Additionally, phage replication only occurs when the bacterial density is above a certain threshold . This threshold is reached in the course of systemic infections [22,48,52], but may be compromised in the case of necrotic lesions, such as those induced by M. ulcerans infections. As described in phage treatment of a local S. aureus infection, even with multiple subcutaneous doses of 10 9 PFU/mouse, phages significantly reduced but did not eliminate the bacterial load in abscesses induced by bacteria . A possible concern about phagetherapy is the emergence of phage-resistant bacteria [22,24,48,53]. Although in this study we do not provide data related to the emergence of M. ulcerans phage- resistance, it has been described, in experimental models of other bacterial diseases, namely with Pseudomonas aeruginosa, Escherichia coli and S. aureus, that phage resistance is a rare event [22,24,48], even represent the mean 6 SD (n = 5). Significant differences between treated and non-treated mice were performed using Student’s t test (*, p#0.05, **, p#0.01, ***, p#0.001).
The results of this study strongly suggest that the VP-2 phage can protect fish larvae against Vibrio infections. Although infecting fish with V. anguillarum in the absence of the phage resulted in low fish mortality (17%), the survival of infected fish larvae in the presence of the phage increased significantly, reaching values similar to those obtained in the control treatment (fish control). Addition of the phage lysate to the fish larvae without bacterial challenge did not decrease larvae fish survival. The mortality in this group was similar to that of controls (fish control). Although phage VP-2 produced no observable effects in larval fish (morphological alterations or mortality), it is necessary to evaluate this phage (whole genome sequencing) for the presence of genes encoding toxins and antibiotic resistance. The phage lysate could also potentially contain residual bacterial antigens or endotoxins . In this study, fish larvae experimentally treated only with the phage preparation (phage lysate at ,10 9 PFU mL- 1 diluted 10-fold) did not show any negative effect on fish health, therefore, it is likely that the VP-2 phage lysate has few or no toxins. Moreover, as infecting fish with V. anguillarum in the absence of the phage resulted in low fish mortality (17%), future experiments using several concentrations of bacteria at a MOI of 100 in order to get a higher fish killing rate or to reach a plateau, would be very useful to confirm the potential of phagetherapy to inactivate pathogenic bacteria in fish.
Whatever the case may be, the success of phagetherapy depends on enough phage generations in the locality of the target to bring about the eradication of pathogen bacteria from the body. The increase in phage concentration to sufficient densities can be attained by two means. Firstly, with in situ replication, that is called active treatment, or as a result of pharmacologically conventional dosing, the passive treatment. The ways of rising phage concentration must be adequately stout so that they compensate the mechanism of phage reduction. The goal is thus to achieve minimum phage concentration at the target site, that is necessary for the desired levels of bacterial eradication. Also, worth mentioning is the necessity for phage preparations to be adequately purified (e.g., to remove bacterial debris). Such purification should be substantial (e.g., to remove most bacterial components, including endo-toxins), particularly when phages are to be delivered directly to an animal’s systemic circulation (Chan, Abedon, Loc-Carrillo, 2013).
In the type of synergy considered here, the growth of one phage augments the infection properties of a second phage on the same bacterium (which may be contrasted with two phages having somewhat complementary host ranges). This augmentation can operate in one direction or both. For a lytic phage, growth is primarily determined via effects on any of three properties (Adams, 1959): the rate the phage infects cells (adsorption rate), the number of progeny per infection (burst), and the time from infection until progeny are released (lysis time or latent period). In theory, synergy could operate via effects on any of them, but documented examples are so far restricted to effects on infection rate: one phage produces a depolymerase that strips the bacterial capsule (Sutherland, 1995; Hughes et al., 1998; Azeredo & Sutherland, 2008) enhancing infection by another phage (Bull, Vimr & Molineux, 2010). Phages can also interfere with each other, as is well documented at the intracellular level in which coinfection by two phages reduces the burst size of one (Delbruck, 1945; Adams, 1959).
Moreover, we observed that bacterial debris released during bacteriophage treatments were not pro-inflammatory, as produc- tion of cytokines was not strongly stimulated in either the curative or the preventive treatments. A pro-inflammatory response is often used as an argument against phagetherapy. Four days after administration of the preventative treatment that consisted of intranasal bacteriophage solution only, the lung concentrations of IL-6 and KC were low (Figure 2A and B). The main cause of bacterial destruction can therefore be attributed to the bacterio- phage itself. However, the cytokine concentrations were still significantly higher than those measured 24 h after administration of a PBS control solution, and partial involvement of a weak immune response stimulated by the bacteriophage solution cannot thus be ruled out. Indeed, the quality of the bacteriophage preparation, particularly the elimination of endotoxins, influenced the extent of the immune response following pre-infection treatments (data not shown). This is in contrast to post-infection treatments, where the immune system has already been primed by bacteria when the bacteriophages were administered.
