Search

Biocatalytic systems based on enzyme cascade reactions have attracted growing interest in the field of biocatalytic medicine. However, it is a major challenge to reasonably construct enzyme cascade reactions with high stability, selectivity, and catalytic efficiency for the in vivo biocatalytic application. Herein, two-in-one engineered glucose oxidase (GOx-Fe 0 ) is fabricated by a biomineralization strategy, through which a nanozyme (Fe 0 NP) is anchored within the inner cavity of GOx. Then, GOx-Fe 0 is immobilized in a pH-sensitive metal-organic framework (MOF) zeolitic imidazolate framework-8 (ZIF-8) to establish a stable and effective MOF-immobilized two-in-one engineered enzyme, GOx-Fe 0 @ZIF-8. In vitro studies show that GOx-Fe 0 @ZIF-8 exhibits excellent stability and high pH/glucose selectivity, and the shorter spacing between cascade enzymes can increase the cascade throughput and effectively improve the reaction efficiency of the enzyme cascade. In vivo experiments exhibit that GOx-Fe 0 @ZIF-8 solves the instability and systemic toxicity of free enzymes, and achieves deep tumor penetration and significant chemodynamic therapeutic efficacy through a pH/glucose-selective enzyme cascade reaction in tumor site. Taken together, such an orchestrated enzyme engineering strategy can effectively improve enzyme stability, selectivity, and enzyme cascade reaction efficiency via chemical transformations, and also provide a promising strategy for the application of biocatalytic cascade reactions in vivo., (© 2023 Wiley-VCH GmbH.)

Phenylketonuria (PKU), a disease resulting in the disability to degrade phenylalanine (Phe) is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity. As an potential alternative to a protein-restricted diet, oral intake of engineered probiotics degrading Phe inside the body is a promising treatment, currently at clinical stage II (Isabella, et al., 2018). However, limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic's activity. Here, we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL) as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917 (EcN) which overcomes the transportation problem. The metabolic engineering strategy was also combined with strengthening of Phe transportation, transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia. Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah F263S PKU mouse model reduced blood Phe concentration by 44.4% compared to the control EcN, independent of dietary protein intake. TYS8500 shows great potential in future applications for PKU therapy., (© 2022. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Multimodal nanotherapeutic cancer treatments are widely studied but are often limited by their costly and complex syntheses that are not easily scaled up. Herein, a simple formulation of glucose-oxidase-coated CuS nanoparticles was demonstrated to be highly effective for melanoma treatment, acting through a synergistic combination of glucose starvation, photothermal therapy, and synergistic advanced chemodynamic therapy enabled by near-infrared irradiation coupled with Fenton-like reactions that were enhanced by endogenous chloride.

Nanozyme has been regarded as one of the antibacterial agents to kill bacteria via a Fenton-like reaction in the presence of H 2 O 2 . However, it still suffers drawbacks such as insufficient catalytic activity in near-neutral conditions and the requirement of high H 2 O 2 levels, which would minimize the side effects to healthy tissues. Herein, a mesoporous ceria hollow sphere/enzyme nanoreactor is constructed by loading glucose oxidase in the mesoporous ceria hollow sphere nanozyme. Due to the mesoporous framework, large internal voids, and high specific surface area, the obtained nanoreactor can effectively convert the nontoxic glucose into highly toxic hydroxyl radicals via a cascade catalytic reaction. Moreover, the generated glucose acid can decrease the localized pH value, further boosting the peroxidase-like catalytic performance of mesoporous ceria. The generated hydroxyl radicals could damage severely the cell structure of the bacteria and prevent biofilm formation. Moreover, the in vivo experiments demonstrate that the nanoreactor can efficiently eliminate 99.9% of bacteria in the wound tissues and prevent persistent inflammation without damage to normal tissues in mice. This work provides a rational design of a nanoreactor with enhanced catalytic activity, which can covert glucose to hydroxyl radicals and exhibits potential applications in antibacterial therapy.

We demonstrate a MnO2-based nanoreactor to achieve continuous oxygen generation and efficient conversion from glucose to singlet oxygen for combined photodynamic-starvation therapy.

