Carcinogenic effect of human tumor-derived cell-free filtrates in nude mice.

Cancer remains a worldwide cause of morbidity and mortality. Investigational research efforts have included the administration of tumor-derived extracts to healthy animals. Having previously demonstrated that the administration of non-transmissible, human cancer-derived homogenates induced malignant tumors in mice, here, we examined the consequences of administering 50 or 100 µg of protein of crude homogenates from mammary carcinoma, pancreatic adenocarcinoma, and melanoma samples in 6 inoculations per week during 2 months. The concurrent control mice received homogenates of healthy donor-skin cosmetic surgery fragments. Mammary carcinoma homogenate administration did not provoke the deterioration or mortality of the animals. Multiple foci of lung adenocarcinomas with a broad expression of malignity histomarkers coexisting with small cell-like carcinomas were found. Disseminated cells, positive to classic epithelial markers, were detected in lymphoid nodes. The administration of pancreatic tumor and melanoma homogenates progressively deteriorated animal health. Pancreatic tumor induced poorly differentiated lung adenocarcinomas and pancreatic islet hyperplasia. Melanoma affected lungs with solid pseudopapillary adenocarcinomas. Giant atypical hepatocytes were also observed. The kidney exhibited dispersed foci of neoplastic cells within a desmoplastic matrix. Nuclear overlapping with hyperchromatic nuclei, mitotic figures, and prominent nuclear atypia was identified in epidermal cells. None of these changes were ever detected in the control mice. Furthermore, the incubation of zebrafish embryos with breast tumor homogenates induced the expression of c-Myc and HER-2 as tumor markers, contrasting to embryos exposed to healthy tissue-derived material. This study confirms and extends our hypothesis that tumor homogenates contain and may act as vectors for "malignancy drivers," which ultimately implement a carcinogenesis process in otherwise healthy mice.

Endogenous Biological Drivers in Diabetic Lower Limb Wounds Recurrence: Hypothetical Reflections.

An impaired healing response underlies diabetic foot wound chronicity, frequently translating to amputation, disability, and mortality. Diabetics suffer from underappreciated episodes of post-epithelization ulcer recurrence. Recurrence epidemiological data are alarmingly high, so the ulcer is considered in "remission" and not healed from the time it remains epithelialized. Recurrence may result from the combined effects of behavioral and endogenous biological factors. Although the damaging role of behavioral, clinical predisposing factors is undebatable, it still remains elusive in the identification of endogenous biological culprits that may prime the residual scar tissue for recurrence. Furthermore, the event of ulcer recurrence still waits for the identification of a molecular predictor. We propose that ulcer recurrence is deeply impinged by chronic hyperglycemia and its downstream biological effectors, which originate epigenetic drivers that enforce abnormal pathologic phenotypes to dermal fibroblasts and keratinocytes as memory cells. Hyperglycemia-derived cytotoxic reactants accumulate and modify dermal proteins, reduce scar tissue mechanical tolerance, and disrupt fibroblast-secretory activity. Accordingly, the combination of epigenetic and local and systemic cytotoxic signalers induce the onset of "at-risk phenotypes" such as premature skin cell aging, dysmetabolism, inflammatory, pro-degradative, and oxidative programs that may ultimately converge to scar cell demise. Post-epithelialization recurrence rate data are missing in clinical studies of reputed ulcer healing therapies during follow-up periods. Intra-ulcer infiltration of epidermal growth factor exhibits the most consistent remission data with the lowest recurrences during 12-month follow-up. Recurrence data should be regarded as a valuable clinical endpoint during the investigational period for each emergent healing candidate.

Evidence of SARS-CoV-2 infection in postmortem lung, kidney, and liver samples, revealing cellular targets involved in COVID-19 pathogenesis.

There is an urgent need to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions involved in virus spread and pathogenesis, which might contribute to the identification of new therapeutic targets. In this study, we investigated the presence of SARS-CoV-2 in postmortem lung, kidney, and liver samples of patients who died with coronavirus disease (COVID-19) and its relationship with host factors involved in virus spread and pathogenesis, using microscopy-based methods. The cases analyzed showed advanced stages of diffuse acute alveolar damage and fibrosis. We identified the SARS-CoV-2 nucleocapsid (NC) in a variety of cells, colocalizing with mitochondrial proteins, lipid droplets (LDs), and key host proteins that have been implicated in inflammation, tissue repair, and the SARS-CoV-2 life cycle (vimentin, NLRP3, fibronectin, LC3B, DDX3X, and PPARγ), pointing to vimentin and LDs as platforms involved not only in the viral life cycle but also in inflammation and pathogenesis. SARS-CoV-2 isolated from a patient´s nasal swab was grown in cell culture and used to infect hamsters. Target cells identified in human tissue samples included lung epithelial and endothelial cells; lipogenic fibroblast-like cells (FLCs) showing features of lipofibroblasts such as activated PPARγ signaling and LDs; lung FLCs expressing fibronectin and vimentin and macrophages, both with evidence of NLRP3- and IL1ß-induced responses; regulatory cells expressing immune-checkpoint proteins involved in lung repair responses and contributing to inflammatory responses in the lung; CD34+ liver endothelial cells and hepatocytes expressing vimentin; renal interstitial cells; and the juxtaglomerular apparatus. This suggests that SARS-CoV-2 may directly interfere with critical lung, renal, and liver functions involved in COVID-19-pathogenesis.