In this study, virulent phage with lytic activity against MRSA strain was isolated. Morphologically, the phage is member of the family Myoviridae, and exhibited an identical host range. Adsorption studies showed 100% adsorption of MRSA-Phage after 35 minutes of exposure. SDS-PAGE indicated that the phage particles contain one major structural protein (about 30 Kda). Our results indicated good efficacy for this phage, which consistent with data cited by Kaźmierczak et al., 2014. They studied the phages which able to kill S. aureus in the treatment of various human diseases, e.g., venous leg ulcers and eye infections, septicemia, staphylococcal lung infections, and others. Because there are not enough chemotherapeutics to destroy bacteria and to counteract the problem of infections in the human population, however the field of phagetherapy as antibacterial agents has advanced considerably as an alternative to antibiotics (Kwiatek et al., 2012; Chhibber et al., 2008; Kim et al., 2007 and Westwater et al., 2003). Finally, phage treatment may help to reduce the frequency of potentially lethal infections in the hospital environment, with related costs that can be significantly lower than those of antibiotics.
It is generally recognized that bacteria predominantly live in biofilms. With regard to phagetherapy, phages with viral- attached EPS depolymerases should therefore preferably be selected for, as they are after all able to break down biofilms by attacking two of their main constituting parts, bacterial cells and EPS material. However, one of the major hurdles with regard to phagetherapy still to overcome is their narrow host range, conferred by the high specificity of their associated EPS depolymerases [43,60,61]. This restricts the phage in infecting only a very few strains of one single species. As observed in our study, phage Q15 only infects seven P. putida strains out of a selection of 53 but conferred also a marked difference in breaking down PpG1 and RD5PR2 biofilms. Therefore, one should first carefully identify the bacterial pathogens before therapy with a highly lytic phage with a proper viral attached EPS depolymerase can be started. The chance of finding such a specific phage is however likely to be low, and nonetheless bacteria easily develop resistance to phages. From this point of view, application of ‘phage cocktails’ directed at numerous strains of the target species in combination with the concept of ‘engineering’ highly lytic phages with the appropriate EPS degrading enzymes are powerful tools opening new perspectives in phagetherapy.
nated from the body, resulting in a drastic reduction of therapeutic efﬁcacy; and (vii) bacteria developed many different types of mech- anisms that confer resistance to phages (Hermoso et al., 2007; O’Flaherty et al., 2009; Parracho et al., 2012; Chan and Abedon, 2012; Wittebole et al., 2013). Phages are protein-based entities totally devoid of metabolic machinery that can potentially inter- act with the immune system within the human body, can actively replicate only in the presence of viable host bacterial cells, and can even evolve during manufacture or use, but are far from being unique in these regards (Loc-Carrillo and Abedon, 2011). For exam- ple, many protein-based (bio)pharmaceuticals can stimulate the immune system, chemical antibiotics that lyse bacteria will release in situ bacterial (endo)toxins, and live-attenuated vaccines both actively replicate and evolve within the body tissues. Protein-based drugs, chemical antibiotics, and whole vaccines have previously been approved for use despite these various properties. There are no antimicrobials displaying selective toxicity that will affect all possible microbial targets. Typically the narrowness of phage host ranges – few strains or species of bacteria – will at a minimum place limitations on presumptive treatment, i.e., treatment courses that begin prior to the identiﬁcation of the pathogen’s suscep- tibility to antibacterials such as to speciﬁc phages. However, as phages can often be employed in combination with other antibac- terial agents, including other phages (so-called phage cocktails), the lytic spectrum of phage products can be much broader than the spectrum of activity of individual phage types (Loc-Carrillo and Abedon, 2011). Even phage cocktails with broad spectrum of action are normally more selective in their spectrum of activity than typ- ical “narrow-spectrum” antibiotics, a property that can be viewed as an additional advantage of phagetherapy. When using bacterio- phage particles
for proteins with unknown function. A more thorough analysis of these proteins was completed using HHPred. With Pfam, for protein motif search, an e value threshold of <1x10-5 was always used. The main obtained results are illustrated in Attachment V. As mentioned previously, some proteins had known functions, however 28 proteins had unknown function. In certain cases, when no significant resemblances were found, conserved domains or protein families were searched for. With the e value obtained, some homologues of the 28 proteins were obtained. Most of them were hypothetical proteins from other Salmonella phages, although some of the results obtained were putative proteins or not possible to find the best homologue. Then, Pfam database enabled to go farther and obtain a possible function to some proteins, but still some of proteins presented unknown function. From these results stands out a phage lysozyme (CDS 7 with a e value of 1.3e-33), a proteasome subunit (CDS 10 with a e value of 1.8e-20), some proteins responsible for tail components (CDS 32 with a e value of 4.9e-10 and CDS 43 with a e value of 9.4e-15), gp6 and gp20 (CDS 57 with a e value of 4.1e-29 and CDS 51 with a e value of 2.3e-07, respectively). In the end, only a few protein functions were predicted, and with BLASTP and HHpred no virulence genes were found, which is important for choosing a good candidate for later studies in phagetherapy (Loc-Carrillo & Abedon, 2011).
The sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), is the most important insect pest of sugarcane grown in Brazil and others localities in the Americas, causing stalk damage to sugarcane plants which results in loss of efficiency in both sugar and alcohol production. The extensive damage caused by this pest in the sugarcane cultivation and its difficult control by conventional insecticides justify the search for new strategies that can help to reduce the use of harmful chemicals to humans and the environment. In order to generate new molecules with insecticidal activity against the sugarcane borer, the present work aimed to build a scFv antibody fragment library through Phage Display technology for selection and characterization of molecules with binding properties against D. saccharalis larval midgut proteins. The methodology used was efficient, allowing the selection of a scFv with immunoreactivity for the insect intestinal epithelium and with potential insecticidal activity.
In this century, cancer incidence has become one of the most significant problems concerning human. Conventional radiotherapy damage healthy tissue and in some cases may cause new primary cancers. This problem can be partially solved by hadron therapy which would be more effective and less harmful compared to other forms of radiotherapies used to treat some cancers. Although carbon ion and proton therapy both are effective treatments, they have serious differences which are mentioned in this paper and compared between the two methods. Furthermore, various treatments have been performed on head and neck cancer with hadrons so far will be discussed. Keywords: cancer; proton therapy; carbon ion therapy; Boron neutron capture therapy (BNCT); head; neck
serotypes isolated from patients is not uncommon and has been reported in both O157 and non-O157 isolates [12,29]. In studies where the mechanism of stx gene loss has been investigated, it has been shown that the stx-negative strains lack the entire Stx- encoding bacteriophage. This has been demonstrated by the presence or reappearance of an intact integration site for the Stx phage and an altered PFGE pattern [29–32]. Such stx-negative isolates from patients are believed to be progenies of an EHEC that lost the stx genes during the course of illness, and might be referred to as EHEC-LST (lost Shiga toxin) . The EHEC-LST model is supported by the finding that EHEC are difficult to isolate from patients late in illness , and the theory is further confirmed by Mellmann and Karch who demonstrated the presence of stx-negative strains of O26:H11/NM and sorbitol fermenting (SF) O157:NM subsequent to stx 2 -positive isogenic
Leprosy presents a clinical spectrum that spans from a strong cellular mediated immunity and bacili growth control of Mycobacterium leprae at the tuberculoid pole to a poor T cell immunity with extensive bacterial load at the lepromatous pole. The antigenic profile characterization in both clinical forms is a necessary step for discover and evaluation of recombinant peptides that are mimotopes of M. leprae antigens, which may present immunogenic potential, in order to obtain a beTer understanding of the disease evolution, and allowing the improvement of diagnosis, prognosis and therapeutics. Mimotopes of M. leprae antigens were selected from a random heptapeptide conformational library expressed in fusion with the pIII protein of the M13 phage, using as the ligand target the total purified IgM from leprosy patients of clinical forms, tuberculoid (TT) and lepromatous (LL), and a healthy control population. Recombinant peptides were selected by glycine elution (unspecific) and by mitsudin elution (specific). All clones, after bioinformatic analyses, were validated by immunoenzymatic and colony reduction assays. For the glycine selection, the TT pole presented 24 distinct fagotypes, while the LL pole presented only 14 fagotypes. For the mitsudin selection, only 4 fagotypes were obtained in the LL pole, and 8 fagotypes in the TT pole. The two most frequent mimotopes, in both TT and LL poles and in both elution protocols, were coincidents. The peptides LFPAMHQ and VERHPST were more frequent in the LL pole, and the peptides KNPTTGT and ETHPTTR were more frequent in the TT pole. The three cited first mimotopes had an accentuated plaque reduction with incubation of sera from LL patients; however, the mimotope ETHPTTR presented the highest reduction with sera from TT patients. The immunogenic potential of these mimotopes may be useful in the development of new diagnostic and therapeutic strategies for leprosy control in the future.
