and EGF receptor levels by EGF was also studied to reveal the mechanism of the growth inhibitory effects of EGF. EGF exhibited a dose-dependent growth inhibitory effect on MKN-28 human gastric carcinoma transplanted into nude mice. Local injection of 2 ng of EGF moderately inhibited the growth of MKN-28 gastric carcinoma, while injections of 20 ng, 200 ng and 2 μg of EGF significantly inhibited tumour growth. EGF decreased tissue cAMP levels in a dose-dependent manner within 24 h after EGF injection. On the other hand, EGF increased the EGF receptor levels up to two or three fold within 24 h after EGF injection. Conversely, the EGF receptor affinity for EGF decreased according to the increase in EGF receptor levels.

We investigated the effects of epidermal growth factor on the survival and growth of caprine preantral follicles. Ovarian fragments were cultured for 1 and 7 days in enriched minimal essential medium with epidermal growth factor (0, 1, 10, 50, 100, or 200 ng/mL). Non-cultured and cultured tissues were processed for histological and ultrastructural studies. Results showed that after 7 days, the epidermal growth factor (1 and 10 ng/mL) maintained the percentage of normal follicles similar to control. An increase in the percentage of primary follicles was observed with 1, 10, and 50 ng/mL of epidermal growth factor compared to enriched minimal essential medium. Ultrastructural studies confirmed follicular integrity after 7 days in epidermal growth factor (1 and 10 ng/mL). In conclusion, the low concentrations of epidermal growth factor maintain caprine follicular viability and promote the transition from primordial to primary follicles.

This rare ulcer was treated with a human recombinant epidermal growth factor-based formulation and antihypertensive drugs. Full granulation was achieved in 49 days and complete wound closure in 79 days. No adverse events were reported

We investigated the influence of dehydration-rehydration vesicles (DRV) phospholipid composition and the addition of other components on human recombinant epidermal growth factor (hrEGF) encapsulation efficiency and its release from liposomes. Encapsulation of EGF into DRV composed of phosphatidylcholine with different unsaturation levels was around 20-35%. The best result was obtained with dipalmitoyl phosphatidylcholine: cholesterol (DPPC:Ch) liposomes (35%) corresponding to the lowest hrEGF release during one month of storage. Even with this phospholipid composition, modification of the DRV procedure by including an extrusion step did not improve hrEGF encapsulation efficiency, rendering less stable particles. The inclusion of recombinant P64k from Neisseria meningitidis (rP64k), as such or conjugated to hrEGF, decreased the encapsulation efficiency of the latter protein into DRV or freeze and thaw multilamellar vesicles (FATMLV). The hrEGF release from liposomes could be related to the interaction between this polypeptide and the bilayer, as evidenced by increased carboxyfluorescein release from hrEGF-DRV; less susceptibility to fluorescence quenching by acrylamide in the presence of liposomes; and a measurable decrease of phospholipid phase transition Delta enthalpy (DeltaH). DRV comprising saturated phospholipids (DPPC:Ch or distearoyl phosphatidylcholine [DSPC]:Ch) and containing the conjugate EGF-P64k induced a more efficient immune response against hrEGF than unsaturated phospholipid and alum in terms of total IgG, IgG(2a), and IgG(2b) subclasses and the ability of antibody to inhibit the interaction of the EGF receptor with hrEGF.