ABSTRACT
Influenza hemagglutinin (HA) is considered a major protective antigen of seasonal influenza vaccine but antigenic drift of HA necessitates annual immunizations using new circulating HA versions. Low variation found within conserved non-HA influenza virus (INFV) antigens may maintain protection with less frequent immunizations. Conserved antigens of influenza A virus (INFV A) that can generate cross protection against multiple INFV strains were evaluated in BALB/c mice using modified Vaccinia virus Ankara (MVA)-vectored vaccines that expressed INFV A antigens hemagglutinin (HA), matrix protein 1 (M1), nucleoprotein (NP), matrix protein 2 (M2), repeats of the external portion of M2 (M2e) or as tandem repeats (METR), and M2e with transmembrane region and cytoplasmic loop (M2eTML). Protection by combinations of non-HA antigens was equivalent to that of subtype-matched HA. Combinations of NP and forms of M2e generated serum antibody responses and protected mice against lethal INFV A challenge using PR8, pandemic H1N1 A/Mexico/4108/2009 (pH1N1) or H5N1 A/Vietnam/1203/2004 (H5N1) viruses, as demonstrated by reduced lung viral burden and protection against weight loss. The highest levels of protection were obtained with NP and M2e antigens delivered as MVA inserts, resulting in broadly protective immunity in mice and enhancement of previous natural immunity to INFV A.
Subject(s)
Antibodies, Viral/blood , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/immunology , Nucleocapsid Proteins/immunology , Orthomyxoviridae Infections/prevention & control , Viral Matrix Proteins/immunology , Viroporin Proteins/immunology , Animals , Antigens, Viral/immunology , Cross Protection , Female , Genetic Vectors , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza Vaccines/administration & dosage , Mice, Inbred BALB C , Nucleocapsid Proteins/administration & dosage , Orthomyxoviridae Infections/immunology , Pandemics , Vaccination , Viral Matrix Proteins/administration & dosage , Viral Matrix Proteins/genetics , Viroporin Proteins/administration & dosageABSTRACT
The U.S. Food and Drug Administration (FDA) hosted a public workshop entitled "Advancing Animal Models for Antibacterial Drug Development" on 5 March 2020. The workshop mainly focused on models of pneumonia caused by Pseudomonas aeruginosa and Acinetobacter baumannii The program included discussions from academic investigators, industry, and U.S. government scientists. The potential use of mouse, rabbit, and pig models for antibacterial drug development was presented and discussed.
Subject(s)
Acinetobacter baumannii , Anti-Bacterial Agents , Animals , Anti-Bacterial Agents/therapeutic use , Drug Development , Mice , Models, Animal , Rabbits , Swine , United States , United States Food and Drug AdministrationABSTRACT
Francisella tularensis is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans. The incidence of tularemia is very low with a lack of comprehensive data that describe disease in humans due to difficulty in understanding time and routes of exposure. Under the title Operation Whitecoat, researchers at Ft. Detrick, MD conducted 40 clinical studies of tularemia from 1958 to 1968. In these studies, one of the objectives was to evaluate candidate countermeasures for treatment or prophylaxis of disease after exposure to Francisella tularensis strain Schu S4 by inhalation. These studies were reviewed retrospectively to delineate the early signs and symptoms or natural history of pneumonic tularemia and examine the efficacy of tetracycline in controlled human clinical studies. Using vital signs, onset of fever was objectively defined and calculated for each subject, while Adverse Events reported after exposure were also used to define the timing of disease onset and symptoms of early disease. There was a dose response relationship between time to fever onset and exposed dose at 200 cfu (172.8 h), 700 cfu (163.2 h), 2,500 cfu (105.3 h), and 25,000 cfu (75.5 h). Onset of fever was typically the earliest sign of disease at all doses but was often accompanied by symptoms such as headache, myalgia, chest pain, and nausea, irrespective of dose except at 200 cfu where only 50% of subjects exhibited fever onset or symptoms. Examining the efficacy of different treatment regimens of tetracycline, ineffective treatments were indicated by relapse of disease (fever and Adverse Events) after cessation of antibiotic treatment. Stratification of the data suggested that treatment for <14 days or doses <2g/day was associated with increased percentage of subjects with relapse of disease symptoms. Although these types of human challenge studies would not be ethically possible now, the climate post-World War II supported human testing under rigorous conditions with informed consent. Thus, going back and analyzing these unique clinical human challenge studies has helped describe the course of infection and disease induced by a biothreat pathogen and possible countermeasures for treatment under controlled conditions.