Cell-Free Filtrates (CFF) as Vectors of a Transmissible Pathologic Tissue Memory Code: A Hypothetical and Narrative Review.

Cellular memory is a controversial concept representing the ability of cells to "write and memorize" stressful experiences via epigenetic operators. The progressive course of chronic, non-communicable diseases such as type 2 diabetes mellitus, cancer, and arteriosclerosis, is likely driven through an abnormal epigenetic reprogramming, fostering the hypothesis of a cellular pathologic memory. Accordingly, cultured diabetic and cancer patient-derived cells recall behavioral traits as when in the donor's organism irrespective to culture time and conditions. Here, we analyze the data of studies conducted by our group and led by a cascade of hypothesis, in which we aimed to validate the hypothetical existence and transmissibility of a cellular pathologic memory in diabetes, arteriosclerotic peripheral arterial disease, and cancer. These experiments were based on the administration to otherwise healthy animals of cell-free filtrates prepared from human pathologic tissue samples representative of each disease condition. The administration of each pathologic tissue homogenate consistently induced the faithful recapitulation of: (1) Diabetic archetypical changes in cutaneous arterioles and nerves. (2) Non-thrombotic arteriosclerotic thickening, collagenous arterial encroachment, aberrant angiogenesis, and vascular remodeling. (3) Pre-malignant and malignant epithelial and mesenchymal tumors in different organs; all evocative of the donor's tissue histopathology and with no barriers for interspecies transmission. We hypothesize that homogenates contain pathologic tissue memory codes represented in soluble drivers that "infiltrate" host's animal cells, and ultimately impose their phenotypic signatures. The identification and validation of the actors in behind may pave the way for future therapies.

Diagnostic Accuracy of Blood-Based Biomarker Panels: A Systematic Review.

Background: Because of high prevalence of Alzheimer's disease (AD) in low- and middle-income countries (LMICs), there is an urgent need for inexpensive and minimally invasive diagnostic tests to detect biomarkers in the earliest and asymptomatic stages of the disease. Blood-based biomarkers are predicted to have the most impact for use as a screening tool and predict the onset of AD, especially in LMICs. Furthermore, it has been suggested that panels of markers may perform better than single protein candidates. Methods: Medline/Pubmed was searched to identify current relevant studies published from January 2016 to December 2020. We included all full-text articles examining blood-based biomarkers as a set of protein markers or panels to aid in AD's early diagnosis, prognosis, and characterization. Results: Seventy-six articles met the inclusion criteria for systematic review. Majority of the studies reported plasma and serum as the main source for biomarker determination in blood. Protein-based biomarker panels were reported to aid in AD diagnosis and prognosis with better accuracy than individual biomarkers. Conventional (amyloid-beta and tau) and neuroinflammatory biomarkers, such as amyloid beta-42, amyloid beta-40, total tau, phosphorylated tau-181, and other tau isoforms, were the most represented. We found the combination of amyloid beta-42/amyloid beta-40 ratio and APOEε4 status to be most represented with high accuracy for predicting amyloid beta-positron emission tomography status. Conclusion: Assessment of Alzheimer's disease biomarkers in blood as a non-invasive and cost-effective alternative will potentially contribute to early diagnosis and improvement of therapeutic interventions. Given the heterogeneous nature of AD, combination of markers seems to perform better in the diagnosis and prognosis of the disease than individual biomarkers.

Epidermal growth factor effect on lipopolysaccharide-induced inflammation in fibroblasts derived from diabetic foot ulcer.