Cancer cells possess some inherent characteristics, such as glucose-dependence and intolerance to heat and exogenous reactive oxygen species (ROS). In this study, a strategy has been developed to target these vulnerable weaknesses of cancer cells using glucose oxidase (GOx) and polydopamine (PDA) functionalized iron oxide nanoparticles (Fe 3 O 4 @PDA/GOx NPs). PDA is first deposited on the surfaces of iron oxide NPs through self-polymerization, and then GOx is covalently linked with PDA upon mixing the enzyme and Fe 3 O 4 @PDA under alkaline conditions. In this system, the PDA layer along with iron oxide NPs serves as a photothermal transfer material converting near infrared (NIR) radiation into heat. The covalently linked GOx can competitively consume glucose and spontaneously generate ROS H 2 O 2 that can be further converted by the iron oxide NPs into more toxic ˙OH, inducing apoptosis of cancer cells. The selective toxicity of Fe 3 O 4 @PDA/GOx NPs on cancer cells is demonstrated both in vitro and in vivo. In particular, a single injection rather than multiple doses results in significant suppression of tumors, and does not induce apparent histological lesions in the 4T1 tumor-bearing Balb/c mice. The versatility of the functionalization strategy reported in this study will contribute to developing efficient therapies for selective cancer treatment.

It was demonstrated for the first time that immobilized lysozyme can efficiently remove Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (endotoxins) from solutions. Experimentally confirmed sorption capacity for the developed sorbent was at least 400 ng of endotoxin per ml sorbent. The new sorbent is compatible with the whole human blood and can be potentially used in extracorporeal therapy in the treatment of sepsis.

L-asparaginase, a therapeutic involved in cancer therapy, from Bacillus tequilensis PV9W (ansA gene) was cloned and over expressed in Escherichia coli BL21 (DE3), achieved the aim of maximizing the yield of the recombinant enzyme (6.02 ± 1.77 IU/mL) within 12 h. The native L-asparaginase of B. tequilensis PV9W was encapsulated using solid lipid particles by hot lipid emulsion method, which is reported for first time in this study. Subsequently, the lipid encapsulated L-asparaginase (LPE) was characterized by SEM, UV-Vis spectroscopy, FT-IR, SDS-PAGE and its thermo stability was also analyzed by TGA. Further characterization of LPE revealed that enzyme was highly stable for 25 days when stored at 25 °C, showed high pH (9) tolerance and longer trypsin half-life (120 min). In addition, the cytotoxic ability of LPE on HeLa cells was highly enhanced compared to the native L-asparaginase from Bacillus tequilensis PV9W. Moreover, better kinetic velocity and lower K m values of LPE aided to detect L-asparagine in cell extracts by Differential Pulse Voltammetry (DPV) method. The LPE preparation also showed least immunogenic reaction when tested on normal macrophage cell lines. This LPE preparation might thus pave way for efficient drug delivery and enhancing the stability of L-asparaginase for its therapeutic applications.

This clinical observation describes the enteral nutrition (EN) management of 2 toddlers at high nutrition risk due to cystic fibrosis (CF), exocrine pancreatic insufficiency, and comorbid medical conditions. The first case report describes a boy with severe malabsorption after intestinal resection. The second case report reviews a boy with CF and neuroblastoma. When pancreatic enzyme replacement therapy with EN was not effective or appropriate, use of an in-line digestive cartridge was initiated. While using the digestive cartridge, both children showed improvements in their anthropometric measures. This observation reviews the nutrition management throughout their clinical course and describes the use of a digestive cartridge with EN., (© 2018 The Authors. Nutrition in Clinical Practice published by Wiley Periodicals, Inc. on behalf of American Society for Parenteral and Enteral Nutrition.)

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.