ABSTRACT
In June 2017, the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, organized a workshop entitled "Pharmacokinetics-Pharmacodynamics (PK/PD) for Development of Therapeutics against Bacterial Pathogens." The aims were to discuss details of various PK/PD models and identify sound practices for deriving and utilizing PK/PD relationships to design optimal dosage regimens for patients. Workshop participants encompassed individuals from academia, industry, and government, including the United States Food and Drug Administration. This and the accompanying review on clinical PK/PD summarize the workshop discussions and recommendations. Nonclinical PK/PD models play a critical role in designing human dosage regimens and are essential tools for drug development. These include in vitro and in vivo efficacy models that provide valuable and complementary information for dose selection and translation from the laboratory to human. It is crucial that studies be designed, conducted, and interpreted appropriately. For antibacterial PK/PD, extensive published data and expertise are available. These have been leveraged to develop recommendations, identify common pitfalls, and describe the applications, strengths, and limitations of various nonclinical infection models and translational approaches. Despite these robust tools and published guidance, characterizing nonclinical PK/PD relationships may not be straightforward, especially for a new drug or new class. Antimicrobial PK/PD is an evolving discipline that needs to adapt to future research and development needs. Open communication between academia, pharmaceutical industry, government, and regulatory bodies is essential to share perspectives and collectively solve future challenges.
Subject(s)
Anti-Bacterial Agents/pharmacokinetics , Animals , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Bacterial Infections/metabolism , Humans , MiceABSTRACT
Francisella tularensis is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans and a Tier 1 select agent. The natural incidence of pneumonic tularemia worldwide is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCM) in human populations. Development and licensure of tularemia therapeutics and vaccines need to occur under the Food and Drug Administration's (FDA's) Animal Rule under which efficacy studies are conducted in well-characterized animal models that reflect the pathophysiology of human disease. The Tularemia Animal Model Qualification (AMQ) Working Group is seeking qualification of the cynomolgus macaque (Macaca fascicularis) model of pneumonic tularemia under Drug Development Tools Qualification Programs with the FDA based upon the results of studies described in this manuscript. Analysis of data on survival, average time to death, average time to fever onset, average interval between fever and death, and bacteremia; together with summaries of clinical signs, necropsy findings, and histopathology from the animals exposed to aerosolized F. tularensis Schu S4 in five natural history studies and one antibiotic efficacy study form the basis for the proposed cynomolgus macaque model. Results support the conclusion that signs of pneumonic tularemia in cynomolgus macaques exposed to 300-3,000 colony forming units (cfu) aerosolized F. tularensis Schu S4, under the conditions described herein, and human pneumonic tularemia cases are highly similar. Animal age, weight, and sex of animals challenged with 300-3,000 cfu Schu S4 did not impact fever onset in studies described herein. This study summarizes critical parameters and endpoints of a well-characterized cynomolgus macaque model of pneumonic tularemia and demonstrates this model is appropriate for qualification, and for testing efficacy of tularemia therapeutics under Animal Rule.