BACKGROUND: Diabetic foot ulcers (DFU) are characterised by high levels of inflammatory mediators, resulting from sustained hyperglycaemic insult and the local microbial biofilm. The intralesional administration of epidermal growth factor (EGF) has emerged as an effective treatment that stimulates granulation and closure of DFU, reducing the risk of amputation. Within the wound, fibroblasts play key roles during the healing process, promoting granulation and contraction. The aim of the present study was to examine the anti-inflammatory effect of EGF in DFU-derived fibroblasts, challenged with lipopolysaccharide (LPS), under hyperglycaemic conditions, recreating in vitro what happens in a clinical scenario. METHODS: Healthy skin (HS) and DFU granulation tissue biopsies were used to isolate primary fibroblasts. The effect of LPS on cell proliferation was analysed. Transcriptional expression of toll-like receptor (TLR) pathway mediators (TLR4, TLR2, CD14, MYD88 and NFKB) and pro-inflammatory cytokines (TNF, IL-6 and IL-1B) were measured by semi-quantitative polymerase chain reaction (qPCR), in cells treated with appropriate concentrations of LPS, EGF and their combination. IL-6 protein concentration was quantified by ELISA. RESULTS: LPS stimulated proliferation of HS-derived fibroblasts, while inhibiting the proliferation of cells derived from DFU at the highest assayed concentration of 1 µg/mL. Regarding the TLR signalling pathway, LPS increased messenger RNA levels of mediators and pro-inflammatory genes, while EGF, alone or in the presence of LPS, downregulated them, except for IL-1B. CONCLUSION: The results suggest that EGF might elicit an anti-inflammatory response in LPS-challenged fibroblasts, even in a hyperglycaemic milieu. Collectively, our findings contribute to explain newly observed effects of EGF in the clinical arena. LAY SUMMARY: In this research article, we analyse the putative anti-inflammatory effect of epidermal growth factor (EGF) on fibroblast isolated from diabetic foot ulcer (DFU) granulation tissue. To induce the inflammatory response, the cells were treated with lipopolysaccharide (LPS), simulating the gram-negative bacterial infection that takes place in the wounds of diabetic patients. We studied the expression of genes involved in bacterial recognition receptors signalling pathway and those that code for different pro-inflammatory cytokines.We obtained primary fibroblasts from biopsies of a neuropathic diabetic ulcer and from healthy skin, the former was used as the control. Cells were isolated and grown in high glucose Dulbecco's Modified Eagle Medium (DMEM) culture medium, to simulate the hyperglycaemic insult. The effect of increasing concentrations of LPS on cell proliferation was analysed. Relative transcriptional expression of genes in the study was quantified by quantitative polymerase chain reaction (qPCR) in cells treated with LPS, EGF or a combination. Untreated cells served to normalise the expression.In the present study, we demonstrated that EGF modulated the primary immune response by reducing the activation of pathogen-recognition receptors and common genes involved in these signalling pathways, even in hyperglycaemic conditions. This effect translated in a decreased expression of pro-inflammatory cytokines. These results contribute to explain our previous observations about the reduction of circulating levels of inflammatory cytokines after local administration of human recombinant EGF in DFU. Further molecular studies should be carried out to fully understand the biological mechanisms elicited by EGF in this clinical scenario.

Intralesional Infiltrations of Arteriosclerotic Tissue Cells-Free Filtrate Reproduce Vascular Pathology in Healthy Recipient Rats.

Lower-extremity arterial disease is a major health problem with increasing prevalence, often leading to non-traumatic amputation, disability and mortality. The molecular mechanisms underpinning abnormal vascular wall remodeling are not fully understood. We hypothesized on the existence of a vascular tissue memory that may be transmitted through soluble signaling messengers, transferred from humans to healthy recipient animals, and consequently drive the recapitulation of arterial wall thickening and other vascular pathologies. We examined the effects of the intralesional infiltration for 6 days of arteriosclerotic popliteal artery-derived homogenates (100 µg of protein) into rats' full-thickness wounds granulation tissue. Animals infiltrated with normal saline solution or healthy brachial arterial tissue homogenate obtained from traumatic amputation served as controls. The significant thickening of arteriolar walls was the constant outcome in two independent experiments for animals receiving arteriosclerotic tissue homogenates. This material induced other vascular morphological changes including an endothelial cell phenotypic reprogramming that mirrored the donor's vascular histopathology. The immunohistochemical expression pattern of relevant vascular markers appeared to match between the human tissue and the corresponding recipient rats. These changes occurred within days of administration, and with no cross-species limitation. The identification of these "vascular disease drivers" may pave novel research avenues for atherosclerosis pathobiology.

Wound Chronicity, Impaired Immunity and Infection in Diabetic Patients.