The utilization of scaffolds for enzyme immobilization involves advanced bionanotechnology applications in biorefinery fields, which can be achieved by optimizing the function of various enzymes. This review presents various current scaffolding techniques based on proteins, microbes and nanomaterials for enzyme immobilization, as well as the impact of these techniques on the biorefinery of lignocellulosic materials. Among them, architectural scaffolds have applied to useful strategies for protein engineering to improve the performance of immobilized enzymes in several industrial and research fields. In complexed enzyme systems that have critical roles in carbon metabolism, scaffolding proteins assemble different proteins in relatively durable configurations and facilitate collaborative protein interactions and functions. Additionally, a microbial strain, combined with designer enzyme complexes, can be applied to the immobilizing scaffold because the in vivo immobilizing technique has several benefits in enzymatic reaction systems related to both synthetic biology and metabolic engineering. Furthermore, with the advent of nanotechnology, nanomaterials possessing ideal physicochemical characteristics, such as mass transfer resistance, specific surface area and efficient enzyme loading, can be applied as novel and interesting scaffolds for enzyme immobilization. Intelligent application of various scaffolds to couple with nanoscale engineering tools and metabolic engineering technology may offer particular benefits in research., (© 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nitric oxide (NO) is an important signaling molecule in cardiovascular system, and the sustained release of NO by endothelial cells plays a vital role in maintaining patency and homeostasis. In contrast, lack of endogenous NO in artificial blood vessel is believed to be the main cause of thrombus formation. In this study, enzyme prodrug therapy (EPT) technique was employed to construct a functional vascular graft by immobilization of galactosidase on the graft surface. The enzyme-functionalized grafts exhibited excellent catalytic property in decomposition of the exogenously administrated NO prodrug. Localized and on-demand release of NO was demonstrated by in vitro release assay and fluorescent probe tracing in an ex vivo model. The immobilized enzyme retained catalytic property even after subcutaneous implantation of the grafts for one month. The functional vascular grafts were implanted into the rat abdominal aorta with a 1-month monitoring period. Results showed effective inhibition of thrombus formation in vivo and enhancement of vascular tissue regeneration and remodeling on the grafts. Thus, we create an enzyme-functionalized vascular graft that can catalyze prodrug to release NO locally and sustainably, indicating that this approach may be useful to develop new cell-free vascular grafts for treatment of vascular diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The results of treatment of 77 patients, ageing 18-71 yrs old, for an acute paraproctitis in 2010-2014 yrs were analyzed. A preventive puncture-flush enzymosanation of purulent foci, using immobilized bacterial proteinases (imozimase), metrogyl P in conjunction with low-intensive laser irradiation have permitted to conduct the optimal preoperative preparation of patients, to improve their state, to reduce the local inflammatory reactions intensity significantly.

In this study, a series of semi-interpenetrating polymer network (IPN) hydrogels were prepared as a support material for lipase immobilization. Hydrogels were synthesized via free radical polymerization in different compositions of chitosan (Cs), acrylamide (AAm), and citraconic acid (CA). The swelling values of the hydrogels were found to be 240-400%. Depending on the swelling results, Cs-P(AAm-co-CA)-2 hydrogel was chosen for lipase immobilization. Three different types of immobilization technique were carried out. Lipase release behaviors were investigated, and immobilization yields of three immobilization methods were compared, and the maximum immobilization yield value was determined for entrapment method.

The possibility of boosting antifibrotic activity of testicular hyaluronidase immobilized on polyethylene oxide with spiperone was studied on the bleomycin models of a single (partially reversible pneumofibrosis) and repeated (irreversible pneumofibrosis) injuries to the alveolar epithelium in C57Bl/6 mice. The antifibrotic effect was more pronounced after successive treatment with immobilized hyaluronidase and spiperone than after individual treatment with each of the compounds: no collagen deposition in the parenchyma of bleomycin-damaged lungs was found. The decrease in inflammatory cell (lymphocytes, macrophages, neutrophils, plasma cells) infiltration of the alveoli and alveolar tracts interstitium in mice treated by immobilized hyaluronidase and spiperone did not differ from the anti-inflammatory effect of spiperone monotherapy.

Gout is an abnormality in the body resulting in the accumulation of uric acid mainly in joints. Dissolution of uric acid crystals into soluble allantoin by the enzyme uricase might provide a better alternative for the treatment of gout. This work aims to investigate the feasibility of a transdermal patch loaded with uricase for better patient compliance. Mesoporous silica (SBA-15) was chosen as the matrix for immobilisation of uricase. Highly oriented mesoporous SBA-15 was synthesized, characterized and uricase was physisorbed in the mesoporous material. The percentage adsorption and release of enzyme in borate buffer was monitored. The release followed linear kinetics and greater than 80% enzyme activity was retained indicating the potential of this system as an effective enzyme immobilization matrix. The enzyme permeability was studied with Wistar rat skin and human cadaver skin. It was found that in case of untreated rat skin 10% of enzyme permeated through skin in 100 h. The permeation increased by adding permeation enhancer (combination of oleic acid in propylene glycol (OA in PG)). The permeation enhancement was studied under two concentrations of OA in PG (1%, 5%) in both rat and human cadaver skin and it was found that 1% OA in PG showed better result in rat skin and 5% OA in PG showed good results in human cadaver skin.

