EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation.

Introduction: Colorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations. Methods: We generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells. Results: Using flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture. Discussion: We conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity.

Epidermal Growth Factor Modulates Palmitic Acid-Induced Inflammatory and Lipid Signaling Pathways in SZ95 Sebocytes.

Epidermal growth factor (EGF) acts as a paracrine and autocrine mediator of cell proliferation and differentiation in various types of epithelial cells, such as sebocytes, which produce the lipid-rich sebum to moisturize the skin. However, sebum lipids via direct contact and by penetrating through the epidermis may have regulatory roles on epidermal and dermal cells as well. As EGF receptor (EGFR) is expressed throughout the proliferating and the lipid-producing layers of sebaceous glands (SGs) in healthy and acne-involved skin, we investigated the effect of EGF on SZ95 sebocytes and how it may alter the changes induced by palmitic acid (PA), a major sebum component with bioactive roles. We found that EGF is not only a potent stimulator of sebocyte proliferation, but also induces the secretion of interleukin (IL)6 and down-regulates the expression of genes involved in steroid and retinoid metabolism. Importantly, when applied in combination with PA, the PA-induced lipid accumulation was decreased and the cells secreted increased IL6 levels. Functional clustering of the differentially regulated genes in SZ95 sebocytes treated with EGF, PA or co-treated with EGF+PA further confirmed that EGF may be a potent inducer of hyperproliferative/inflammatory pathways (IL1 signaling), an effect being more pronounced in the presence of PA. However, while a group of inflammatory genes was up-regulated significantly in EGF+PA co-treated sebocytes, PA treatment in the absence of EGF, regulated genes only related to cell homeostasis. Meta-analysis of the gene expression profiles of whole acne tissue samples and EGF- and EGF+PA -treated SZ95 sebocytes showed that the EGF+PA co-activation of sebocytes may also have implications in disease. Altogether, our results reveal that PA-induced lipid accumulation and inflammation can be modulated by EGF in sebocytes, which also highlights the need for system biological approaches to better understand sebaceous (immuno)biology.

Blockade of epidermal growth factor and its receptor and axial elongation in experimental myopia.

To examine the influence of epidermal growth factor (EGF) and its receptor (EGFR) on axial ocular elongation, we intraocularly injected an EGF antibody and an EGFR antibody into young guinea pigs with lens-induced axial elongation (myopization). Mean axial elongation was reduced in the eyes injected with the EGF/EGFR-antibody compared with the contralateral control eyes injected with PBS (phosphate-buffered solution) (0.43 ± 0.13 mm vs 0.53 ± 0.13 mm; P < .001). The intereye difference in axial length increased (P = .005) as the doses of the EGF antibody and EGFR antibody increased. As a corollary, the thickness of the retina at the posterior pole was dose-dependently increased in the injected eyes compared to the contralateral control eyes. Immunohistochemical staining for EGF and the relative mRNA expression of EGF and EGFR were the highest in eyes not injected with the EGF antibody or EGFR antibody and decreased (P < .05) as the dose of EGF antibody or EGFR antibody increased. In an in vitro study, EGF had a stimulating effect and the EGF antibody had an inhibitory effect on the proliferation and migration of RPE cells. The findings showed that the intravitreal application of an EGF antibody and EGFR antibody is associated with a dose-dependent reduction in lens-induced axial elongation in young guinea pigs. The EGFR family may play a role in axial elongation of the eye and in the development of myopia.

Identifying predictive biomarkers of CIMAvaxEGF success in non-small cell lung cancer patients.

