RESUMO
The accelerated rise in antimicrobial resistance (AMR) poses a significant global health risk, necessitating the exploration of alternative strategies to combat pathogenic infections. Biofilm-related infections that are unresponsive to standard antibiotics often require the use of higher-order antimicrobials with toxic side effects and the potential to disrupt the microbiome. Probiotic therapy, with its diverse benefits and inherent safety, is emerging as a promising approach to prevent and treat various infections, and as an alternative to antibiotic therapy. In this study, we isolated novel probiotic bacteria from the gut of domestic goats (Capra hircus) and evaluated their antimicrobial and anti-biofilm activities against the 'ESKAPE' group of pathogens. We performed comprehensive microbiological, biochemical, and molecular characterizations, including analysis of the 16S-rRNA gene V1-V3 region and the 16S-23S ISR region, on 20 caprine gut-derived lactic acid bacteria (LAB). Among these, six selected Lactobacillus isolates demonstrated substantial biofilm formation under anaerobic conditions and exhibited robust cell surface hydrophobicity and autoaggregation, and epithelial cell adhesion properties highlighting their superior enteric colonization capability. Notably, these Lactobacillus isolates exhibited broad-spectrum growth inhibitory and anti-biofilm properties against 'ESKAPE' pathogens. Additionally, the Lactobacillus isolates were susceptible to antibiotics listed by the European Food Safety Authority (EFSA) within the prescribed Minimum Inhibitory Concentration limits, suggesting their safety as feed additives. The remarkable probiotic characteristics exhibited by the caprine gut-derived Lactobacillus isolates in this study strongly endorse their potential as compelling alternatives to antibiotics and direct-fed microbial (DFM) feed supplements in the livestock industry, addressing the escalating need for antibiotic-free animal products.
RESUMO
Endometritis is a uterine inflammatory disease that causes reduced livestock fertility, milk production and lifespan leading to significant economic losses to the dairy industry. Mesenchymal stem cells (MSC) may act as an alternative for inefficacy of antibiotics and rising antibiotic resistance in endometritis. The present study aimed to cure the chronic endometritic buffaloes using allogenic adipose-derived MSCs (AD-MSC). AD-MSCs were isolated from buffalo adipose tissue and characterized by multilineage differentiation as well as MSC-specific markers. The in vivo safety and efficacy were assessed after infusion of AD-MSCs. In safety trial, cells were administered in healthy buffaloes via different routes (IV and IC) followed by examination of clinical and hematological parameters. In efficacy study, AD-MSCs treatments (IV and IC) and antibiotic therapy (ABT) in endometritic buffaloes were comparatively evaluated. AD-MSCs did not induced any immunological reaction in treated buffaloes. PMN count, CRP levels and VDS were significantly (p ≤ 0.05) reduced after AD-MSCs infusions in IV and IC groups and no significant difference was observed in antibiotic group. The IV group was marked with 50% absolute risk reduction in endometritis and 50% live calf births after artificial insemination in comparison with ABT group. Anti-inflammatory cytokines (IL4 and IL10) and anti-microbial peptides (PI3, CATHL4, LCN2 and CST3) expressions were significantly (p ≤ 0.05) upregulated in IV group. The calf delivery rate after the treatments in IV group was higher (50%, 3 calves) than the other groups (IC: 33.3%, 2 calves; ABT: 16.6%, 1 calf). In conclusion, the administration of AD-MSCs through IV route was found to be safe and efficacious for alleviating chronic endometritis in dairy buffaloes.
Assuntos
Bison , Endometrite , Doença Inflamatória Pélvica , Feminino , Humanos , Animais , Endometrite/terapia , Endometrite/veterinária , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , BúfalosRESUMO
BACKGROUND: Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin alpha5beta1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine alpha5beta1, precluding its use in standard mouse xenograft models. METHODS: We generated a panel of rat-anti-mouse alpha5beta1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for alpha5beta1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models. RESULTS: A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin alpha5beta1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40-60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density. CONCLUSION: The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-alpha5beta1 activity and confirms that inhibition of integrin alpha5beta1 impedes angiogenesis and slows tumor growth in vivo.