BACKGROUND: Diabetic foot ulcers are a common diabetic complication leading to alarming figures of amputation, disability, and early mortality. The diabetic glucooxidative environment impairs the healing response, promoting the onset of a 'wound chronicity phenotype'. In 50% of ulcers, these non-healing wounds act as an open door for developing infections, a process facilitated by diabetic patients' dysimmunity. Infection can elicit biofilm formation that worsens wound prognosis. How this microorganism community is able to take advantage of underlying diabetic conditions and thrive both within the wound and the diabetic host is an expanding research field. OBJECTIVES: 1) Offer an overview of the major cellular and molecular derangements of the diabetic healing process versus physiological cascades in a non-diabetic host. 2) Describe the main immunopathological aspects of diabetics' immune response and explore how these contribute to wound infection susceptibility. 3) Conceptualize infection and biofilim in diabetic foot ulcers and analyze their dynamic interactions with wound bed cells and matrices, and their systemic effects at the organism level. 4) Offer an integrative conceptual framework of wound-dysimmunity-infection-organism damage. EVIDENCE AQUISITION: We retrieved 683 articles indexed in Medline/PubMed, SciELO, Bioline International and Google Scholar. 280 articles were selected for discussion under four major subheadings: 1) normal healing processes, 2) impaired healing processes in the diabetic population, 3) diabetic dysimmunity and 4) diabetic foot infection and its interaction with the host. DEVELOPMENT: The diabetic healing response is heterogeneous, torpid and asynchronous, leading to wound chronicity. The accumulation of senescent cells and a protracted inflammatory profile with a pro-catabolic balance hinder the proliferative response and delay re-epithelialization. Diabetes reduces the immune system's abilities to orchestrate an appropriate antimicrobial response and offers ideal conditions for microbiota establishment and biofilm formation. Biofilm-microbial entrenchment hinders antimicrobial therapy effectiveness, amplifies the host's pre-existing immunodepression, arrests the wound's proliferative phase, increases localized catabolism, prolongs pathogenic inflammation and perpetuates wound chronicity. In such circumstances the infected wound may act as a proinflammatory and pro-oxidant organ superimposed onto the host, which eventually intensifies peripheral insulin resistance and disrupts homeostasis. CONCLUSIONS: The number of lower-limb amputations remains high worldwide despite continued research efforts on diabetic foot ulcers. Identifying and manipulating the molecular drivers underlying diabetic wound healing failure, and dysimmunity-driven susceptibility to infection will offer more effective therapeutic tools for the diabetic population.

Registro sanitario de Heberprot-P®, medicamento biotecnológico innovador para el tratamiento de las úlceras del pie diabético / Sanitary registration of Heberprot-P®, an innovative biotechnological drug for the treatment of diabetic foot ulcers

Introducción: Heberprot-P® obtuvo su primer registro sanitario en Cuba en el año 2006, actualmente está aprobado en otros 26 países. Objetivo: Describir el proceso de registro sanitario en México, del medicamento biotecnológico Heberprot-P® para el tratamiento de las úlceras del pie diabético. Métodos: El proceso de registro sanitario de Heberprot-P® siguió las pautas de la reglamentación sanitaria de México sobre la base de la Ley general de salud y el Reglamento de insumos para la salud. Se revisaron además la Farmacopea de los Estados Unidos Mexicanos y las normas oficiales mexicanas en función de cumplir las exigencias para la comercialización de medicamentos en este territorio. Resultados: El proceso de registro se inició en junio de 2017 en México con acciones en función de completar los documentos e informaciones exigidas en el expediente de registro sanitario a presentarse. Entre ellos resaltan las consideraciones del Subcomité de Evaluación de Productos Biotecnológicos y el Comité de Moléculas Nuevas, la evaluación del expediente por un Tercero Autorizado y documentos emitidos por el Centro Nacional de Farmacovigilancia e Instituto Mexicano de la Propiedad Industrial. Se presentó la solicitud del registro sanitario ante Cofepris y esta se aprobó en mayo de 2018. Conclusiones: El trabajo con grupos de expertos permitió a la autoridad mexicana hacer un trabajo más expedito basado en las evidencias de las evaluaciones realizadas que son parte de la información del registro sanitario. Como resultado de este proceso, se otorgó el Registro Sanitario a Heberprot-P® en mayo de 2018 y Cofepris lo reconoció como un medicamento biotecnológico innovador(AU)