BACKGROUND: Immunosenescence biomarkers and peripheral blood parameters are evaluated separately as possible predictive markers of immunotherapy. Here, we illustrate the use of a causal inference model to identify predictive biomarkers of CIMAvaxEGF success in the treatment of Non-Small Cell Lung Cancer Patients. METHODS: Data from a controlled clinical trial evaluating the effect of CIMAvax-EGF were analyzed retrospectively, following a causal inference approach. Pre-treatment potential predictive biomarkers included basal serum EGF concentration, peripheral blood parameters and immunosenescence biomarkers. The proportion of CD8 + CD28- T cells, CD4+ and CD8+ T cells, CD4/CD8 ratio and CD19+ B cells. The 33 patients with complete information were included. The predictive causal information (PCI) was calculated for all possible models. The model with a minimum number of predictors, but with high prediction accuracy (PCI > 0.7) was selected. Good, rare and poor responder patients were identified using the predictive probability of treatment success. RESULTS: The mean of PCI increased from 0.486, when only one predictor is considered, to 0.98 using the multivariate approach with all predictors. The model considering the proportion of CD4+ T cell, basal Epidermal Growth Factor (EGF) concentration, neutrophil to lymphocyte ratio, Monocytes, and Neutrophils as predictors were selected (PCI > 0.74). Patients predicted as good responders according to the pre-treatment biomarkers values treated with CIMAvax-EGF had a significant higher observed survival compared with the control group (p = 0.03). No difference was observed for bad responders. CONCLUSIONS: Peripheral blood parameters and immunosenescence biomarkers together with basal EGF concentration in serum resulted in good predictors of the CIMAvax-EGF success in advanced NSCLC. Future research should explore molecular and genetic profile as biomarkers for CIMAvax-EGF and it combination with immune-checkpoint inhibitors. The study illustrates the application of a new methodology, based on causal inference, to evaluate multivariate pre-treatment predictors. The multivariate approach allows realistic predictions of the clinical benefit of patients and should be introduced in daily clinical practice.

Epidermal growth factor stimulates exosomal microRNA-21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells.

Regulatory T cells (Tregs), a subset of CD4+ T cells, may exert inhibitory effects on alloimmune responses including acute graft-versus-host disease (aGVHD), and several microRNAs are implicated in the pathophysiological process of GVHD. Therefore, we aimed in the present study to characterize the functional relevance of epidermal growth factor (EGF)-stimulated microRNA-21 (miR-21) in regulating bone marrow-derived mesenchymal stem cells (BMSCs) in a mouse model of aGVHD. We first isolated and cultured BMSCs and Tregs. Then, we examined effects of miR-21 knockdown or overexpression and EGF on cell activities of BMSCs and the expression of PTEN, Foxp3, AKT phosphorylation, and extent of c-jun phosphorylation by gain- and loss-of-function approaches. The results showed that miR-21 promoted the proliferation, invasion, and migration of BMSCs. Furthermore, miR-21 in BMSCs-derived exosomes inhibited PTEN, but enhanced AKT phosphorylation and Foxp3 expression in Tregs. In addition, EGF enhanced c-jun phosphorylation to elevate the miR-21 expression. Furthermore, EGF significantly increased the efficacy of BMSCs in a mouse model of aGVHD, manifesting in reduced IFN-γ expression and lesser organ damage. Moreover, EGF treatment promoted the Foxp3 expression of Tregs in BMSCs-treated aGVHD mice. Taken together, EGF induced the BMSCs-derived exosomal miR-21 expression, which enhanced Foxp3 expression in Tregs, thereby improving the therapeutic effect of BMSCs on aGVHD.

Eosinophilic recruitment in thermally injured older animals is associated with worse outcomes and higher conversion to full thickness burn.