Assuntos
Inibidores da Angiogênese/imunologia , Anticorpos Monoclonais/uso terapêutico , Integrina alfa5beta1/imunologia , Animais , Anexina A5/imunologia , Antineoplásicos/uso terapêutico , Morte Celular , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Feminino , Fibronectinas/antagonistas & inibidores , Fibronectinas/imunologia , Citometria de Fluxo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Integrina alfa5beta1/genética , Integrina alfa5beta1/uso terapêutico , Camundongos , Camundongos SCID , Placenta/imunologia , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/imunologia , Transplante HeterólogoRESUMO
A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. We previously showed that the Drosophila melanogaster rel family factor, Dorsal, is a substrate for Smt3 conjugation. The conjugation machinery was found to enhance Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. In this report, we show that Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, we examined the role of Smt3 conjugation in the immune response. We find that the conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Núcleo Celular/metabolismo , DNA/genética , Drosophila melanogaster/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Ativação Transcricional , Transfecção , Ubiquitina/metabolismoRESUMO
Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Integrina alfa5beta1/imunologia , Animais , Anticorpos Monoclonais Murinos , Células COS , Chlorocebus aethiops , Matriz Extracelular/metabolismo , Humanos , Concentração Inibidora 50 , Integrina alfa5beta1/química , Cinética , Macaca fascicularis , Degeneração Macular/tratamento farmacológico , Neovascularização Patológica , RituximabRESUMO
We have used the Eos Hu03 GeneChip array, which represents over 92% of the transcribed human genome, to measure gene expression in a panel of normal and diseased human tissues. This analysis revealed that E-selectin mRNA is selectively overexpressed in prostate cancer epithelium, a finding that correlated strongly with E-selectin protein expression as assessed by immunohistochemistry. Antibodies against E-selectin that blocked function failed to impede cancer cell growth, suggesting that overexpression of E-selectin was not essential for cell growth. However, a novel auristatin E-based antibody drug conjugate (ADC), E-selectin antibody valine-citrulline monomethyl-auristatin E, was a potent and selective agent against E-selectin-expressing cancer cell lines in vitro, with the degree of cytotoxicity varying with surface antigen density. Interestingly, sensitivity to the ADC differed among cell lines from different tissues expressing similar amounts of E-selectin and was found to correlate with sensitivity to free auristatin E. Furthermore, E-selectin-expressing tumors grown as xenografts in severe combined immunodeficient mice were responsive to treatment with E-selectin antibody valine-citrulline monomethyl-auristatin E in vivo, with more than 85% inhibition of tumor growth observed in treated mice. These findings demonstrate that an E-selectin-targeting ADC has potential as a prostate cancer therapy and validates a genomics-based paradigm for the identification of cancer-specific antigens suitable for targeted therapy.
Assuntos
Antineoplásicos/administração & dosagem , Selectina E/biossíntese , Imunotoxinas/metabolismo , Oligopeptídeos/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Selectina E/genética , Selectina E/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition. METHODS: Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured. RESULTS: XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects. CONCLUSIONS: Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency.
Assuntos
Glicemia/metabolismo , Leptina/metabolismo , Leptina/fisiologia , Obesidade/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptores para Leptina/metabolismo , Regulação Alostérica , Animais , Peso Corporal , Diabetes Mellitus/metabolismo , Meia-Vida , Hipotálamo/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Current treatments for advanced stage, hormone-resistant prostate cancer are largely ineffective, leading to high patient mortality and morbidity. To fulfill this unmet medical need, we used global gene expression profiling to identify new potential antibody-drug conjugate (ADC) targets that showed maximal prostate cancer-specific expression. TMEFF2, a gene encoding a plasma membrane protein with two follistatin-like domains and one epidermal growth factor-like domain, had limited normal tissue distribution and was highly overexpressed in prostate cancer. Immunohistochemistry analysis using a specific monoclonal antibody (mAb) to human TMEFF2 showed significant protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease. To evaluate anti-TMEFF2 mAbs as potential ADCs, one mAb was conjugated to the cytotoxic agent auristatin E via a cathepsin B-sensitive valine-citrulline linker. This ADC, Pr1-vcMMAE, was used to treat male severe combined immunodeficient mice bearing xenografted LNCaP and CWR22 prostate cancers expressing TMEFF2. Doses of 3 to 10 mg/kg of this specific ADC resulted in significant and sustained tumor growth inhibition, whereas an isotype control ADC had no significant effect. Similar efficacy and specificity was shown with huPr1-vcMMAE, a humanized anti-TMEFF2 ADC. No overt in vivo toxicity was observed with either murine or human ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with the murine TMEFF2 protein, implying minimal toxicity to other body tissues. These data support the further evaluation and clinical testing of huPr1-vcMMAE as a novel therapeutic for the treatment of metastatic and hormone-resistant prostate cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/imunologia , Oligopeptídeos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Antineoplásicos/química , Encéfalo/metabolismo , Membrana Celular/metabolismo , Proliferação de Células , Clonagem Molecular , DNA Complementar/metabolismo , Citometria de Fluxo , Folistatina/química , Humanos , Hibridomas/química , Imuno-Histoquímica , Cinética , Metástase Linfática , Masculino , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/química , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Fatores de Tempo , TransfecçãoRESUMO
The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. We have identified a novel protein named Dorsal-interacting protein 3 (Dip3) that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. We assess the possibility that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators.