Subject(s)
Disease Models, Animal , Francisella tularensis/physiology , Macaca fascicularis/physiology , Pneumonia/microbiology , Tularemia/microbiology , Animals , Body Temperature , Female , Humans , Macaca fascicularis/genetics , Male , Pneumonia/complications , Pneumonia/pathology , Pneumonia/physiopathology , Treatment Outcome , Tularemia/complications , Tularemia/pathology , Tularemia/physiopathologyABSTRACT
Dietary oxysterols are cholesterol auto-oxidation products widely present in cholesterol-rich foods. They are thought to affect the intestinal barrier function, playing a role in gut inflammation. This study has characterized specific cell signals that are up-regulated in differentiated CaCo-2 colonic epithelial cells by a mixture of oxysterols representative of a hyper-cholesterolemic diet. p38 MAPK activation plays a major role, while other signal branches, i.e. the JNK and ERK pathways, make minor contributions to the intestinal inflammation induced by dietary oxysterols. p38 transduction might be the missing link connecting the known NADPH oxidase activation, and the induction of NF-κB-dependent inflammatory events related to oxysterols' action in the intestine. A NOX1/p38 MAPK/NF-κB signaling axis was demonstrated by the quenched inflammation observed on blocking individual branches of this signal with specific chemical inhibitors. Furthermore, all these signaling sites were prevented when CaCo-2 cells were pre-incubated with phenolic compounds extracted from selected wines made of typical Sardinian grape varieties: red Cannonau and white Vermentino. Notably, Cannonau was more effective than Vermentino. The effect of Sardinian wine extracts on intestinal inflammation induced by dietary oxysterols might mainly be due to their phenolic content, more abundant in Cannonau than in Vermentino. Furthermore, among different phenolic components of both wines, epicatechin and caffeic acid exerted the strongest effects. These findings show a major role of the NOX1/p38 MAPK/NF-κB signaling axis in the activation of oxysterol-dependent intestinal inflammation, and confirm the concept that phenolics act as modulators at different sites of pro-oxidant and pro-inflammatory cell signals.
Subject(s)
Cholesterol/analogs & derivatives , Hydroxycholesterols/adverse effects , Intestines/drug effects , Ketocholesterols/adverse effects , Phenols/chemistry , p38 Mitogen-Activated Protein Kinases/metabolism , Caco-2 Cells , Caffeic Acids/analysis , Cell Survival/drug effects , Cholesterol/adverse effects , Epithelial Cells/drug effects , Humans , Inflammation/chemically induced , Interleukin-8/metabolism , Intestinal Mucosa/metabolism , Intestines/cytology , NADPH Oxidase 1 , NADPH Oxidases/metabolism , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Up-Regulation , Vitis/chemistry , Wine/analysisABSTRACT
It has been established that there is a relationship between chronic inflammation and cancer development. The constant colonic inflammation typical of inflammatory bowel diseases is now considered a risk factor for colorectal carcinoma (CRC) development. The inflammatory network of signaling molecules is also required during the late phases of carcinogenesis, to enable cancer cells to survive and to metastasize. Oxidative reactions are an integral part of the inflammatory response, and are generally associated with CRC development. However, when the malignant phenotype is acquired, increased oxidative status induces antioxidant defenses in cancer cells, favoring their aggressiveness. This contradictory behavior of cancer cells toward redox status is of great significance for potential anticancer therapies. This paper summarizes the essential background information relating to the molecules involved in regulating oxidative stress and inflammation during carcinogenesis. Understanding more of their function in CRC stages might provide the foundation for future developments in CRC treatment.
Subject(s)
Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/metabolism , Inflammation Mediators/metabolism , Oxidative Stress/physiology , Animals , Humans , Oxidation-Reduction , Risk FactorsABSTRACT
Regular consumption of moderate doses of wine is an integral part of the Mediterranean diet, which has long been considered to provide remarkable health benefits. Wine's beneficial effect has been attributed principally to its non-alcoholic portion, which has antioxidant properties, and contains a wide variety of phenolics, generally called polyphenols. Wine phenolics may prevent or delay the progression of intestinal diseases characterized by oxidative stress and inflammation, especially because they reach higher concentrations in the gut than in other tissues. They act as both free radical scavengers and modulators of specific inflammation-related genes involved in cellular redox signaling. In addition, the importance of wine polyphenols has recently been stressed for their ability to act as prebiotics and antimicrobial agents. Wine components have been proposed as an alternative natural approach to prevent or treat inflammatory bowel diseases. The difficulty remains to distinguish whether these positive properties are due only to polyphenols in wine or also to the alcohol intake, since many studies have reported ethanol to possess various beneficial effects. Our knowledge of the use of wine components in managing human intestinal inflammatory diseases is still quite limited, and further clinical studies may afford more solid evidence of their beneficial effects.