BACKGROUND: Partial burn injury in older patients is associated with higher rates of morbidity, mortality, and conversion to full thickness burn (Finnerty et al., 2009; Pham et al., 2009). Both human and mouse models demonstrate an altered systemic immune response in older subjects, however less is known about the localized response (Jeschke et al., 2016; Farinas et al., 2018; Mohs et al., 2017). We hypothesized that a mouse model could demonstrate differences in the localized inflammatory response of the old. METHODS: Six old (66 weeks) and young (8 weeks) mice received partial thickness thermal burns. Localized and systemic expression of nine chemokines (TNFalpha, MCP-1, MIP-2, S100A9, EGF, IL-10, RANTES, G-CSF, and EOTAXIN) were evaluated at day 3 after burn using Luminex analysis. Vimentin immunostaining was used to evaluate injury depth. RESULTS: Vimentin staining demonstrated increased burn depth in old mice (449±38µm) as compared to young (166±18µm) (p<0.05). Both groups exhibited increased localized expression of EOTAXIN after burn (p<0.05), however expression in old mice (83.6±6.1pg/ml) was lower than that of young (126.8±18.7pg/ml) (p<0.05). Systemically, however, old mice had increased baseline EOTAXIN expression (1332.40±110.78pg/ml) compared to young (666.12±45.8pg/ml) (p<0.005). CONCLUSIONS: EOTAXIN is one of the primary chemoattractants for selective eosinophilic recruitment and activation. While eosinophils are important for wound healing, a hyperactive eosinophilic response can result in tissue damage. We hypothesize that the increased baseline serum EOTAXIN in the old may prime their hyperactive response, and may contribute to their worse clinical outcomes. Long-term eosinophil activation requires further study, however our findings indicate a role for EOTAXIN and eosinophils in burn response.

Inhibition of parasite invasion by monoclonal antibody against epidermal growth factor-like domain of Plasmodium vivax merozoite surface protein 1 paralog.

The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P), which has epidermal growth factor (EGF)-like domains, was identified as a novel erythrocyte adhesive molecule. This EGF-like domain (PvMSP1P-19) elicited high level of acquired immune response in patients. Antibodies against PvMSP1P significantly reduced erythrocyte adhesion activity to its unknown receptor. To determine PvMSP1P-19-specific antibody function and B-cell epitopes in vivax patients, five monoclonal antibodies (mAbs) and 18-mer peptides were generated. The mAb functions were determined by erythrocyte-binding inhibition assay and invasion inhibition assay with P. knowlesi. B-cell epitopes of PvMSP1P-19 domains were evaluated by peptide microarray. The pvmsp1p-19 sequences showed limited polymorphism in P. vivax worldwide isolates. The 1BH9-A10 showed erythrocyte binding inhibitory by interaction with the N-terminus of PvMSP1P-19, while this mAb failed to recognize PkMSP1P-19 suggesting the species-specific for P. vivax. Other mAbs showed cross-reactivity with PkMSP1P-19. Among them, the 2AF4-A2 and 2AF4-A6 mAb significantly reduced parasite invasion through C-terminal recognition. The linear B-cell epitope in naturally exposed P. vivax patient was identified at three linear epitopes. In this study, PvMSP1P-19 N-terminal-specific 1BH9-A10 and C-terminal-specific 2AF4 mAbs showed functional activity for epitope recognition suggesting that PvMSP1P may be useful for vaccine development strategy for specific single epitope to prevent P. vivax invasion.

CIMAvax-EGF: Toward long-term survival of advanced NSCLC.

Lung cancer remains one of the leading causes of cancer-related deaths. Non-small cell lung cancer (NSCLC) is the most common histologic type of lung cancer. Medical and scientific progress has led to longer survival in an increasing number of patients suffering from cancer. Concerning patients with advanced NSCLC, there is a subgroup with long-term survival. The human epidermal growth factor receptor (EGFR) family plays a key role in tumor development. This cluster of genes is associated with augmented angiogenesis and enhanced proliferation, survival, and migration of tumor cells. The CIMAvax-EGF vaccine consists of a chemical conjugate of the EGF with the P64 protein derived from the Meningitis B bacteria and the Montanide ISA 51, as adjuvant. The vaccine induces antibodies against EGF that results in EGF withdrawal. CIMAvax-EGF has been demonstrated to be safe and immunogenic in advanced NSCLC patients. Here we summarize the current knowledge of the mechanism of action of CIMAvax-EGF, highlighting the impact of this anti-EGF-based vaccine on the long-term survival of advanced NSCLC patients.

Effect of HTST and Holder Pasteurization on the Concentration of Immunoglobulins, Growth Factors, and Hormones in Donor Human Milk.