Assuntos
Proteínas de Drosophila/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Fluorescência Verde , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Fosfoproteínas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a TwistRESUMO
Many therapeutic monoclonal antibodies act as antagonists to receptors by targeting and blocking the natural ligand binding site (orthosteric site). In contrast, the use of antibodies to target receptors at allosteric sites (distinct from the orthosteric site) has not been extensively studied. This approach is especially important in metabolic diseases in which endogenous ligand levels are dysregulated. Herein, we review our investigations of 3 categories of human monoclonal antibodies that bind allosterically to the insulin receptor (INSR) and affect its activity: XMetA, XMetS and XMetD. XMetA directly activates the INSR either alone or in combination with insulin. XMetS, in contrast, does not directly activate the INSR but markedly enhances the receptor's ability to bind insulin and potentiate insulin signaling. Both XMetA and XMetS are effective in controlling hyperglycemia in mouse models of diabetes. A third allosteric antibody, XMetD, is an inhibitor of INSR signaling. This antibody reverses insulin-induced hypoglycemia in a mouse model of hyperinsulinemia. These studies indicate, therefore, that allosteric antibodies to INSR can modulate its signaling and correct conditions of glucose dysregulation. These studies also raise the possibility that the use of allosteric antibodies can be expanded to other receptors for the treatment of metabolic disorders.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Hiperglicemia/tratamento farmacológico , Hipoglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Receptor de Insulina/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Insulina/sangue , Insulina/metabolismo , Proteína Oncogênica v-akt/metabolismo , FosforilaçãoRESUMO
Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling â¼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory hyperinsulinism in insulin resistant conditions.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD/metabolismo , Glucose/metabolismo , Receptor de Insulina/metabolismo , Sítio Alostérico , Animais , Peptídeo C/química , Células CHO , Separação Celular , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Citometria de Fluxo , Humanos , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Insulina/química , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Biblioteca de Peptídeos , Fosforilação , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.
Assuntos
Anticorpos Monoclonais/imunologia , Hiperinsulinismo Congênito/imunologia , Receptor de Insulina/antagonistas & inibidores , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/imunologia , Células CHO , Hiperinsulinismo Congênito/tratamento farmacológico , Hiperinsulinismo Congênito/patologia , Cricetinae , Cricetulus , Glucose/imunologia , Resistência à Insulina/imunologia , Camundongos , Ratos , Receptor de Insulina/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
Many patients with diabetes mellitus (both type 1 and type 2) require therapy to maintain normal fasting glucose levels. To develop a novel treatment for these individuals, we used phage display technology to target the insulin receptor (INSR) complexed with insulin and identified a high affinity, allosteric, human monoclonal antibody, XMetA, which mimicked the glucoregulatory, but not the mitogenic, actions of insulin. Biophysical studies with cultured cells expressing human INSR demonstrated that XMetA acted allosterically and did not compete with insulin for binding to its receptor. XMetA was found to function as a specific partial agonist of INSR, eliciting tyrosine phosphorylation of INSR but not the IGF-IR. Although this antibody activated metabolic signaling, leading to enhanced glucose uptake, it neither activated Erk nor induced proliferation of cancer cells. In an insulin resistant, insulinopenic model of diabetes, XMetA markedly reduced elevated fasting blood glucose and normalized glucose tolerance. After 6 weeks, significant improvements in HbA(1c), dyslipidemia, and other manifestations of diabetes were observed. It is noteworthy that hypoglycemia and weight gain were not observed during these studies. These studies indicate, therefore, that allosteric monoclonal antibodies have the potential to be novel, ultra-long acting, agents for the regulation of hyperglycemia in diabetes.