Subject(s)
Intestinal Mucosa/metabolism , Wine/analysis , Animals , Antioxidants/chemistry , Antioxidants/metabolism , Antioxidants/pharmacology , Homeostasis/drug effects , Humans , Intestines/chemistry , Intestines/microbiology , Microbiota/drug effects , Oxidation-Reduction , Polyphenols/chemistry , Polyphenols/pharmacology , Signal Transduction/drug effectsABSTRACT
Chronic inflammatory events appear to play a fundamental role in Alzheimer's disease (AD)-related neuropathological changes, and to result in neuronal dysfunction and death. The inflammatory responses observed in the AD brain include activation and proliferation of glial cells, together with up-regulation of inflammatory mediators and of free radicals. Along with glial cells, neurons themselves can also react and contribute to neuroinflammatory changes in the AD brain, by serving as sources of inflammatory mediators. Because excess cholesterol cannot be degraded in the brain, it must be excreted from that organ as cholesterol oxidation products (oxysterols), in order to prevent its accumulation. Among risk factors for this neurodegenerative disease, a mechanistic link between altered cholesterol metabolism and AD has been suggested; oxysterols appear to be the missing linkers between the two, because of their neurotoxic effects. This study shows that 24-hydroxycholesterol, 27-hydroxycholesterol, and 7ß-hydroxycholesterol, the three oxysterols potentially implicated in AD pathogenesis, induce some pro-inflammatory mediator expression in human neuroblastoma SH-SY5Y cells, via Toll-like receptor-4/cyclooxygenase-2/membrane bound prostaglandin E synthase (TLR4/COX-2/mPGES-1); this clearly indicates that oxysterols may promote neuroinflammatory changes in AD. To confirm this evidence, cells were incubated with the anti-inflammatory flavonoid quercetin; remarkably, its anti-inflammatory effects in SH-SY5Y cells were enhanced when it was loaded into ß-cyclodextrin-dodecylcarbonate nanoparticles, versus cells pretreated with free quercetin. The goal of loading quercetin into nanoparticles was to improve its permeation across the blood-brain barrier into the brain, and its bioavailability to reach target cells. The findings show that this drug delivery system might be a new therapeutic strategy for preventing or reducing AD progression.
Subject(s)
Antioxidants/pharmacology , Gene Expression Regulation/drug effects , Hydroxycholesterols/pharmacology , Nanoparticles/chemistry , Quercetin/pharmacology , Antioxidants/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Humans , Inflammation/prevention & control , Inflammation Mediators/metabolism , Integrin beta1/genetics , Integrin beta1/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Quercetin/chemistry , beta-Cyclodextrins/chemistryABSTRACT
Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.
Subject(s)
Anthrax Vaccines/administration & dosage , Anthrax Vaccines/immunology , Anthrax/prevention & control , Post-Exposure Prophylaxis/methods , Vaccination/methods , Adolescent , Adult , Aged , Animals , Anthrax Vaccines/adverse effects , Antibodies, Bacterial/blood , Antibodies, Neutralizing/blood , Antitoxins/blood , Disease Models, Animal , Female , Humans , Macaca fascicularis , Male , Middle Aged , Rabbits , Survival Analysis , Vaccination/adverse effects , Young AdultABSTRACT
Cholesterol auto-oxidation products, namely oxysterols, are widely present in cholesterol-rich foods. They are thought to potentially interfere with homeostasis of the human digestive tract, playing a role in intestinal mucosal damage. This report concerns the marked up-regulation in differentiated CaCo-2 colonic epithelial cells of two key inflammatory interleukins, IL-6 and IL-8, caused by a mixture of oxysterols representative of a high cholesterol diet. This strong pro-inflammatory effect appeared to be dependent on the net imbalance of red-ox equilibrium with the production of excessive levels of reactive oxygen species through the colonic NADPH-oxidase NOX1 activation. Induction of NOX1 was markedly while not fully inhibited by CaCo-2 cell pre-incubation with phenolic extracts obtained from well-selected wines from typical grape varieties grown in Sardinia. Oxysterol-dependent NOX1 activation, as well as interleukin synthesis, were completely prevented by Cannonau red wine extract that contains an abundant phenolic fraction, in particular phenolic acids and flavonoids. Conversely, cell pre-treatment with Vermentino white wine extract with smaller phenolic fraction showed only a partial NOX1 down-regulation and was ineffective in interleukin synthesis induced by dietary oxysterols. It is thus likely that the effects of Sardinian wine extracts against intestinal inflammation induced by dietary oxysterols are mainly due to their high phenolic content: low doses of phenolics would be responsible only for direct scavenging oxysterol-dependent ROS production. Besides this direct activity, an excess of phenolic compounds detectable in red wine, may exert an additional indirect action by blocking oxysterol-related NOX1 induction, thus totally preventing the pro-oxidant and pro-inflammatory events triggered by dietary oxysterols.