Donor human milk (DHM) is submitted to Holder pasteurization (HoP) to ensure its microbiological safety in human milk banks but this treatment affects some of its bioactive compounds. The objective of this work was to compare the effects of HoP and high temperature short time (HTST) treatments on some bioactive compounds found in DHM. A total of 24 DHM batches were processed in a continuous HTST system (70, 72, and 75°C for 5-25 s) and by HoP (62.5°C for 30 min). The concentrations of immunoglobulins (Igs) A, G, and M, transforming growth factor-beta 2 (TGF-ß2), adiponectine, ghrelin, and leptin were measured using a multiplex system, whereas the concentration of epidermal growth factor (EGF) was determined by ELISA. In relation to Igs, IgG showed the highest preservation rates (87-101%) after HTST treatments, followed by IgA (54-88%) and IgM (25-73%). Ig retention after any of the HTST treatments was higher than after HoP (p < 0.001). Treatment times required to reduce the concentration of IgM by 90% (D-value) were 130, 88, and 49 s at 70, 72, and 75°C, while the number of degrees Celsius required to change the D-value by one factor of 10 (z-value) was 11.79°C. None of the heat treatments had a significant effect on the concentrations of TGF-ß2, EGF, adiponectin, and ghrelin. In contrast, leptin was detected only in 4 of the samples submitted to HoP, whereas it was present in all samples after the different HTST treatments, with retention rates ranging between 34 and 68%. Globally, the concentration of IgA, IgG, IgM, and leptin in DHM was significantly higher after HTST pasteurization performed in a continuous system designed to be used in human milk banks than after the HoP procedure that is routinely applied at present.

EGF ligand fused to truncated Pseudomonas aeruginosa exotoxin A specifically targets and inhibits EGFR­positive cancer cells.

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple­targeted therapies have been developed, with a recent clinical application of the antibody­mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N­terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF­ETA), which has ADP­ribosylation activity and induces apoptosis. The EGF­ETA protein was expressed in E. coli as a His­tagged fusion. Our results showed that EGF­ETA significantly inhibited the proliferation of EGFR­positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549­IC50 1,000 ng/ml; MCF­7­IC50 >10,000 ng/ml). Compared to cetuximab, EGF­ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF­ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild­type HT29 colon cancer cells. Finally, co­incubation of EGF­ETA with an anti­EGF antibody abrogated its effect on the EGFR­positive A431 cells. Our results show that the chimeric EGF­ETA toxin is extremely effective against EGFR­positive cancers and raises the potential to further develop this chimera for use in targeting EGFR­positive tumours resistant to monoclonal antibodies.

Modulation of Apoptosis by Cytotoxic Mediators and Cell-Survival Molecules in Sjögren's Syndrome.

The pathogenesis of Sjögren's syndrome (SS) involves multiple factors including genetic background, cell death, and exocrine dysfunction. We here discuss apoptotic control in exocrine glands in SS by showing various pro- and anti-apoptotic pathways. Although the membrane-bound and soluble form of the Fas/Fas ligand system is a leading player with activation of the death domain and caspase 8/3 cleavage, the role of soluble Fas/FasL (including its polymorphism) in apoptosis is controversial. The tumor necrosis factor related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of salivary gland epithelial cells (SGECs) involves a mitochondrial pathway that includes caspase 9 cleavage. The involvement of innate immunity cells such as toll-like receptors (TLRs) has been investigated; TLR2-4 and TLR7-9 are associated with the induction of inflammation in exocrine glands of SS patients. TLR3 has the potential to induce the apoptosis of SS patients' SGECs. Linkage of epidermal growth factor (EGF) was shown in exocrine glands in SS, and it inhibited the Fas/FasL system with the help of cell-survival factors. TLR3 has dual actions to cause inflammation as well as apoptosis, which are inhibited by EGF. In conclusion, apoptosis in exocrine glands of SS patients is tightly controlled by balance of pro-apoptotic signals and growth factor.

Cripto-1 Plasmid DNA Vaccination Targets Metastasis and Cancer Stem Cells in Murine Mammary Carcinoma.