Neste trabalho foi selecionado um novo antígeno, através da técnica de Phage Display, para o imunodiagnóstico da neurocisticercose humana. Trata-se de um epitopo que mimetiza um epitopo conformacional que é reconhecido pelos anticorpos presentes no soro de pacientes com neurocisticercose. Este antígeno foi sintetizado, purificado e sua massa foi confirmada através da espectrometria de massa. O antígeno produzido foi avaliado em ensaio de ELISA indireto frente aos soros de pacientes com neurocisticercose, de indivíduos normais e de indivíduos portadores de outras doenças. Como parâmetro para comparação foi utilizado o antígeno bruto do escólex de cisticerco. O ensaio de ELISA utilizando peptídeo sintético e o antígeno bruto do escólex de cisticerco apresentou índice de sensibilidade de 92 e 89%, respectivamente, e ambos apresentaram índice de especificidade de 100%, quando foram utilizados os soros de pacientes com neurocisticercose e do grupo controle. Quando foram testados os soros dos pacientes com diversas infecções, o ensaio de ELISA utilizando o peptídeo sintético e o antígeno bruto de escólex de cisticerco apresentou especificidades de 94.5 e 34.2% respectivamente. Isto demonstra que o imunodiagnóstico utilizando o peptídeo sintético assegura alta sensibilidade e especificidade a baixos custos. A partir destes resultados é possível dizer que foi selecionado um mimotopo com grande potencial para ser utilizado no imunodiagnóstico da neurocisticercose humana.
Juvenile Idiopathic Arthritis (JIA) is a set of chronic diseases characterized by persistent inflammation of joints. "Idiopathic" means that no one knows the cause of illness and "Juvenile" in this case means that the onset of symptoms occurs before 16 years of age. The diagnosis of JIA is clinical and is based on the finding of arthritis in one or more joints, lasting less than six weeks. It is essential that several diseases such as infections are investigated and removed, since arthritis is a common manifestation of various rheumatic and non-rheumatic diseases. Therefore, studies that are aimed at investigating new biomarkers become of great importance for the diagnosis of JIA. The aim of this study was to select and identify peptides recognized by antibodies purified from the serum of patients with JIA by Phage Display technology. For selection of peptides was performed a bioppaning using a peptide library Ph.D.-C7C expressed on the surface of filamentous phage M13. The DNA from selected clones was sequenced, translated, and several clones were subjected to ELISA assays and bioinformatics. Among the 192 clones sequenced, 100 had valid sequences, which were identified 40 different sequences. The bioinformatics analysis showed that there are similarities between most of the selected peptides and proteins highly expressed in patients with JIA. ELISA allowed the pre-validation of a clone with a potential biomarker for the serological immunodiagnosis of JIA.
Neurocysticercosis (NC), presence of Taenia solium metacestodes in the central nervous system is the most serious form of cysticercosis and is the cause of epilepsy in countries under development. The improvement of immunodiagnostic tests which use recombinant antigens is importance because of the difficulty of obtaining parasites from naturally infected pigs for the preparation of metacestodes T. solium metacestodes crude antigen. The aim of this study was to select phagotopes by phage display peptide library specific to IgG present in serum samples from patients with NC and confirm the immunoreactivity from the selected phagetopes in the ELISA test for the diagnosis of human neurocysticercosis in serum samples. We used in the selection procedure a phage display peptide library in a selection strategy against the immunoglobulins (IgG) purified from serum samples positively diagnosed for NC, other parasitoses and apparently health individuals. The DNA sequences corresponding to the inserts of the selected phage clones were sequenced, translated and analyzed by bioinformatics. ELISA tests were performed with the ten phagetopes selected against the three groups of patients (NC, other parasitoses and healthy). The binding specificities of the recombinant phages to the pool of serum samples were analyzed by competitive ELISA. Peptides showed significant similarities with important proteins from T. solium. All phagetopes presented satisfactory sensitivities and specificities in the ELISA test, varying from 52.5% to 100% and 86.3% to 100%, respectively. Some phagetopes did not reacted with serum samples from patients infected with Echinococcus granulosus, what is an advance since this cross reaction is very commonly reported. The recognition data indicated that the selected peptides could indeed mimic the epitopes on T. solium and bind specifically to the pool of serum with NC. We concluded that the identified phage clones displayed specific peptides to NC, and are potential biomarkers for NC diagnosis in serum samples.