Subject(s)
Cholesterol/analogs & derivatives , Cholesterol/pharmacology , Phenols/pharmacology , Wine , Caco-2 Cells , Diet , Enzyme Activation , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Italy , NADPH Oxidases/metabolismABSTRACT
Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage. However, the treatment of colon cancer cells with a diet-compatible mixture of oxysterols does not elicit the same responses than individual components added to the cells at the same concentrations at which they are present in the mixture. Sixty µM oxysterol mixture showed a slight pro-apoptotic effect on human colon cancer CaCo-2 cell line, evaluated in terms of caspase-3 and caspase-7 activation; conversely, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and 5α,6α-epoxycholesterol were identified to be able to induce a significant pro-apoptotic effect if added to cell culture singly; 7ß-hydroxycholesterol had stronger action than other compounds. The enhanced production of reactive oxygen species through up-regulation of the colonic NADPH-oxidase isoform NOX1 appeared to be the key event in oxysterol-induced apoptosis in these colon cancer cells. As regards pro-inflammatory effects of oxysterols, IL-8 and MCP-1 were evaluated for their chemotactic activity. Only MCP-1 production was significantly induced by 7ß-hydroxycholesterol, as well as by cholesterol and oxysterol mixture. However, oxysterol-induced inflammation appeared to be NOX1-independent, suggesting a secondary role of this enzyme in inducing inflammation in colon cancer cells. A selective cell death induced by specific oxysterols against colon cancer cells, mainly exploiting their ability to activate NOX1 in generating oxidative reactions, might represent a promising field of investigation in colorectal cancer, and might bring new insights on strategies in anticancer therapy.
Subject(s)
Cholesterol, Dietary/pharmacology , Cholesterol/chemistry , Cholesterol/pharmacology , Colonic Neoplasms/pathology , Apoptosis/drug effects , Caco-2 Cells , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Homeostasis/drug effects , Humans , Oxidation-ReductionABSTRACT
The hypercholesterolemia-atherosclerosis association is now established; hypercholesterolemia may induce vascular-cell activation, subsequently increasing expression of adhesion molecules, cytokines, chemokines, growth factors, and other key inflammatory molecules. Among inflammatory molecules expressed by vascular cells, integrins play a critical role in regulating macrophage activation and migration to the site of inflammation, by mediating cell-cell and cell-extracellular matrix interactions. The main lipid oxidation products present in oxidized LDL that may be responsible for inflammatory processes in atherogenesis, are cholesterol oxidation products, known as oxysterols. This study demonstrates the effect of an oxysterol mixture, compatible with that detectable in human hypercholesterolemic plasma, on the expression and synthesis of ß(1)-integrin in cells of the macrophage lineage. The molecular signaling whereby oxysterols induce ß(1)-integrin up-regulation is also comprehensively investigated. Over-expression of ß(1)-integrin depends on activation of classic and novel members of protein kinase C and extracellular signal-regulated kinases 1 and 2, as well as of the up-stream G-protein (Gq and G13), c-Src, and phospholipase C. In addition, the localization of ß(1)-integrin in advanced human carotid plaques is highlighted, marking its importance in atherosclerotic plaque progression.
