Therapeutic Activity of Type 3 Streptococcus pneumoniae Capsule Degrading Enzyme Pn3Pase.

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.

[Treatment of postprandial discomfort syndrome in the elderly: a multi-centered prospective randomized controlled clinical study].

Objective: To evaluate the efficacy and safety of Oryz-Aspergillus enzyme and pancreatin tablets (Combizym(®)) in the treatment of postprandial distress syndrome (PDS) in the elderly, compared with gastrointestinal motility drugs. Methods: A prospective randomized controlled trial was designed and registered in the China Clinical Trials Registry (ChiCTR-IPR-16008185). The elderly patients with PDS were randomly divided into three groups, including Mosapride group with Mosapride citrate tablets 5 mg 3 times per day for 2 weeks; Combizym(®) group with Combizym tablets 244 mg 3 times per day for 2 weeks; combined treatment group with both drugs and same doses for 2 weeks. The modified Nepean dyspepsia index (NDSI) score, discomfort intensity score and PDS score were calculated on patients before treatment, at the end of first and second week of treatment, as well as 4 weeks after treatment finished, respectively. Adverse effects were evaluated. Results: A total of 323 patients from 16 tertiary hospitals in China were enrolled in this study. Among them, 105 patients were in Mosapride group, 109 in Combizym(®) group and 109 in combined treatment group. There were 148 males (45.8%) and 175 females (54.2%) with median age 71.4±9.0 years (60-100 years). Baseline characteristics of three groups were comparable. After treatment, the NDSI scores in three groups all decreased significantly (P<0.001), while they were similar between groups (P>0.05). The discomfort intensity score and PDS score in three groups showed a significant reduction after treatment (P<0.001), especially in the combined treatment group. Compared with Mosapride group, the scores in Combizym(®) group decreased significantly after one or two weeks [discomfort intensity score: after one week, 4.0(2.5, 8.0) vs. 6.0(3.0, 10.0); after two weeks, 3.0(0.0, 5.0) vs. 4.0(2.0, 6.0); all P<0.05. PDS score: after one week, 6.0(3.0, 9.0) vs. 7.0(3.5, 10.5); after two weeks, 3.0(0.0, 5.0) vs. 4.0(2.0, 7.0); all P<0.05]. The efficacy rate in all patients after first week of treatment was over 15.0%. The efficacy rates after two weeks were 55.2%, 68.8% and 73.4% in Mosapride group, Combizym(®) group and combined treatment group, respectively. After two week treatment, the efficacy rates in Combizym(®) group (P=0.041) and combined group (P=0.006) were higher than that of Mosapride group. The recurrence rate of Mosapride group was 9.5%, which was significantly higher than that of Combizym(®) group (1.8%, P<0.05) and combined treatment group (1.8%, P<0.05). There were no serious adverse effects in the three groups. Conclusions: The efficacy of Oryz-Aspergillus enzyme and pancreatin tablets is comparable with that of Mosapride in elderly PDS patients, with fewer adverse effects and low recurrence rate. Combination regimen indicates better efficacy than that of Oryz-Aspergillus enzyme and pancreatin tablets or Mosapride alone.

In vivo enzymatic modulation of IgG antibodies prevents immune complex-dependent skin injury.

IgG antibodies are potent inducers of proinflammatory responses by cross-linking Fc receptors on innate immune effector cells resulting in tissue injury. The recently discovered enzymes endoglycosidase S (EndoS) and IgG-degrading enzyme (IdeS) of Streptococcus pyogenes are able to modulate the interaction between IgG antibodies and the Fc receptors, by hydrolysis of the glycan associated with the heavy chain of the IgG molecule (EndoS), or cleavage in the hinge region of the heavy IgG chain (IdeS). In this work, we investigated their ability to inhibit damage mediated by skin-bound antibodies in vivo in two different experimental models, the Arthus reaction, and epidermolysis bullosa acquisita, an autoimmune blistering skin disease associated with autoantibodies against type VII collagen. We demonstrate that both enzymes efficiently interfere with IgG-mediated proinflammatory processes, offering a great asset to specifically target pathological IgG antibodies in the skin and holding great promise for future applications in human therapy.