Introduction: Heberprot-P® obtained its first Sanitary Registration in Cuba in 2006, and it is currently approved in 26 other countries. Objective: Describe the sanitary registration process in Mexico of the biotechnological drug Heberprot-P® for the treatment of diabetic foot ulcers. Methods: The sanitary registration process of Heberprot-P® followed the guidelines of the sanitary regulations of Mexico on the basis of the General Health Law and the Regulation of Supplies for Health. The Pharmacopoeia of the United Mexican States and the official Mexican standards were also revised in order to comply with the requirements for the marketing of medicines in this territory. Results: The registration process began in June 2017 in Mexico with actions to complete the documents and information required in the sanitary registration file to be submitted. Among them are the considerations of the Sub-committee on the Evaluation of Biotechnological Products and the Committee on New Molecules, the evaluation of the file by an Authorized Third Party and documents issued by the National Center for Pharmacovigilance and the Mexican Institute of Industrial Property. The application for sanitary registration was submitted to Cofepris and this was approved in May 2018. Conclusions: The work with groups of experts allowed the Mexican authority to do a more expeditious work based on the evidence of the evaluations carried out that are part of the information of the sanitary registry. As a result of this process, Heberprot-P® was granted the Sanitary Registry in May 2018 and COFEPRIS recognized it as an innovative biotechnological medicine(AU)

Revisiting Pleiotropic Effects of Type I Interferons: Rationale for Its Prophylactic and Therapeutic Use Against SARS-CoV-2.

The pandemic distribution of SARS-CoV-2 together with its particular feature of inactivating the interferon-based endogenous response and accordingly, impairing the innate immunity, has become a challenge for the international scientific and medical community. Fortunately, recombinant interferons as therapeutic products have accumulated a long history of beneficial therapeutic results in the treatment of chronic and acute viral diseases and also in the therapy of some types of cancer. One of the first antiviral treatments during the onset of COVID-19 in China was based on the use of recombinant interferon alfa 2b, so many clinicians began to use it, not only as therapy but also as a prophylactic approach, mainly in medical personnel. At the same time, basic research on interferons provided new insights that have contributed to a much better understanding of how treatment with interferons, initially considered as antivirals, actually has a much broader pharmacological scope. In this review, we briefly describe interferons, how they are induced in the event of a viral infection, and how they elicit signaling after contact with their specific receptor on target cells. Additionally, some of the genes stimulated by type I interferons are described, as well as the way interferon-mediated signaling is torpedoed by coronaviruses and in particular by SARS-CoV-2. Angiotensin converting enzyme 2 (ACE2) gene is one of the interferon response genes. Although for many scientists this fact could result in an adverse effect of interferon treatment in COVID-19 patients, ACE2 expression contributes to the balance of the renin-angiotensin system, which is greatly affected by SARS-CoV-2 in its internalization into the cell. This manuscript also includes the relationship between type I interferons and neutrophils, NETosis, and interleukin 17. Finally, under the subtitle of "take-home messages", we discuss the rationale behind a timely treatment with interferons in the context of COVID-19 is emphasized.

Intralesional Infiltrations of Cell-Free Filtrates Derived from Human Diabetic Tissues Delay the Healing Process and Recreate Diabetes Histopathological Changes in Healthy Rats.

Lower limb ulcers in type-2 diabetic patients are a frequent complication that tributes to amputation and reduces survival. We hypothesized that diabetic healing impairment and other histopathologic hallmarks are mediated by a T2DM-induced tissue priming/metabolic memory that can be transferred from humans to healthy recipient animals and consequently reproduce diabetic donor's phenotypes. We examined the effect of human T2DM tissue homogenates injected into non-diabetic rat excisional wounds. Fresh granulation tissue, popliteal artery, and peroneal nerve of patients with T2DM were obtained following amputation. Post-mammoplasty granulation and post-traumatic amputation-tissue of normal subjects acted as controls. The homogenates were intralesionally injected for 6-7 days into rats' excisional thickness wounds. Infiltration with the different homogenates caused impaired wound closure, inflammation, nerve degeneration, and arterial thickening (all P < 0.01 vs relevant control) resembling histopathology of diabetic donor tissues. Control materials caused marginal inflammation only. Infiltration with glycated bovine albumin provoked inflammation and wound healing delay but did not induce arterial thickening. The reproduction of human diabetic traits in healthy recipient animals through a tissue homogenate support the notion on the existence of tissue metabolic memory-associated and transmissible factors, involved in the pathogenesis of diabetic complications. These may have futuristic clinical implications for medical interventions.

Insulin Resistance at the Crossroad of Alzheimer Disease Pathology: A Review.