Subject(s)
Gene Expression Regulation/drug effects , Integrin beta1/genetics , Integrin beta1/metabolism , Macrophages/drug effects , Macrophages/metabolism , Signal Transduction , Steroids/pharmacology , Atherosclerosis/genetics , Atherosclerosis/metabolism , GTP-Binding Proteins/metabolism , Humans , MAP Kinase Signaling System , Oxidation-Reduction , Phosphoinositide Phospholipase C/metabolism , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/metabolism , Protein Kinase C/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , RNA Interference , U937 CellsABSTRACT
BACKGROUND: Inflammatory reactions, known to promote tumor growth and invasion, have been found associated with colorectal carcinoma (CRC). Macrophages are the chief component of the inflammatory infiltration that occurs early in the progression from non-invasive to malignant tumor, with a switch from the pro-inflammatory phenotype to the tumor-promoting phenotype. Tumor and stroma are additional sources of inflammation-related molecules. The study aimed to evaluate, during colorectal carcinogenesis from benign to malignant phases: i) the trend of serum levels of IL-8, IL-6, TGFß1, VEGF and MMPs; ii) the parallel trend of CRP serum levels; iii) derangement of the principal TGFß1 receptors (TGFß1RI/RII) in tumor tissues. METHODOLOGY/PRINCIPAL FINDINGS: 96 patients with colon adenomas or CRC at different stages of progression, and 17 controls, were recruited. Serum IL-8, IL-6, TGFß1, VEGF, MMPs and CRP levels were analyzed before endoscopy or surgery. TGFß1 receptors were evaluated in adenoma biopsies and surgically-removed colorectal adenocarcinomas. Serum levels of IL-8 in adenocarcinoma patients were increased from stage II, when also the enzymatic activity of MMP-9 increased. Of note, the increasing trend of the two serum markers was found significantly correlated. Trend of serum CRP was also very similar to that of IL-8 and MMP-9, but just below statistical significance. TGFß1 levels were lower at stage III CRC, while IL-6 and VEGF levels had no significant variations. In tissue specimens, TGFß1 receptors were already absent in about 50% of adenomas, and this percentage of missing receptors markedly increased in CRC stages III and IV. CONCLUSIONS: Combined quantification of serum IL-8, MMP-9 and CRP, appears a reliable and advanced index of inflammation-related processes during malignant phase of colorectal carcinogenesis, since these molecules remain within normal range in colorectal adenoma bearing patients, while consistently increase in the blood of CRC patients, even if from stage II only.
Subject(s)
Colorectal Neoplasms/blood , Colorectal Neoplasms/pathology , Interleukin-8/blood , Matrix Metalloproteinase 9/blood , Adenocarcinoma/blood , Adenocarcinoma/enzymology , Adenocarcinoma/pathology , Adenoma/blood , Adenoma/enzymology , Adenoma/pathology , Aged , Aged, 80 and over , C-Reactive Protein/metabolism , Colorectal Neoplasms/enzymology , Disease Progression , Female , Humans , Interleukin-6/blood , Male , Middle Aged , Neoplasm Staging , Receptors, Transforming Growth Factor beta/blood , Time Factors , Transforming Growth Factor beta1/blood , Vascular Endothelial Growth Factor A/bloodABSTRACT
One of the most common approaches for large-scale protein identification is LC, followed by MS. If more than a few proteins are to be identified, the additional fragmentation of individual peptides has so far been considered as indispensable, and thus, the associated costs, in terms of instrument time and infrastructure, as unavoidable. Here, we present evidence to the contrary. Using a combination of (i) highly accurate and precise mass measurements, (ii) modern retention time prediction, and (iii) a robust scoring algorithm, we were able to identify 257 proteins of Francisella tularensis from a single LC-MS experiment in a fragmentation-free approach (i.e. without experimental fragmentation spectra). This number amounts to 59% of the number of proteins identified in a standard fragmentation-based approach, when executed with the same false discovery rate. Independent evidence supports at least 27 of a set of 31 proteins that were identified only in the fragmentation-free approach. Our results suggest that additional developments in retention time prediction, measurement technology, and scoring algorithms may render fragmentation-free approaches an interesting complement or an alternative to fragmentation-based approaches.