Assuntos
Anticorpos Monoclonais/farmacologia , Glicemia/fisiologia , Diabetes Mellitus Experimental/terapia , Receptor de Insulina/agonistas , Animais , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Biomarcadores , Células CHO , Células Cultivadas , Cricetinae , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Organismos Livres de Patógenos EspecíficosRESUMO
OBJECTIVE: Atherosclerosis is a condition that is increasingly contributing to worldwide mortality through complications such as stroke and myocardial infarction. IL-1ß plays multiple direct, local roles in the formation and stability of the atheroma by eliciting the production of additional cytokines and proteolytic enzymes from macrophages, endothelial cells (EC) and smooth muscle cells (SMC). We therefore tested whether an anti-IL-1ß antibody, XOMA 052, might inhibit the secretion of pro-atherogenic cytokines from macrophages in vitro and affect a positive outcome in the Apolipoprotein E-deficient mouse (ApoE(-/-)) model of atherosclerosis in vivo. METHODS AND RESULTS: In an in vitro co-culture model, XOMA 052 inhibited macrophage-induced secretion of key atherogenic cytokines from EC and SMC, including IL-6, IL-8, MCP-1 and TNFα. The release of degradative enzymes, such as the matrix metalloproteinases MMP-3 and MMP-9, was also decreased by XOMA 052. In addition, XOMA 052 inhibited the secretion of IL-7 from EC and IL-4 from SMC, cytokines not previously reported to be driven by IL-1ß in this context. In vivo, XMA052 MG1K, a chimeric murine version of XOMA 052, inhibited the formation of atherosclerotic lesions in the ApoE(-/-) model at all three doses tested. This effect was comparable to that reported for complete genetic ablation of IL-1ß or IL-1R1 on an ApoE(-/-) background and was associated with decreases in plasma non-HDL/HDL cholesterol ratio and plaque lipid content and macrophage infiltration. CONCLUSIONS: These results demonstrate for the first time that an antibody targeting IL-1ß can inhibit the progression of atherosclerosis in vivo, highlighting the importance of this key cytokine in cardiovascular disease.
Assuntos
Anticorpos Monoclonais/metabolismo , Apolipoproteínas E/genética , Aterosclerose/sangue , Biomarcadores/metabolismo , Interleucina-1beta/metabolismo , Placa Aterosclerótica/sangue , Animais , Apolipoproteínas E/sangue , Aterosclerose/imunologia , Peso Corporal , Técnicas de Cocultura , Citocinas/metabolismo , Células Endoteliais/citologia , Humanos , Lipídeos/química , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/citologia , Placa Aterosclerótica/imunologiaRESUMO
Angiogenesis, the process by which new blood vessels form from existing vasculature, is critical for tumor growth and invasion. Growth factors, such as VEGF, initiate signaling cascades resulting in the proliferation of resting endothelial cells. Blockade of growth factor pathways has proven effective in inhibiting angiogenesis and tumor growth in vivo. Integrins, including the integrin alpha5beta1, are also important mediators of angiogenesis and these adhesion molecules also regulate cancer cell growth and migration in vitro. Volociximab is a high affinity, function-blocking antibody against integrin alpha5beta1 that is currently in multiple Phase II oncology clinical trials. Volociximab displays potent anti-angiogenic activity in a monkey model of choroidal neovascularization. In this study, we explored the consequences of integrin alpha5beta1 blockade on tumorigenesis. Because volociximab does not cross-react with rodent alpha5beta1, the syngeneic rabbit VX2 carcinoma model was utilized as an alternative to standard mouse xenograft models for the assessment of anti-tumor activity of volociximab. Volociximab administered intravenously to rabbits bearing VX2 tumors is detectable on tumor cells and vasculature 45 min post-administration. Volociximab was found to significantly inhibit the growth of tumors growing subcutaneously or intramuscularly, despite a 20-fold lower affinity for rabbit integrin, relative to human. This effect was found to correlate with decreased blood vessel density within these tumors. These results support the use of volociximab in the intervention of malignant disease.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Integrina alfa5beta1/antagonistas & inibidores , Neoplasias Experimentais/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Ensaios Clínicos Fase III como Assunto , Relação Dose-Resposta a Droga , Fibronectinas/metabolismo , Humanos , Imunoquímica , Injeções Intravenosas , Integrina alfa5beta1/imunologia , Integrina alfa5beta1/metabolismo , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ligação Proteica , Coelhos , Especificidade da Espécie , Ressonância de Plasmônio de Superfície/métodos , Carga Tumoral/efeitos dos fármacosRESUMO
E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of alpha(5)-integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an alpha(5)beta(1)-integrin-blocking antibody. When E-cadherin is silenced, alpha(5)-integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogen-activated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/beta-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of alpha(5)-integrin, i.p. treatment with an alpha(5)beta(1)-integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). alpha(5)-Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had alpha(5)-integrin overexpression, and 39% had some level of alpha(5)-integrin expression. The median survival for patients with high alpha(5)-integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified alpha(5)-integrin up-regulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.
Assuntos
Caderinas/antagonistas & inibidores , Integrina alfa5/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/metabolismo , Western Blotting , Caderinas/biossíntese , Caderinas/metabolismo , Adesão Celular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa5/biossíntese , Integrina alfa5/genética , Integrina alfa5beta1/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/enzimologia , Neoplasias Peritoneais/metabolismo , Prognóstico , TransfecçãoRESUMO
SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substrate-specific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.