Enzymes in therapy of biofilm-related oral diseases.

Biofilm-related infections of the oral cavity, including dental caries and periodontitis, represent the most prevalent health problems. For years, the treatment thereof was largely based on antibacterial chemical agents. Recently, however, there has been growing interest in the application of more preventive and minimally invasive biotechnological methods. This review focuses on the potential applications of enzymes in the treatment and prevention of oral diseases. Dental plaque is a microbial community that develops on the tooth surface, embedded in a matrix of extracellular polymeric substances of bacterial and host origin. Both cariogenic microorganisms and the key components of oral biofilm matrix may be the targets of the enzymes. Oxidative salivary enzymes inhibit or limit the growth of oral pathogens, thereby supporting the natural host defense system; polysaccharide hydrolases (mutanases and dextranases) degrade important carbohydrate components of the biofilm matrix, whereas proteases disrupt bacterial adhesion to oral surfaces or affect cell-cell interactions. The efficiency of the enzymes in in vitro and in vivo studies, advantages and limitations, as well as future perspectives for improving the enzymatic strategy are discussed.

Anti-inflammatory and anti-apoptotic effects of (RS)-glucoraphanin bioactivated with myrosinase in murine sub-acute and acute MPTP-induced Parkinson's disease.

This study was focused on the possible neuroprotective role of (RS)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive RS-GRA), in an experimental mouse model of Parkinson's disease (PD). RS-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale seeds. RS-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration and analyzed by HPLC. Following, pure RS-GRA was characterized by (1)H and (13)C NMR spectrometry and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method. The obtained purity has been of 99%. To evaluate the possible pharmacological efficacy of bioactive RS-GRA (administrated at the dose of 10mg/kg, ip +5µl/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experiment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an acute (2 injections·40mg/kg MPTP) and a sub-acute (5 injections·20mg/kg MPTP) model of PD. Behavioural test, body weight changes measures and immunohistochemical localization of the main PD markers were performed and post-hoc analysis has shown as bioactive RS-GRA is able to reduce dopamine transporter degradation, tyrosine hydroxylase expression, IL-1ß release, as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP immunolocalization). These effects have been correlated with the release of neurotrophic factors, such as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioactive RS-GRA. Moreover, after PD-induction mice treated with bioactive RS-GRA are appeared more in health than animals that did not received the treatment both for phenotypic behaviour and for general condition (movement coordination, presence of tremors, nutrition). Overall, our results suggest that bioactive RS-GRA can protect neurons against the neurotoxicity involved in PD via an anti-apoptotic/anti-inflammatory action.

Successful treatment of experimental glomerulonephritis with IdeS and EndoS, IgG-degrading streptococcal enzymes.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) disease often results in end-stage renal failure despite therapy with plasma exchange and immunosuppressive drugs. The newly discovered streptococcal enzymes IgG-degrading enzyme of S.pyogenes (IdeS) and endoglycosidase S (EndoS) act with remarkable specificity on circulating IgG. In this study, we investigate their ability in vivo to prevent damage mediated by kidney-bound antibodies in a mouse model of anti-GBM disease. METHODS: Anti-GBM disease was induced in mice by injection of subnephritogenic doses of rabbit anti-mouse GBM, followed a week later by injection of monoclonal mouse anti-rabbit IgG antibodies. By administrating IdeS or EndoS as fusion partners with GST between these antibody injections, we tested their ability to prevent damage by acting on kidney-bound rabbit anti-GBM. Control animals received placebo injections. RESULTS: All animals in the positive control groups developed severe albuminuria immediately after the second antibody injection (mean, 2.51 mg/24 h; range, 0.13-8.20). This was significantly diminished by EndoS (1.3 +/- 1.3 mg/24 h) and completely prevented by IdeS (0.017 +/- 0.014 mg/24 h). Immunofluorescence studies showed that IdeS treatment effectively removed the Fc fragments of the rabbit IgG. This was accompanied by a significant reduction of the deposition of the complement components C3 and C1q, and this diminished the recruitment of leukocytes to the glomeruli. CONCLUSION: IdeS degrades IgG bound to the GBM in vivo, thereby preventing renal damage in this animal model. Most likely, IdeS would degrade both circulating and kidney-bound anti-GBM in patients with Goodpasture's disease. Whether this would lead to a halt in disease progression and a better prognosis remains to be determined.