Subject(s)
Chromatography, High Pressure Liquid , Computational Biology , Tandem Mass Spectrometry/methods , Francisella tularensis/metabolism , Peptides/metabolism , Proteins/metabolismABSTRACT
The severe and fatal human disease, tularemia, results from infection with the Gram-negative pathogen Francisella tularensis. Identification of surface outer membrane proteins, specifically lipoproteins, has been of interest for vaccine development and understanding the initiation of disease. We sought to identify Francisella live vaccine strain lipoproteins that could be a component of a subunit vaccine and have adjuvant properties as TLR2 agonists. We have identified a membrane lipoprotein of Francisella LVS isolated by sarkosyl extraction and gel filtration chromatography that is recognized by sera from LVS-vaccinated individuals and tularemia patients, indicating its potential diagnostic value. Sequencing of the protein by mass spectrometry indicated that it encodes the FTL_0645 open reading frame of F. holarctica LVS, which is 100% identical/homologous to FTT1416c of F. tularensis Schu S4. The predicted 137 amino acid lipoprotein encoded by FTL_0645 ORF, was expressed in Escherichia coli, purified, and demonstrated to be a lipoprotein. This recombinant lipoprotein, named Flpp3, was able to activate TLR2 and induce an immunogenic response in mice, suggesting that the E. coli-expressed Flpp3 is palmitoylated and closely resembles the native protein in structure and immunogenicity. Taken together, these data suggest that Flpp3 could be a candidate for inclusion in a F. tularensis vaccine.
Subject(s)
Bacterial Proteins/immunology , Bacterial Proteins/isolation & purification , Cloning, Molecular , Francisella tularensis/immunology , Gene Expression , Lipoproteins/immunology , Lipoproteins/isolation & purification , Tularemia/immunology , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cell Line , Female , Francisella tularensis/chemistry , Francisella tularensis/genetics , Humans , Lipoproteins/chemistry , Lipoproteins/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sequence Alignment , Tularemia/microbiologyABSTRACT
Infliximab is an anti-tumor necrosis factor-monoclonal antibody shown to be effective in the treatment of moderate-to-severe psoriasis and psoriatic arthritis. We report on the first two patients in Croatia in which the efficacy of infliximab therapy was monitored and evaluated primarily on the basis of cutaneous manifestations of psoriasis. Both patients had severe, treatment-resistant chronic plaque psoriasis and psoriatic arthritis and were on methotrexate therapy before the initiation and throughout the course of infliximab treatment. Infliximab was administered intravenously at a dose of 4 or 5 mg/kg at week 0, 2, 6 and every 8 weeks thereafter. Disease severity was measured before each infusion by means of Psoriasis Area and Severity Index (PASI) score. A remarkable clinical response was achieved in both patients with a 50% or greater improvement in baseline PASI at week 2 after therapy initiation and a 90% or greater improvement at week 6 in one patient and at week 14 in the other. Both patients also reported a significant decline in their arthritis symptoms shortly after the introduction of infliximab. The concomitant use of infliximab and methotrexate in these two patients resulted in rapid and sustained remission of psoriasis with no major adverse effects detected.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Psoriasis/drug therapy , Adult , Antibodies, Monoclonal/administration & dosage , Female , Humans , Infliximab , Infusions, Intravenous , Male , Middle AgedABSTRACT
Nutritional support, addressing the specific needs of this patient group, is required to help improve prognosis, and reduce the consequences of cancer-associated nutritional decline. Early intervention with nutritional supplementation has been shown to halt malnutrition, and may improve outcome in some patients. In our study we tried to assess the influence of nutritional support (counseling, oral liquids, megestrol acetate) on nutritional status and symptoms prevalence in patients with colorectal cancer during chemotherapy. Group I consisted of 215 (55%) patients with medium age 68 +/- 2.6 years who were monitored prospectively and were given nutritional support. Group II included 173 (45%) patients (medium age 67 +/- 2.9 years) without the proper nutritional counseling, in whom the data were collected retrospectively during a 6 years period of time. After evaluation Nottingham Screening Tool Score, Appetite Loss Scale and Karnofsky Performance Status) all patients in the group I received nutritional counseling, 153 of them (72%) were taking form of enteral food supplement and 103 (48%) patients were using megestrol acetate. Evaluating the initial risk measurements according to BMI, decrease in weight gain and NST, we did not find any significant difference between the two groups. After chemotherapy completion, patients in group I had a 15.3% drop of those who's BMI was < 20.65% patients increased their body weight, with an average weight gain of 1.5 kg (0.6-2.8 kg). Contrary, in group II we found increase in weight loss > or = 2 kg/month in 39% of patients. The appetite improvement was detected on Appetite Loss Scale from 3.1 (pre-chemotherapy) to 4.7 (post-chemotherapy) in group I, especially in those receiving megestrol acetate. In both groups Karnofsky Performance Status didn't change significantly reflecting the impact of the disease itself and chemotherapy procedures to the patient's condition. Nutritional counseling, supplemental feeding and pharmacological support do temporarily stop weight loss and improve appetite, social life and quality of life in those groups of patients. However, this improvement have no implications on patients KPS and course of their disease.