Hyaluronidase immobilized on polyethylenoxide obtained by electron bean synthesis was administered intranasally and intravenously to C57Bl/6 mice after intratracheal bleomycin and the enzyme effects on the development of pneumofibrosis in animals were studied. Intranasal immobilized hyaluronidase prevented connective tissue growth in the lungs exposed to bleomycin and virtually did not modulate the infiltration of the alveolar and alveolar duct interstitium by inflammatory cells (lymphocytes, macrophages, neutrophils, plasma cells). The antifibrotic effect developed sooner after intranasal inoculation of immobilized hyaluronidase and was more pronounced than after intranasal native hyaluronidase. Intravenous injection of immobilized hyaluronidase did not modify the inflammatory process and deposition of collagen fibrils in the lung parenchyma in pneumofibrosis.

Using the model of lung fibrosis induced by intratracheal administration of bleomycin we studied anti-fibrotic activity of combined treatment with neuroleptic haloperidol and hyaluronidase immobilized on polyethylene oxide using electron-beam synthesis. It was shown that successive administration of immobilized hyaluronidase and the neuroleptic drug inhibits deposition of collagen fibers in the bleomycin-treated lungs. Combined treatment with the test compounds reduced swelling of the alveolar epithelium, exudation and infiltration of the alveolar interstitium and alveolar passages by inflammatory cells, and desquamation of alveolocytes into alveolar lumen, so that the alveolar-capillary membrane function was preserved.

Gout is an inflammatory arthritis characterized by sudden, painful inflammation. Gout can affect any joint in an asymmetric distribution. Gouty attacks may be isolated or can be followed by years of recurrent flares. Over time, elevated serum urate levels and tophaceous deposits can lead to deformity and disability from underlying bony erosion. The concept of 'treatment-failure gout' describes a unique population that has been either unable to tolerate allopurinol or who have not experienced normalization of serum urate levels on allopurinol. It is estimated that approximately 1-1.5% of the estimated 3-8 million people with gout in the USA have treatment-failure gout. Pegloticase is an US FDA-approved intravenous medication that is a mammalian recombinant uricase conjugated to monomethoxy polyethylene glycol. Two recent Phase III trials have found pegloticase to be effective in the management of treatment-failure gout. These studies also highlight safety concerns regarding the drug's immunogenicity.

High hepatoprotective activity of granulocytic CSF and hyaluronidase immobilized using electron-beam immobilization technology was demonstrated on the model of CCl(4)-induced hepatitis: the preparations produced anticholestatic, anti-inflammatory, and antisclerotic effects. These effects developed against the background of stimulation of bone marrow multipotent precursor cells and their mobilization into circulation accompanied by an increase in the content of parenchymatous progenitor cells in the liver. The most pronounced positive effect was observed in combined treatment with the test preparations.

Hypoglycemic effect of hyaluronate-endo-β-N-acetylhexosaminidase immobilized by electron-beam synthesis nanotechnology (imHEA-HA) was studied in experimental insulin-dependent and insulin-independent diabetes mellitus. The drug exhibited a hypoglycemic effect of its own and potentiated the pharmacological effect of exogenous insulin injected in vivo. Studies on liver cell culture demonstrated an increase of cell sensitivity to insulin after treatment with imHEA-HA.

Immobilized hyaluronidase (nanotechnology method of electron-beam synthesis) exhibited high hepatoprotective activity on the model of Cl4-induced hepatitis. This agent produced anticholestatic, anti-inflammatory, and antisclerotic effects. These effects were shown to accompany stimulation of multipotent bone marrow precursors, mobilization of these cells into the peripheral blood, and cell migration to the target organ increasing the number of parenchymal progenitor cells in the liver. The mechanisms for targeted migration of progenitor cells suggest a decrease in SDF-1 production by bone marrow stromal cells and increase in the synthesis of this factor by microenvironmental cells of the liver tissue.