Enzyme replacement in Fabry's disease, an inborn error of metabolism.

Two patients with Fabry's disease were infused with normal plasma to provide active enzyme (ceramide trihexosidase) for hydrolysis of the plasma substrate, galactosylgalactosylglucosylceramide. Maximum ceramide trihexosidase activity occurred 6 hours after infusion of the plasma, attaining a level approximately 150 percent of that in normal plasma; enzymatic activity was detectable for 7 days. The amount of accumulated substrate in the plasma of these recipients decreased about 50 percent on day 10 after infusion. Thus, periodic replacement of ceramide trihexosidase activity in the plasma of patients with Fabry's disease might lead to consistently lower amounts of substrate in the plasma and a decrease in its rate of accumulation in tissues.

Therapeutic potential of biofilm-dispersing enzymes.

Surface-attached colonies of bacteria known as biofilms play a major role in the pathogenesis of medical device infections. Biofilm colonies are notorious for their resistance to antibiotics and host defenses, which makes most device infections difficult or impossible to eradicate. Bacterial cells in a biofilm are held together by an extracellular polymeric matrix that is synthesized by the bacteria themselves. Enzymes that degrade biofilm matrix polymers have been shown to inhibit biofilm formation, detach established biofilm colonies, and render biofilm cells sensitive to killing by antimicrobial agents. This review discusses the potential use of biofilm matrix-degrading enzymes as anti-biofilm agents for the treatment and prevention of device infections. Two enzymes, deoxyribonuclease I and the glycoside hydrolase dispersin B, will be reviewed in detail. In vitro and in vivo studies demonstrating the anti-biofilm activities of these two enzymes will be summarized, and the therapeutic potential and possible drawbacks of using these enzymes as clinical agents will be discussed.

Protective and therapeutic application of the depolymerase derived from a novel KN1 genotype of Klebsiella pneumoniae bacteriophage in mice.

Klebsiella pneumoniae is one of the major Gram-negative bacterial pathogens causing hospital-acquired multidrug-resistant infections, and the antimicrobial treatment options are scarce. The lack of available antimicrobials has prompted the development of alternative strategies for the treatment of these infections. In this study, a K. pneumoniae bacteriophage (vB_KpnP_IME321) targeting a KN1 capsular type strain, Kp409, was isolated, characterized and sequenced. This bacteriophage has a latent period of 20 min and a burst size of approximately 410 pfu/cell. It contained 49 predicted open reading frames, of which ORF42 was identified as encoding the putative capsule depolymerase. The enzyme expressed and purified in the Escherichia coli BL21 system, namely Dp42, could depolymerize the capsular polysaccharide of Kp409 and form translucent halos on the plates. The phage-encoded depolymerase could increase the inhibitory effect of serum on the growth of bacteria in vitro. Pre-treated with Dp42 rescued 100% of mice following lethal Kp409 challenge, and administration of this enzyme after infection significantly increased survival rates of infected mice in the animal experiment. In conclusion, the phage-encoded depolymerase Dp42 represents a potential alternative strategy for controlling infections mediated by K. pneumoniae expressing the KN1 capsular polysaccharide.

Staphylococcus biofilm components as targets for vaccines and drugs.

Staphylococci have become the most common cause of nosocomial infections, especially in patients with predisposing factors such as indwelling or implanted foreign polymer bodies. The pathogenesis of foreign-body associated infections with S.aureus and S. epidermidis is mainly related to the ability of these bacteria to form thick, adherent multilayered biofilms. In a biofilm, staphylococci are protected against antibiotic treatment and attack from the immune system, thus making eradication of the infections problematic. This necessitates the discovery of novel prophylactic and therapeutic strategies to treat these infections. In this review, we provide an overview of staphylococcal biofilm components and discuss new possible approaches to controlling these persistent biofilm-dwelling bacteria.

