Choroidal Thickness and Ganglion Cell Complex in Pubescent Children with Type 1 Diabetes without Diabetic Retinopathy Analyzed by Spectral Domain Optical Coherence Tomography.

AIM: To assess the retinal and choroidal thickness and ganglion cell complex (GCC) in pubescent children with type 1 diabetes (T1D) without diabetic retinopathy (DR), using spectral domain optical coherence tomography (SD-OCT). MATERIALS AND METHOD: Sixty-four right eyes of 64 subjects with T1D and 45 right eyes of 45 age-matched healthy volunteers (control group) were enrolled in this study. The mean age of the subjects and controls was 15.3 (±SD = 2.2) and 14.6 (±SD = 1.5), respectively. SD-OCT was performed using RTVue XR Avanti. Ganglion cell complex (GCC), GCC focal loss volume (FLV), GCC global loss volume (GLV), choroidal thickness (CT), foveal (FT) and parafoveal thickness (PFT), and foveal (FV) and parafoveal volume (PFV) data were analyzed. RESULTS: There was no significant difference between subjects and controls in the CT in the fovea and nasal, temporal, superior, and inferior quadrants of the macula. There were no significant correlations between CT, duration of diabetes, and HbA1C level (p = 0.272 and p = 0.197, resp.). GCC thickness did not differ significantly between the groups (p = 0.448), but there was a significant difference in FLV (p = 0.037). Significant differences between the groups were found in the PFT and PFV (p = 0.004 and p = 0.005, resp.). There was a significant negative correlation between PFT, PFV, and HbA1C level (p = 0.002 and p = 0.001, resp.). CONCLUSIONS: Choroidal thickness remains unchanged in children with T1D. Increased GCC FLV might suggest an early alteration in neuroretinal tissue. Parafoveal retinal thickness is decreased in pubescent T1D children and correlates with HbA1C level. OCT can be considered a part of noninvasive screening in children with T1D and a tool for early detection of retinal and choroidal abnormalities. Further OCT follow-up is needed to determine whether any of the discussed OCT measurements are predictive of future DR severity.

Optical Coherence Tomography-Based Angiography in Retinal Artery Occlusion in Children.

PURPOSE: To evaluate the role of optical coherence tomography (OCT)-based angiography (OCTA) in the diagnosis and monitoring of pediatric patients with retinal artery occlusion (RAO). MATERIAL AND METHODS: This retrospective study was conducted at the Department of Ophthalmology, The Children's Memorial Health Institute, in Warsaw between March 2015 and May 2016. Every patient underwent a complete ophthalmological examination. The diagnosis of the disease was based on fundus examination and fluorescein angiography (FA). OCT and OCTA were performed at baseline and every follow-up visit. RESULTS: Four patients (4 eyes) (2 boys/2 girls, age 8-16 years) with RAO were enrolled in the study. In all cases, initial OCTA images revealed typical ischemic changes in superficial and deep retinal capillary plexuses. Follow-up OCTA revealed increasing areas of ischemia in the RAO region and persistent narrowing of the arteries. The loss of capillary network and the darker, smooth background due to ischemia were visible on OCTA images. CONCLUSIONS: OCTA enables clear visualization of progressive impairment of the retinal vascular perfusion in children with RAO and may be an alternative to the standard FA. Further studies are needed to confirm our results and establish the role of OCTA in pediatric patients.

Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood.

A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

Bacteriophages displaying anticancer peptides in combined antibacterial and anticancer treatment.

AIMS: Novel anticancer strategies have employed bacteriophages as drug carriers and display platforms for anticancer agents; however, bacteriophage-based platforms maintain their natural antibacterial activity. This study provides the assessment of combined anticancer (engineered) and antibacterial (natural) phage activity in therapies. MATERIALS & METHODS: An in vivo BALB/c mouse model of 4T1 tumor growth accompanied by surgical wound infection was applied. The wounds were located in the areas of tumors. Bacteriophages (T4) were modified with anticancer Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides by phage display and injected intraperitoneally. RESULTS & CONCLUSION: Tumor growth was decreased in mice treated with YIGSR-displaying phages. The acuteness of wounds, bacterial load and inflammatory markers in phages-treated mice were markedly decreased. Thus, engineered bacteriophages combine antibacterial and anticancer activity.

Immunogenicity studies of proteins forming the T4 phage head surface.

UNLABELLED: Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc, and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary responses in mice, and the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice. In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivities of other head proteins were noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages. IMPORTANCE: Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete; thus, the central problem of this work is timely and may have strong practical implications. Here is presented the very first description of the contribution of bacteriophage proteins to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere.

Molecular imaging of T4 phage in mammalian tissues and cells.

Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. Here we propose a new green fluorescent protein (GFP)-based method for T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Molecular imaging provides innovative techniques that have brought substantial progress in life sciences. We propose it as a useful tool for studies of phage biology.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

T4 phage and its head surface proteins do not stimulate inflammatory mediator production.

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1ß, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.

Purification of phage display-modified bacteriophage T4 by affinity chromatography.

BACKGROUND: Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. RESULTS: Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. CONCLUSIONS: Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.