Insulin plays a major neuroprotective and trophic function for cerebral cell population, thus countering apoptosis, beta-amyloid toxicity, and oxidative stress; favoring neuronal survival; and enhancing memory and learning processes. Insulin resistance and impaired cerebral glucose metabolism are invariantly reported in Alzheimer's disease (AD) and other neurodegenerative processes. AD is a fatal neurodegenerative disorder in which progressive glucose hypometabolism parallels to cognitive impairment. Although AD may appear and progress in virtue of multifactorial nosogenic ingredients, multiple interperpetuative and interconnected vicious circles appear to drive disease pathophysiology. The disease is primarily a metabolic/energetic disorder in which amyloid accumulation may appear as a by-product of more proximal events, especially in the late-onset form. As a bridge between AD and type 2 diabetes, activation of c-Jun N-terminal kinase (JNK) pathway with the ensued serine phosphorylation of the insulin response substrate (IRS)-1/2 may be at the crossroads of insulin resistance and its subsequent dysmetabolic consequences. Central insulin axis bankruptcy translates in neuronal vulnerability and demise. As a link in the chain of pathogenic vicious circles, mitochondrial dysfunction, oxidative stress, and peripheral/central immune-inflammation are increasingly advocated as major pathology drivers. Pharmacological interventions addressed to preserve insulin axis physiology, mitochondrial biogenesis-integral functionality, and mitophagy of diseased organelles may attenuate the adjacent spillover of free radicals that further perpetuate mitochondrial damages and catalyze inflammation. Central and/or peripheral inflammation may account for a local flood of proinflammatory cytokines that along with astrogliosis amplify insulin resistance, mitochondrial dysfunction, and oxidative stress. All these elements are endogenous stressor, pro-senescent factors that contribute to JNK activation. Taken together, these evidences incite to identify novel multi-mechanistic approaches to succeed in ameliorating this pandemic affliction.

Cellular Senescence as the Pathogenic Hub of Diabetes-Related Wound Chronicity.

Diabetes is constantly increasing at a rate that outpaces genetic variation and approaches to pandemic magnitude. Skin cells physiology and the cutaneous healing response are progressively undermined in diabetes which predisposes to lower limb ulceration, recidivism, and subsequent lower extremities amputation as a frightened complication. The molecular operators whereby diabetes reduces tissues resilience and hampers the repair mechanisms remain elusive. We have accrued the notion that diabetic environment embraces preconditioning factors that definitively propel premature cellular senescence, and that ulcer cells senescence impair the healing response. Hyperglycemia/oxidative stress/mitochondrial and DNA damage may act as major drivers sculpturing the senescent phenotype. We review here historical and recent evidences that substantiate the hypothesis that diabetic foot ulcers healing trajectory, is definitively impinged by a self-expanding and self-perpetuative senescent cells society that drives wound chronicity. This society may be fostered by a diabetic archetypal secretome that induces replicative senescence in dermal fibroblasts, endothelial cells, and keratinocytes. Mesenchymal stem cells are also susceptible to major diabetic senescence drivers, which accounts for the inability of these cells to appropriately assist in diabetics wound healing. Thus, the use of autologous stem cells has not translated in significant clinical outcomes. Novel and multifaceted therapeutic approaches are required to pharmacologically mitigate the diabetic cellular senescence operators and reduce the secondary multi-organs complications. The senescent cells society and its adjunctive secretome could be an ideal local target to manipulate diabetic ulcers and prevent wound chronification and acute recidivism. This futuristic goal demands harnessing the diabetic wound chronicity epigenomic signature.

Epidermal Growth Factor in Healing Diabetic Foot Ulcers: From Gene Expression to Tissue Healing and Systemic Biomarker Circulation.

Lower-extremity diabetic ulcers are responsible for 80% of annual worldwide nontraumatic amputations. Epidermal growth factor (EGF) reduction is one of the molecular pillars of diabetic ulcer chronicity, thus EGF administration may be considered a type of replacement therapy. Topical EGF ad-ministration to improve and speed wound healing began in 1989 on burn patients as part of an acute-healing therapy. Further clinical studies based on topically administering EGF to different chronic wounds resulted in disappointing out-comes. An analysis of the literature on unsuccessful clinical trials identifi ed a lack of knowledge concerning: (I) molecular and cellular foundations of wound chronicity and (II) the phar-macodynamic requisites governing EGF interaction with its receptor to promote cell response. Yet, EGF intra- and perile-sional infi ltration were shown to circumvent the pharmacody-namic limitations of topical application. Since the fi rst studies, the following decades of basic and clinical research on EGF therapy for problem wounds have shed light on potential uses of growth factors in regenerative medicine. EGF's molecular and biochemical effects at both local and systemic levels are diverse: (1) downregulation of genes encoding infl ammation mediators and increased expression of genes involved in cell proliferation, angiogenesis and matrix secretion; (2) EGF in-tervention positively impacts both mesenchymal and epithelial cells, reducing infl ammation and stimulating the recruitment of precursor circulating cells that promote the formation of new blood vessels; (3) at the subcellular level, upregulation of the EGF receptor with subsequent intracellular traffi cking, includ-ing mitochondrial allocation along with restored morphology of multiple organelles; and (4) local EGF infi ltration resulting in a systemic, organismal repercussion, thus contributing to attenuation of circulating infl ammatory and catabolic reac-tants, restored reduction-oxidation balance, and decreased toxic glycation products and soluble apoptogenic effectors. It is likely that EGF treatment may rearrange critical epigenetic drivers of diabetic metabolic memory. KEYWORDS Epidermal Growth Factor, diabetes, diabetes complications, wound healing, diabetic foot, amputation, ulcer, Cuba.

