Sex-dependent differences in behavioral and immunological responses to antibiotic and bacteriophage administration in mice

Introduction: The problem of antibiotic resistance is a global one, involving many industries and entailing huge financial outlays. Therefore, the search for alternative methods to combat drug-resistant bacteria has a priority status. Great potential is seen in bacteriophages which have the natural ability to kill bacterial cells. Bacteriophages also have several advantages over antibiotics. Firstly, they are considered ecologically safe (harmless to humans, plants and animals). Secondly, bacteriophages preparations are readily producible and easy to apply. However, before bacteriophages can be authorized for medical and veterinary use, they must be accurately characterized in vitro and in vivo to determinate safety. Methods: Therefore, the aim of this study was to verify for the first time the behavioral and immunological responses of both male and female mice (C57BL/6J) to bacteriophage cocktail, composed of two bacteriophages, and to two commonly used antibiotics, enrofloxacin and tetracycline. Animal behavior, the percentage of lymphocyte populations and subpopulations, cytokine concentrations, blood hematological parameters, gastrointestinal microbiome analysis and the size of internal organs, were evaluated. Results: Unexpectedly, we observed a sex-dependent, negative effect of antibiotic therapy, which not only involved the functioning of the immune system, but could also significantly impaired the activity of the central nervous system, as manifested by disruption of the behavioral pattern, especially exacerbated in females. In contrast to antibiotics, complex behavioral and immunological analyses confirmed the lack of adverse effects during the bacteriophage cocktail administration. Discussion: The mechanism of the differences between males and females in appearance of adverse effects, related to the behavioral and immune functions, in the response to antibiotic treatment remains to be elucidated. One might imagine that differences in hormones and/or different permeability of the blood-brain barrier can be important factors, however, extensive studies are required to find the real reason(s)

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein

The impairment of the alternative complement pathway contributes to rare kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). We recently described an aHUS patient carrying an exceptional gain-of-function (GoF) mutation (S250C) in the classical complement pathway component C2 leading to the formation of hyperactive classical convertases. We now report the identification of the same mutation and another C2 GoF mutation R249C in two other patients with a glomerulopathy of uncertain etiology. Both mutations stabilize the classical C3 convertases by a similar mechanism. The presence of R249C and S250C variants in serum increases complement-dependent cytotoxicity (CDC) in antibody-sensitized human cells and elevates deposition of C3 on ELISA plates coated with C-reactive protein (CRP), as well as on the surface of glomerular endothelial cells. Our data justify the inclusion of classical pathway genes in the genetic analysis of patients suspected of complement-driven renal disorders. Also, we point out CRP as a potential antibody-independent trigger capable of driving excessive complement activation in carriers of the GoF mutations in complement C2

Biological aspects of phage therapy versus antibiotics against Salmonella enterica serovar Typhimurium infection of chickens

Phage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed

New perspectives on complement mediated immunotherapy

Tumor-specific monoclonal antibodies (mAbs) offer several modes of tumor cell killing, from direct cytotoxic activity to indirect mechanisms employing the host immune system, particularly its innate branch. The latter effector functions seem to dominate among clinically approved anti-cancer mAbs and major efforts are being undertaken by both academia and the pharmaceutical industry with the aim to improve complement activation, antibody-dependent cellular cytotoxicity (ADCC) and Fc/opsonin-mediated phagocytosis. On one hand, there are a variety of available effector mechanisms to allow multistep elimination of tumor cells. On the other hand, tumor cells adopt a number of strategies to evade immune attack, such as overexpression of complement inhibitors, trogocytosis, shedding or internalization of mAb-targeted epitopes, which all contribute to their resistance against host defense mechanisms. Another problem recognized only recently is the depletion of immune effectors during the first round of treatment, which in concordance with delayed turnover of immune components renders subsequent rounds of therapy ineffective. Herein, we discuss newly identified limiting factors but also novel mechanistic data on complement activation by antitumor antibodies as issues important for guidance towards the next generations of immunotherapeutics

Efficacy and safety of phage therapy against Salmonella enterica serovars Typhimurium and Enteritidis estimated by using a battery of in vitro tests and the Galleria mellonella animal model

In light of spreading antibiotic resistance among pathogenic bacteria, the development of novel approaches to combat such microorganisms is crucial. Salmonella enterica is pathogenic to humans, however, it can also infect poultry, being a potential foodborne pathogen when poultry-derived food is contaminated by this bacterium. Phage therapy is one of the alternative ways to treat Salmonella-infected animals while the establishment of this method and its introduction to a general practice requires detailed studies on safety and efficacy. Here, we present the results of such studies with two previously isolated and characterized bacteriophages, vB_SenM2 and vB_Sen-TO17, and four strains of S. enterica belonging to two serovars, Typhimurium and Enteritidis. We demonstrated effective reduction of bacterial cell number and cell culture density when using each phage alone, and in combination (as a cocktail). These phages were also effective in reducing bacterial biofilm. The efficacy of this in vitro phage therapy was compared to the action of known antibiotics, as was the efficiency of appearance of bacteria resistant to both these types of antibacterial agents. Safety of the use of bacteriophages was demonstrated using the LAL chromogenic test and the chicken fibroblast viability assay. Finally, the efficacy of phage therapy was assessed with the in vivo model of S. enterica-infected Galleria mellonella larvae, showing a significant improvement in the survival of the animals. In conclusion, we demonstrated high efficacy and acceptable safety profiles of phage therapy against S. enterica strains using vB_SenM-2 and vB_Sen-TO17 phages (both alone and in a cocktail). These results open a possibility for a trial with the use of poultry and these phages which might potentially allow to introduce of this method for practical use in poultry farming