Metastatic breast cancer is a fatal disease that responds poorly to treatment. Cancer vaccines targeting antigens expressed by metastatic breast cancer cells and cancer stem cells could function as anticancer therapies. Cripto-1 is an oncofetal protein overexpressed in invasive breast cancer and cancer-initiating cells. In this study, we explored the potential of a Cripto-1-encoding DNA vaccine to target breast cancer in preclinical mouse models. BALB/c mice and BALB-neuT mice were treated with a DNA vaccine encoding mouse Cripto-1 (mCr-1). BALB/c mice were challenged with murine breast cancer 4T1 cells or TUBO spheres; BALB-neuT mice spontaneously developed breast cancer. Tumor growth was followed in all mouse models and lung metastases were evaluated. In vitro assays were performed to identify the immune response elicited by vaccination. Vaccination against mCr-1 reduced primary tumor growth in the 4T1 metastatic breast cancer model and reduced lung metastatic burden. In BALB-neuT mice, because the primary tumors are Cripto-1 negative, vaccination against mCr-1 did not affect primary tumors but did reduce lung metastatic burden. Spheroid-cultured TUBO cells, derived from a BALB/neuT primary tumor, develop a cancer stem cell-like phenotype and express mCr-1. We observed reduced tumor growth in vaccinated mice after challenge with TUBO spheres. Our data indicate that vaccination against Cripto-1 results in a protective immune response against mCr-1 expressing and metastasizing cells. Targeting Cripto-1 by vaccination holds promise as an immunotherapy for treatment of metastatic breast cancer. Cancer Immunol Res; 6(11); 1417-25. ©2018 AACR.

HCV modifies EGF signalling and upregulates production of CXCR2 ligands: Role in inflammation and antiviral immune response.

BACKGROUND & AIMS: To affect immune response and inflammation, the hepatitis C virus (HCV) substantially influences intercellular communication pathways that are decisive for immune cell recruitment. The present study investigates mechanisms by which HCV modulates chemokine-mediated intercellular communication from infected cells. METHODS: Chemokine expression was studied in HCVcc-infected cell lines or cell lines harbouring a subgenomic replicon, as well as in serum samples from patients. Expression or activity of mediators and signalling intermediates was manipulated using knockdown approaches or specific inhibitors. RESULTS: HCV enhances expression of CXCR2 ligands in its host cell via the induction of epidermal growth factor (EGF) production. Knockdown of EGF or of the p65 subunit of the NF-κB complex results in a substantial downregulation of HCV-induced CXCR2 ligand expression, supporting the involvement of an EGF-dependent mechanism as well as activation of NF-κB. Furthermore, HCV upregulates expression of CXCR2 ligands in response to EGF stimulation via downregulation of the T-cell protein tyrosine phosphatase (TC-PTP [PTPN2]), activation of NF-κB, and enhancement of EGF-inducible signal transduction via MEK1 (MAP2K1). This results in the production of a cytokine/chemokine pattern by the HCV-infected cell that can recruit neutrophils but not monocytes. CONCLUSIONS: These data reveal a novel EGF-dependent mechanism by which HCV influences chemokine-mediated intercellular communication. We propose that this mechanism contributes to modulation of the HCV-induced inflammation and the antiviral immune response. LAY SUMMARY: In most cases hepatitis C virus (HCV) results in chronic infection and persistent viral replication, taking decades until development of overt disease. To achieve such a course, the respective virus must have developed mechanisms to circumvent antiviral response, to modulate the inflammatory response and to utilise the infrastructure of its host with moderate effect on its viability. The present study provides novel data indicating that HCV induces epidermal growth factor production in its host cell, enhancing epidermal growth factor-inducible expression of chemokines that bind to the CXCR2 receptor and recruit neutrophile granulocytes. Importantly, chemokines are critical mediators determining the pattern of immune cells recruited to the site of injury and thereby the local inflammatory and immunological milieu. These data strongly suggest that HCV triggers mechanisms that enable the virus to influence the inflammatory and immunological processes of its host.

Therapeutic cancer vaccine: phase I clinical tolerance study of Hu-rhEGF-rP64k/Mont in patients with newly diagnosed advanced non-small cell lung cancer.