Review: Insulin resistance and mitochondrial dysfunction following severe burn injury.

The insulin signaling pathway plays a pivotal role in glucose metabolism and metabolic homeostasis. Disruption of this pathway is commonly seen in critical illness such as following severe burn injuries where homeostatic control is lost, leading to "insulin resistance" with poor blood glucose control. The aberrant signaling pathways involved in insulin resistance following burn injury include increases in hyperglycemic stress hormones, pro-inflammatory cytokines and free radical production. Leakage of mitochondrial sequestered self-antigens and signaling between mitochondria and endoplasmic reticulum also contribute to insulin resistance. Greater understanding of molecular processes involved in burn-related insulin resistance could potentially lead to the development of novel therapeutic approaches to improve patient management.

Systemic translation of locally infiltrated epidermal growth factor in diabetic lower extremity wounds.

Diabetic foot ulcer is one of the most frightened diabetic complications leading to amputation disability and early mortality. Diabetic wounds exhibit a complex networking of inflammatory cytokines, local proteases, and reactive oxygen and nitrogen species as a pathogenic polymicrobial biofilm, overall contributing to wound chronification and host homeostasis imbalance. Intralesional infiltration of epidermal growth factor (EGF) has emerged as a therapeutic alternative to diabetic wound healing, reaching responsive cells while avoiding the deleterious effect of proteases and the biofilm on the wound's surface. The present study shows that intralesional therapy with EGF is associated with the systemic attenuation of pro-inflammatory markers along with redox balance recovery. A total of 11 diabetic patients with neuropathic foot ulcers were studied before and 3 weeks after starting EGF treatment. Evaluations comprised plasma levels of pro-inflammatory, redox balance, and glycation markers. Pro-inflammatory markers such as erythrosedimentation rate, C-reactive protein, interleukin-6, soluble FAS, and macrophage inflammatory protein 1-alpha were significantly reduced by EGF therapy. Oxidative capacity, nitrite/nitrate ratio, and pentosidine were also reduced, while soluble receptor for advanced glycation end-products significantly increased. Overall, our results indicate that the local intralesional infiltration of EGF translates in systemic anti-inflammatory and antioxidant effects, as in attenuation of the glycation products' negative effects.

Quantitative Studies of Diabetic Foot Ulcer Evolution Under Treatment by Digital Stereotactic Photography.

BACKGROUND: Imaging the lower extremity reproducibly and accurately remains an elusive goal. This is particularly true in the high risk diabetic foot, where tissue loss, edema, and color changes are often concomitant. The purpose of this study was to evaluate the reproducibility of a novel and inexpensive stereotaxic frame in assessment of wound healing. METHODS: The main idea is to keep constant and reproducible the relative position of extremities related to the sensor used for the examination during a serial studies by stereotaxic digital photographic sequence. Ten healthy volunteers were evaluated at 10 different time moments to estimate the foot position variations in the stereotaxic frame. The evolution of 40 of DFU patients under treatment was evaluated before and during the epidemical grow factor intralesional treatment. RESULTS: The wound closing and granulation speeds, the relative contribution of the contraction and tissue restauration mechanism were evaluated by stereotaxic digital photography. CONCLUSIONS: The results of this study suggest that the stereotaxic frame is a robust platform for serial study of the evolution of wound healing which allow to obtain consistent information from a variety of visible and hyperspectral measurement technologies. New stereotaxic digital photography evidences related to the diabetic foot ulcer healing process under treatment has been presented.

Growth hormone-releasing peptide 6 prevents cutaneous hypertrophic scarring: early mechanistic data from a proteome study.