Phage display de peptídeos foi descrito pela primeira vez por George Smith (1985) ao demonstrar que fagos filamentosos (vírus de bactérias) poderiam ser utilizados para apresentar peptídeos antigênicos fundidos a uma proteína do capsídeo viral denominada de pIII. Esta metodologia foi inicialmente empregada para caracterização de epitopos de anticorpos monoclonais e policlonais (SMITH G, 1985), na qual fagos "apresentando" peptídeos poderiam então ser imuno-capturados pelo respectivo anti-soro dirigido contra o antígeno contendo os peptídeos presentes na superfície do capsídeo viral (SMITH G, 1985). Assim, surgiu o conceito de "Peptide Phage Display", que permitiu que bibliotecas de phage display fossem amplamente utilizadas na identificação de peptídeos ligantes de virtualmente qualquer alvo biológico, tais como anticorpos, receptores, enzimas, moléculas de adesão, carboidratos, entre outros.
Com o objetivo de identificar modificações na expressão de moléculas em células melan-a após contato com linfócitos B-1 (melan-a transformadas) - potencialmente envolvidas na interação entre células e nas mudanças fenotípicas observadas no melanoma, foi adotada a metodologia de phage display de peptídeos. O método BRASIL (Biopanning and rapid analysis of selective interactive ligands - Giordano et al., 2001), permite que proteínas diferencialmente expressas em nosso sistema celular sejam identificadas, além de predizer os possíveis receptores presentes em células melan-a transformadas. Na Figura 2, foram esquematicamente representadas as principais etapas adotadas para a seleção de fagos carregadores de peptídeos que se ligam especificamente a células melan-a transformadas. Resumidamente, uma biblioteca de bacteriófagos que expressam peptídeos compostos de 7 aminoácidos aleatórios flanqueados por duas cisteínas (CX 7 C) foi previamente incubada com células melan-a, a fim de eliminar os fagos
Finalizado o Phage Display, as sequências dos clones VL selecionados foram alinhadas e, posteriormente, determinada a sua similaridade. A partir desta análise e, tendo em conta todas as sequências obtidas, foi encontrada uma sequência consensus. Esta sequência consensus permitiu a construção de uma árvore filogenética capaz de determinar a similaridade entre os diversos clones e, consequentemente, avaliar quantos clones diferentes foram selecionados após seleção por Phage Display. Tendo em conta esta análise e com base na nomenclatura de Kabat, foi possível encontrar em cada sequência as regiões correspondentes aos CDRs . A identificação dos resíduos que compõem estes últimos tem por base a localização das FRs, regiões altamente conservadas que antecedem e precedem os CDRs, facilitando a sua identificação. Nos domínios VL o CDR1 é precedido por uma cisteína conservada (C) e seguido por um triptofano (W) o qual é normalmente seguido por uma tirosina (Y). O CDR2 é quase sempre composto por sete resíduos e é precedido e seguido por uma tirosina e glicina (G), respetivamente. O CDR3 é precedido por uma cisteína conservada e seguido por um par de resíduos fenilalanina e glicina (FG). Como espectável, os CDRs são as regiões que apresentam maior variabilidade entre os clones, sendo a CDR3 a região mais variável, seguida pela CDR1 e, por último, a CDR2. A elevada variabilidade destas regiões é resultante do processo de maturação por afinidade que ocorreu no sistema imunológico do coelho . As sequências dos diferentes clones, a árvore filogenética construída com base nessas
growth, when aerobic respiration is highly expressed. On the contrary, phage amplification is a limited process during stationary phase, where anaerobic bacterial respiration takes place [56–58]. The mostly common E. coli strain for M13KE manipulation is ER2738, a lacZα-complementing strain. While M13KE phage has the lacZ alpha gene fragment (lacZα), its host bacteria have the complementary lacZ omega fragment (lacZω). Thus, when infected bacteria are plated in an IPTG (Isopropyl β-D-1-thiogalactopyranoside) and Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) selective media, a blue color appears in infected bacteria, while non-infected bacteria remain white [46, 55, 59]. Briefly, in this blue/white screening technique, E. coli has a lac operon that contains 3 structural genes – lacZ, lacY and lacA –, where lacZ gene encodes the beta-galactosidase enzyme (β-galactosidase). At the IPTG presence, the lac repressor protein is inactivated, allowing the lac operon transcription and the lacZω fragment expression. During phage infection, transcription of the lacZ gene of the phage allows the expression of lacZα fragment. LacZω and lacZα fragments per se are not functional; when both fragments are present, an alpha complementation is accomplished and the β-galactosidade enzyme becomes functional. Xgal is a histochemical dye, hydrolyzed through β-galactosidade activity. Thus, when both fragments are present, Xgal cleavages the β- galactosidade enzyme and accumulation of Xgal product results in a blue coloration. When phage infection is not effective, β-galactosidade enzyme is not functional because the lacZα fragment is not present and white colonies appear .