Subject(s)
Colorectal Neoplasms/drug therapy , Nutritional Support , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Appetite , Body Mass Index , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Fluorouracil/administration & dosage , Humans , Leucovorin/administration & dosage , Nutritional Status , Quality of Life , Retrospective Studies , Weight LossABSTRACT
UNLABELLED: The aim of our study was to assess whether the influence of nutritional support, consisting of counseling, enteral liquids support and pharmacologic support, can slow down weight loss and whether the change in weight has the impact on the performance status in our patients. In our study 44 patients with pancreatic cancer were included--26 males (mean age 69 years +/- 2.4 years) and 18 females (mean age 63 +/- 3.2 years). Metastatic disease was found in 21 patients, 15 patients had liver metastasis. Locally advanced disease was found in 24 patients and metastatic and locally advanced disease in 17 patients. Surgery was performed in 34 patients. Forty four (100%) patients underwent nutritional counseling, 33 of them (75%) took supplemental enteral feeding and 44 (100%) took megestrol acetate 400 mg per a day. The patients were followed up during 8 weeks during 5 visits. At first visit we took initial nutritional status of patients. Appetite loss, weight gain and Karnofsky performance status were monitored at every visit. All patients were treated with gemcitabin for a 7 week period. RESULTS: NTS score at initial visit in 44 patients (100%) was > or = 5. Using nutritional counseling, enteral food substitution and pharmacological support, weight gain was observed in 61.1% patients and appetite improved. Average KPS mostly improved after first month of therapy while after two months was again at the basal level. With nutritional counseling, supplemental feeding and pharmacologic support weight loss in our patients slowed down and appetite improved. Despite of that, Karnofsky Performance Status didn't change significantly, reflecting the impact of the disease itself and chemotherapy procedures to the patient's condition. We can conclude that nutritional and pharmacological support can temporarily stop weight loss and improve appetite, social life and quality of life in those groups of patients but have no implications on patients KPS and course of their disease.
Subject(s)
Antineoplastic Agents/therapeutic use , Appetite Stimulants/therapeutic use , Nutritional Support , Pancreatic Neoplasms/therapy , Aged , Deoxycytidine/analogs & derivatives , Deoxycytidine/therapeutic use , Female , Humans , Karnofsky Performance Status , Male , Megestrol Acetate/therapeutic use , Middle Aged , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Weight Loss , GemcitabineABSTRACT
The aim of the study was to analyse psychological characteristics and medical parameters in obese and overweight to identify the possible psychosocial consequences of obesity that may occur along with the numerous medical problems associated with excess body weight. Analysis was made on 296 patients (103 males and 193 females, median age 50, range 16-81) divided in three groups, depending on their Body mass index (BMI). Group I included 41 patients with BMI ranging from 25 to 29.9, group II included 170 patients with BMI from 30 to 34.9, and group III 85 patients with BM > or =35. We compared medical (glucose, cholesterol, triglycerides, HDL-cholesterol, systolic and diastolic blood pressure, body fat percentage) and psychological parameters (anxiety, depression, pros and cons of losing weight, self efficacy and four stages of change) in the patients included in the study. Univariate analysis has shown statistically significant difference among obese and overweight patients in goal weight, systolic and diastolic blood pressure, body fat percentage, glucose and cholesterol serum level. People with higher BMI (>30) found more advantages (pros) over disadvantages (cons) of weight loss but the level of anxiety and depression did not differ significantly among those 3 groups of patients. The results have shown that overweight and obese people have serious medical problems. They also differ in some psychological characteristics which have to be taken into consideration. Therefore, approach to these patients should be multidisciplinary, including dietary care, physical activity, psychological and medical care.