Hypertrophic scars (HTS) and keloids are forms of aberrant cutaneous healing with excessive extracellular matrix (ECM) deposition. Current therapies still fall short and cause undesired effects. We aimed to thoroughly evaluate the ability of growth hormone releasing peptide 6 (GHRP6) to both prevent and reverse cutaneous fibrosis and to acquire the earliest proteome data supporting GHRP6's acute impact on aesthetic wound healing. Two independent sets of experiments addressing prevention and reversion effects were conducted on the classic HTS model in rabbits. In the prevention approach, the wounds were assigned to topically receive GHRP6, triamcinolone acetonide (TA), or vehicle (1% sodium carboxy methylcellulose [CMC]) from day 1 to day 30 post-wounding. The reversion scheme was based on the infiltration of either GHRP6 or sterile saline in mature HTS for 4 consecutive weeks. The incidence and appearance of HTS were systematically monitored. The sub-epidermal fibrotic core area of HTS was ultrasonographically determined, and the scar elevation index was calculated on haematoxylin/eosin-stained, microscopic digitised images. Tissue samples were collected for proteomics after 1 hour of HTS induction and treatment with either GHRP6 or vehicle. GHRP6 prevented the onset of HTS without the untoward reactions induced by the first-line treatment triamcinolone acetonide (TA); however, it failed to significantly reverse mature HTS. The preliminary proteomic study suggests that the anti-fibrotic preventing effect exerted by GHRP6 depends on different pathways involved in lipid metabolism, cytoskeleton arrangements, epidermal cells' differentiation, and ECM dynamics. These results enlighten the potential success of GHRP6 as one of the incoming alternatives for HTS prevention.

Heberprot-P's Effect on Gene Expression in Healing Diabetic Foot Ulcers.

NTRODUCTION Diabetic foot ulcers are a chronic complication in patients with diabetes mellitus. They appear as a result of the combination of diabetic polyneuropathy and angiopathy, and in many cases require amputation of the affected extremity. Clinical trials have demonstrated that repeated local infiltration with Heberprot-P can improve healing of chronic diabetic foot ulcers. Although there is evidence of its effects as a granulation stimulator and on cell migration and proliferation, genetic control mechanisms explaining its anti-inflammatory and oxidative stress reduction properties are not yet thoroughly understood. OBJECTIVE Analyze changes in expression of genes involved in healing after treatment of diabetic foot ulcers with Heberprot-P. METHODS Biopsies were collected from diabetic foot ulcers of 10 responding patients before and after 2 weeks' treatment with Heberprot-P (75-µg applied intralesionally 3 times per week). Total RNA was obtained and quantitative PCR used to determine expression of 26 genes related to inflammation, oxidative stress, cell proliferation, ngiogenesis and extracellular matrix formation. Genetic expression was quantified before and after treatment using REST 2009 v2.0.13. RESULTS After treatment, there was a statistically significant increase in expression of genes related to cell proliferation, angiogenesis and formation of extracellular matrix (PDGFB, CDK4, P21, TP53, ANGPT1, COL1A1, MMP2 and TIMP2). A significant decrease was observed in gene expression related to inflammatory processes and oxidative stress (NFKB1, TNFA and IL-1A). CONCLUSIONS Our findings suggest that Heberprot-P's healing action on diabetic foot ulcers is mediated through changes in genetic expression that reduce hypoxia, inflammation and oxidative stress, and at the same time increase cell proliferation, collagen synthesis and extracellular matrix remodeling. The kinetics of expression of two genes related to extracellular matrix formation needs further exploration. KEYWORDS Epidermal growth factor, EGF, diabetic foot ulcer, wound healing, quantitative real-time PCR, gene expression, Cuba.

Diabetic Foot Ulcers and Epidermal Growth Factor: Revisiting the Local Delivery Route for a Successful Outcome.

Soon after epidermal growth factor (EGF) discovery, some in vivo models appeared demonstrating its property to enhance cutaneous wound healing. EGF was the first growth factor (GF) introduced in the clinical arena as a healing enhancer, exerting its mitogenic effects on epithelial, fibroblastoid, and endothelial cells via a tyrosine kinase membrane receptor. Compelling evidences from the 90s documented that, for EGF, locally prolonged bioavailability and hourly interaction with the receptor were necessary for a successful tissue response. Eventually, the enthusiasm on the clinical use of EGF to steer the healing process was wiped out as the topical route to deliver proteins started to be questioned. The simultaneous in vivo experiments, emphasizing the impact of the parenterally administered EGF on epithelial and nonepithelial organs in terms of mitogenesis and cytoprotection, rendered the theoretical fundamentals for the injectable use of EGF and shaped the hypothesis that locally infiltrating the diabetic ulcers would lead to an effective healing. Although the diabetic chronic wounds microenvironment is hostile for local GFs bioavailability, EGF local infiltration circumvented the limitations of its topical application, thus expanding its therapeutic prospect. Our clinical pharmacovigilance and basic studies attest the significance of the GF local infiltration for chronic wounds healing.