BACKGROUND: Hu-rhEGF-rP64k/Mont is a biotechnology product for the treatment of advanced non-small cell lung cancer (NSCLC). The vaccine induces a neutralizing antibody-mediated immune response, against the normal circulating self-protein antigen epidermal growth factor (EGF), which prevents its binding to and activation of the EGF receptor, inhibiting the transduction of the signals that drive cancer cell proliferation, survival and spread. This phase I study aimed to evaluate the safety and the immunological response of Hu-rhEGF-rP64k vaccine in NSCLC patients. RESULTS: The Hu-rhEGF-rP64k/Mont vaccine showed to be safe and well tolerated, with dizziness, injection-site reactions and tremors being the most commonly reported adverse event. No severe adverse events or death were related to the vaccination. Immune monitoring demonstrated the generation of anti-EGF antibody titers and as a consequence the patients exhibited a decrease in the EGF concentration. In 80% of the vaccinated patients stable disease was achieved. CONCLUSION: Hu-rhEGF-rP64k/Mont elicited a valuable immune response, with good safety profile assuring further clinical development of the vaccine in this population to further confirm the potential benefits on survival. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-OID-17014048 , date 2017/12/20 (retrospectively registered); Chinese Food and Drug Administration, CFDA 2009 L02105, date 2009/04/03; China Drug Trial, CTR20131039 .

Emerging roles of rhomboid-like pseudoproteases in inflammatory and innate immune responses.

Rhomboid-like pseudoproteases are a conserved superfamily of proteins related to the rhomboid intramembrane serine proteases that lack key catalytic residues. iRhom2, a member of the rhomboid-like pseudoprotease superfamily, regulates the maturation and trafficking of ADAM17 and is associated with inflammatory arthritis. Recent studies demonstrate that iRhom2 is also involved in innate immunity by regulating the trafficking and stability of MITA (also called STING), which is a central adaptor in innate antiviral signalling pathways. Here, we summarize recent progress on the roles and mechanisms of iRhom2 and its homologues in innate immunity and also discuss the links between the physiological functions of iRhoms and immunological diseases.

Immune dysregulation in offspring of a bipolar parent. Altered serum levels of immune growth factors at adolescent age.

Immune dysregulation plays a role in the vulnerability for mood disorders. Immune growth factors, such as Stem Cell Factor (SCF), Insulin-like Growth Factor-Binding Protein-2 (IGF-BP2), Epidermal Growth Factor (EGF), IL-7 and sCD25 have repeatedly been reported altered in patients with mood disorders. The aim of this study was to investigate levels of these factors in serum of adolescent bipolar offspring, who have a heightened risk for mood disorder development and to also analyze the data combined with previously published data. Growth factors were assessed by CBA/ELISA in adolescent bipolar offspring (n=96, mean age=16years) and in age- and gender-matched healthy controls (n=50). EGF belonged to a mutually correlating cluster of mainly neurotrophic compounds including S100B and BDNF, which were in general decreased in serum. IL-7, SCF, IGF-BP2 and sCD25, belonged to a different mutually correlating cluster of immune growth factors, which were in general increased: IGF-BP2 significantly in serum of offspring without a mood disorder, IL-7 and SCF in serum of offspring who had experienced a mood episode. This pattern of de- and increases was not different between bipolar offspring that developed or did not develop a mood disorder over time, apart from the IGF-BP2 level, which was near significantly higher in offspring later developing a mood disorder. Correlations with the previously published immune-cellular abnormalities were not found. In conclusion non-affected adolescents at familial mood disorder development risk were characterized by a distinct pattern of a series of compounds operating in a network of hematopoiesis, neurogenesis and inflammation.