Marine Enzymes in Cancer: A New Paradigm.

Over the last decades, the vast chemical and biodiversity of marine environment has been identified as an important source of new anticancer drugs. The evolution of marine life is a result of competition among microorganisms for space and nutrients in the marine environment, which drives marine microorganisms to generate diverse enzyme systems with unique properties to adapt to harsh conditions of ocean. Therefore, marine-derived sources offer novel enzymes endowed with extraordinary properties. Recent advances in cancer therapy have facilitated enzyme therapy as a promising tool. But, the available information on the use of enzymes derived from marine sources as therapeutic agents for cancer therapy is scanty. The potential utility of marine enzymes in cancer therapy will be discussed in this chapter.

Toward Clinical use of the IgG Specific Enzymes IdeS and EndoS against Antibody-Mediated Diseases.

The endoglycosidase EndoS and the protease IdeS from the human pathogen Streptococcus pyogenes are immunomodulating enzymes hydrolyzing human IgG. IdeS cleaves IgG in the lower hinge region, while EndoS hydrolyzes the conserved N-linked glycan in the Fc region. Both enzymes are remarkably specific for human IgG that after hydrolysis loses most of its effector functions, such as binding to leukocytes and complement activation, all contributing to bacterial evasion of adaptive immunity. However, taken out of their infectious context, we and others have shown that IdeS and EndoS can alleviate autoimmune disease in a number of animal models of antibody-mediated disorders. In this chapter, we will briefly describe the discovery and characterization of these unique enzymes, present the findings from a number of animal models of autoimmunity where the enzymes have been tested, and outline the ongoing clinical testing of IdeS. Furthermore, we will discuss the rationale for further development of IdeS and EndoS into novel pharmaceuticals against diseases where IgG antibodies contribute to the pathology, including, but not restricted to, chronic and acute autoimmunity, transplant rejection, and antidrug antibody reactions.

[Status of lysosomal diseases amongst metabolic diseases]. / Place des maladies lysosomales dans les maladies métaboliques.

Lysosomal diseases belong to the group of diseases which disturb the synthesis, catabolism or processing of complex molecules. These are the most common metabolic diseases, as they account for about one-third of all known metabolic diseases. They can be expressed as early as the intra-uterine stage of life, or appear after birth at any age, including during adulthood. They develop gradually, evolve independently of any intercurrent catabolic event and are independent of diet. Although they have long been associated with an inevitably fatal outcome, several can now be treated effectively, either by enzyme replacement therapy which increases catabolism of the overloaded substance thanks to an input of the deficient enzyme, or by drugs which reduce the synthesis of the overloaded substance.

Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy.

The rise of antibiotic-resistant Klebsiella pneumoniae, a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36), from the Siphoviridae family. To gain insights into the catalytic and structural features of depoKP36, we have recombinantly produced this protein of 93.4 kDa and showed that it is able to hydrolyze a crude exopolysaccharide of a K. pneumoniae host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical K. pneumoniae strains, representing the K63 type, and significantly inhibited Klebsiella-induced mortality of Galleria mellonella larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of Klebsiella strains. The activity of this enzyme was retained in a broad range of pH values (4.0-7.0) and temperatures (up to 45 °C). Consistently, the circular dichroism (CD) spectroscopy revealed a highly stability with melting transition temperature (Tm) = 65 °C. In contrast to other phage tailspike proteins, this enzyme was susceptible to sodium dodecyl sulfate (SDS) denaturation and proteolytic cleavage. The structural studies in solution showed a trimeric arrangement with a high ß-sheet content. Our findings identify depoKP36 as a suitable candidate for the development of new treatments for K. pneumoniae infections.

Poly(ADP-ribose) glycohydrolase inhibitor as chemosensitiser of malignant melanoma for temozolomide.