The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability

Interactions of Bacteriophages with Animal and Human Organisms—Safety Issues in the Light of Phage Therapy

Bacteriophages are viruses infecting bacterial cells. Since there is a lack of specific receptors for bacteriophages on eukaryotic cells, these viruses were for a long time considered to be neutral to animals and humans. However, studies of recent years provided clear evidence that bacteriophages can interact with eukaryotic cells, significantly influencing the functions of tissues, organs, and systems of mammals, including humans. In this review article, we summarize and discuss recent discoveries in the field of interactions of phages with animal and human organisms. Possibilities of penetration of bacteriophages into eukaryotic cells, tissues, and organs are discussed, and evidence of the effects of phages on functions of the immune system, respiratory system, central nervous system, gastrointestinal system, urinary tract, and reproductive system are presented and discussed. Modulations of cancer cells by bacteriophages are indicated. Direct and indirect effects of virulent and temperate phages are discussed. We conclude that interactions of bacteriophages with animal and human organisms are robust, and they must be taken under consideration when using these viruses in medicine, especially in phage therapy, and in biotechnological application

Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth, Neovascularization, Angiogenesis and Macrophage Infiltration.

CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A1, A2A, and A3 in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5'-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A1AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A3AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy

In Silico Designed Gain-of-Function Variants of Complement C2 Support Cytocidal Activity of Anticancer Monoclonal Antibodies

The molecular target for the classical complement pathway (CP) is defined by surface-bound immunoglobulins. Therefore, numerous anticancer monoclonal antibodies (mAbs) exploit the CP as their effector mechanism. Conversely, the alternative complement pathway (AP) is spontaneously induced on the host and microbial surfaces, but complement inhibitors on host cells prevent its downstream processing. Gain-of-function (GoF) mutations in the AP components that oppose physiological regulation directly predispose carriers to autoimmune/inflammatory diseases. Based on the homology between AP and CP components, we modified the CP component C2 so that it emulates the known pathogenic mutations in the AP component, factor B. By using tumor cell lines and patient-derived leukemic cells along with a set of clinically approved immunotherapeutics, we showed that the supplementation of serum with recombinant GoF C2 variants not only enhances the cytocidal effect of type I anti-CD20 mAbs rituximab and ofatumumab, but also lowers the threshold of mAbs necessary for the efficient lysis of tumor cells and efficiently exploits the leftovers of the drug accumulated in patients’ sera after the previous infusion. Moreover, we demonstrate that GoF C2 acts in concert with other therapeutic mAbs, such as type II anti-CD20, anti-CD22, and anti-CD38 specimens, for overcoming cancer cells resistance to complement attack

Mutations resulting in the formation of hyperactive complement convertases support cytocidal effect of anti-CD20 immunotherapeutics

Anti-CD20 monoclonal antibodies (mAbs) rituximab and ofatumumab are potent activators of the classical complement pathway, and have been approved for the treatment of B-cell malignancies. However, complement exhaustion and overexpression of complement inhibitors by cancer cells diminish their therapeutic potential. The strategies of targeting membrane complement inhibitors by function-blocking antibodies and the supplementation with fresh frozen plasma have been proposed to overcome tumour cell resistance. We present a novel approach, which utilizes gain-of-function variants of complement factor B (FB), a component of alternative C3/C5 convertases, which augment mAb-activated reactions through a positive feedback mechanism called an amplification loop. If complement concentration is limited, an addition of quadruple gain-of-function FB mutant p.D279G p.F286L p.K323E p.Y363A (or selected single mutants) results in significantly increased complement-mediated lysis of ofatumumab-resistant tumour cells, as well as the complete lysis of moderately sensitive cells. Importantly, this effect cannot be achieved by further increasing ofatumumab concentration. Potentiation of cytotoxic effect towards moderately sensitive cells was less apparent at physiological serum concentration. However, an addition of hyperactive FB could compensate the loss of cytotoxic potential of serum collected from the NHL and CLL patients after infusion of rituximab. Residual levels of rituximab in such sera, in combination with added FB, were able to efficiently lyse tumour cells. We suggest that the administration of gain-of-function variants of FB can restore CDC potential of complement-exhausted serum and maximize the therapeutic effect of circulating anti-CD20 mAbs