Expresión de E-cadherina y factor de crecimiento epidérmico en leucoplasias orales / Expression of E-cadherin and epidermal growth factor in oral leukoplakia

Introducción: se define a las leucoplasias orales como una placa blancaque no puede desprenderse por raspado y que no puede clasifi carse comoninguna otra lesión. Son lesiones con potencial maligno, relacionadascon la presencia de displasia epitelial. Estos cambios preneoplásicospueden ser evidenciados histológicamente como también a travésde técnicas que pongan en evidencia los diferentes cambios a nivelmolecular. La E-cadherina es una glicoproteína membranosa quedesempeña papeles importantes en el mantenimiento de la adhesióncélula-célula, la preservación de la polaridad del tejido epitelial y laintegridad estructural. Los factores de crecimiento epidérmico son unconjunto de moléculas de naturaleza proteica, biorreguladores, cuyafuncionalidad fundamental radica en el control del ciclo celular. Elobjetivo del presente trabajo es identifi car y comparar parámetros histológicosy moleculares predictores de riesgo de transformación malignaen leucoplasias orales. Material y métodos: El estudio correspondea un diseño observacional descriptivo. Se seleccionaron muestras de26 biopsias de leucoplasias orales, las cuales fueron evaluadas contécnica histológica de rutina y tinción con hematoxilina y eosina, luegosometidas a inmunomarcación con factor de crecimiento epidérmico yE-cadherina, donde se evaluó la intensidad de tinción y cambios en laexpresión de cada marcador, así como la localización en los diferentessubtipos celulares. Resultados: De las 26 leucoplasias observadas,16 mostraron histología con cambios hiperplásicos y 10 con cambiosdisplásicos leves a moderados. La expresión de E-cadherina no mostróalteraciones signifi cativas en leucoplasias sin displasia, sólo hubopérdida de expresión en aquellas leucoplasias con cambios displásicosde alto grado, en concordancia a los hallazgos histológicos...

Introduction: oral leukoplakia is defined as a white plaque thatcannot be removed by scraping and cannot be classifi ed as any otherdisease entity. They are potentially malignant lesions related to thepresence of epithelial dysplasia. These preneoplastic changes can bedetected histologically, as well as through techniques that demonstratediff erent changes at the molecular level. E-cadherin is a membraneglycoprotein that plays a major role in maintaining cell-cell adhesion,preserving structural integrity and the polarity of epithelial tissue.Epidermal growth factors are a group of bio-regulatory proteins,whose primary function is to control the cell cycle. The aim of thisstudy is to identify and compare the parameters for histological andmolecular markers for malignant transformation in oral leukoplakia.Material and methods: The study was observational and descriptive indesign. Samples were selected from 26 oral leukoplakia biopsies, whichwere routinely evaluated for histology and stained with hematoxylinand eosin, then subjected to immunostaining with epidermal growthfactor and E-cadherin, with the intensity of staining and changes inthe expression of each marker being evaluated. Results: Of the 26leukoplakia examined, 16 showed hyperplastic changes and 10 mildto moderate dysplastic changes. The expression of E-cadherin showedno signifi cant changes in non-dysplastic leukoplakia, while a lossof expression was found in only those leukoplakias with high-gradedysplastic changes, which was consistent with the histological fi ndings...

An enzyme immunoassay for determining epidermal growth factor (EGF) in human serum samples using an ultramicroanalytical system.

Human epidermal growth factor is a small peptide consisting of 53 amino acid residues, which stimulates cell proliferation and is associated with several human carcinomas. A simple sandwich-type ultramicroELISA assay (UMELISA), based on the advantages of high affinity reaction between streptavidin and biotin has been developed for the measurement of EGF in human serum samples. Strips coated with a high affinity monoclonal antibody directed against EGF are used as solid phase, to ensure the specificity of the assay. The EGF assay was completed in 18 hr, with a measuring range of 39-2500 pg/mL. The intra- and inter-assay coefficients of variation were 4.4-7.3% and 0-5.1%, respectively, depending on the EGF concentrations evaluated. Percentage recovery ranged from 96-104%. Regression analysis showed a good correlation with the commercially available Human EGF Immunoassay Quantikine® ELISA kit (n = 130, r = 0.92, P < 0.01). The analytical performance characteristics of our UMELISA EGF endorse its use for the quantification of EGF in human serum samples.

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1.

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1(-/-)Ifng(-/-) mice with dimethylnitrosamine or carbon tetrachloride. Ifng(-/-) and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1(-/-)Ifng(-/-) mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng(-/-) mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.