Disruption of poly(ADP-ribose) polymerase (PARP) pathways by inhibitors of PARP catalytic domain has been shown to increase the anti-tumour activity of temozolomide (TMZ). Since PARP is inhibited by poly(ADP)ribosylation, herein we tested whether inhibition of poly(ADP-ribose) glycohydrolase (PARG) might enhance TMZ efficacy. The PARG inhibitor N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide (GPI 16552) was administered in combination with TMZ to mice injected subcutaneously or intracranially with B16 melanoma cells. The ability of treatment to reduce melanoma metastatic spreading and invasion of the extracellular matrix was also tested. The results indicated that combined treatment with GPI 16552 and TMZ significantly reduced melanoma growth, increased life-span of mice bearing tumour at the CNS site, and decreased the ability of melanoma cells to form lung metastases and to invade the extracellular matrix. In conclusion, PARG inhibition represents an alternative strategy to enhance TMZ efficacy against melanoma in peripheral as well as at CNS site.

Significance of immune response to enzyme-replacement therapy for patients with a lysosomal storage disorder.

Lysosomal storage disorders are collectively important because they cause significant morbidity and mortality. Patients can present with severe symptoms that include somatic tissue and bone pathology, developmental delay and neurological impairment. Enzyme-replacement therapy has been developed as a treatment strategy for patients with a lysosomal storage disorder, and for many of these disorders this treatment is either in clinical trial or clinical practice. One major complication arising from enzyme infusion into patients with a lysosomal storage disorder is an immune response to the replacement protein. From clinical trials, it is clear that there is considerable variability in the level of immune response to enzyme-replacement therapy, dependent upon the replacement protein being infused and the individual patient. Hypersensitivity reactions, neutralizing antibodies to the replacement protein and altered enzyme targeting or turnover are potential concerns for patients exhibiting an immune response to enzyme-replacement therapy. The relative occurrence and significance of these issues have been appraised.

Non-conventional therapeutics for oral infections.

As our knowledge of host-microbial interactions within the oral cavity increases, future treatments are likely to be more targeted. For example, efforts to target a single species or key virulence factors that they produce, while maintaining the natural balance of the resident oral microbiota that acts to modulate the host immune response would be an advantage. Targeted approaches may be directed at the black-pigmented anaerobes, Porphyromonas gingivalis and Prevotella intermedia, associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Functional inhibition approaches, including the use of enzyme inhibitors, are also being explored to control periodontitis. More general disruption of dental plaque through the use of enzymes and detergents, alone and in combination, shows much promise. The use of probiotics and prebiotics to improve gastrointestinal health has now led to an interest in using these approaches to control oral disease. More recently the potential of antimicrobial peptides and nanotechnology, through the application of nanoparticles with biocidal, anti-adhesive and delivery capabilities, has been explored. The aim of this review is to consider the current status as regards non-conventional treatment approaches for oral infections with particular emphasis on the plaque-related diseases.

(1→3)-α-D-Glucan hydrolases in dental biofilm prevention and control: A review.

Dental plaque is a highly diverse biofilm, which has an important function in maintenance of oral and systemic health but in some conditions becomes a cause of oral diseases. In addition to mechanical plaque removal, current methods of dental plaque control involve the use of chemical agents against biofilm pathogens, which however, given the complexity of the oral microbiome, is not sufficiently effective. Hence, there is a need for development of new anti-biofilm approaches. Polysaccharides, especially (1→3),(1→6)-α-D-glucans, which are key structural and functional constituents of the biofilm matrix, seem to be a good target for future therapeutic strategies. In this review, we have focused on (1→3)-α-glucanases, which can limit the cariogenic properties of the dental plaque extracellular polysaccharides. These enzymes are not widely known and have not been exhaustively described in literature.

Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

[Hyaluronidase as a therapeutic alternative in perforated tympanum]. / La hialuronidasa como alternativa al tratamiento de las perforaciones timpánicas.

There are many causes of tympanic perforation but usually only one treatment, surgical. However, many patients cannot or will not undergo surgery. In such cases, treatment with sodium hyaluronidate may be satisfactory. In our group of 20 patients with 21 tympanic perforations who underwent this treatment, 15 evolved favorably. The drawbacks and patient selection are discussed.