In this paper a device, based on urease-loaded microspheres, is presented. The first task of this work was the optimization of a procedure for the alginate microspheres realization, having a radius as close as possible to the optimal one necessary to achieve the maximum enzyme exploitation. This optimal radius was calculated theoretically through a mathematical model which describes the concentration of substrate (urea) inside the microspheres on the assumption of a diffusion-reaction mechanism. The enzyme-loaded microspheres were successfully tested in a prototypal device aimed at the depletion of urea from a circulating fluid simulating blood flow: the results showed that urea concentration in the circulating fluid drops down to less than 25% of the initial value after 5 h.

Acylated Superoxide Dismutase (Ac-SOD) enzymosomes, liposomal enzymatic systems expressing catalytic activity in the intact form, were previously characterized. The main scope of the present work was to investigate the biological behaviour of Ac-SOD inserted in the lipid bilayer of liposomes, in comparison with SOD located in the aqueous compartment of liposomes. Two types of liposomes were used: conventional liposomes presenting an unmodified external surface and long circulating liposomes coated with poly (ethylene glycol) (PEG). Liposomal formulations of Ac-SOD and SOD were prepared and labelled with indium-111 and their in vivo fate compared. Data obtained led us to the conclusion that, for liposomes coated with PEG the in vivo fate was not influenced by the insertion of Ac-SOD in the lipid bilayers. The potential therapeutic effect of Ac-SOD enzymosomes was compared with SOD liposomes in a rat model of adjuvant arthritis. A faster anti-inflammatory effect was observed for Ac-SOD enzymosomes by monitoring the volume of the inflamed paws. The present results allowed us to conclude that Ac-SOD enzymosomes are nano-carriers combining the advantages of expressing enzymatic activity in intact form and thus being able to exert therapeutic effect even before liposomes disruption, as well as acting as a sustained release of the enzyme.

Bovine serum amine oxidase (BSAO) oxidatively deaminates polyamines containing primary amine groups, spermidine and spermine, to form the cytotoxic products hydrogen peroxide and aldehyde(s). Polyamines are present at elevated levels in many tumor tissues. The aims of the study were to evaluate the anti-tumoral activities of native and immobilized BSAO in mouse melanoma and also to determine the mechanism of tumor cell death. C57BL mice received a subcutaneous injection of B16 melanoma cells to induce formation of tumors, prior to antitumor treatments with native and immobilized BSAO. The enzyme was immobilized in a poly(ethylene glycol) (PEG) biocompatible matrix. Antitumor treatments consisted of a single injection of enzyme into the tumor. When immobilized BSAO (2.5mU) was injected into the tumor, there was a marked decrease of 70% of the tumor growth. This was compared with a decrease of only 32% of tumor size when the same amount of native BSAO was administered. The type of cell death was analysed in tumors that were treated with native or immobilized BSAO. When tumors were treated with immobilized BSAO, there was induction of a high level of apoptosis (around 70%), compared to less than 10% with the native enzyme. Apoptotic cell death was assessed by nuclear chromatin condensation using Hoechst staining and labelling of externalized phosphatidylserine using Annexin V. However, native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of about 40%, compared to less than 10% with immobilized BSAO. In conclusion, the advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumor cell death by apoptosis rather than necrosis.

Phospholipase A2 (PLA2; EC 3.1.1.4) is a lipolytic enzyme that hydrolysis the ester bond in sn-2 position of phospholipids. In this work, the PLA2 from hog pancreas was covalently coupled to porous glass. The properties of free and immobilized enzyme were also investigated and compared. The optimum pH and temperature were found as 8.5 and 50 degrees C, respectively for both free and immobilized enzyme. The immobilized enzyme had good properties that potential for medical application is considerable. Its use in lowering plasma cholesterol concentrations in blood samples was also demonstrated.

Background: Multiple myeloma remains an incurable disease. New agents are needed to improve therapy for patients with this disease. Previous investigators evaluated in vitro sensitivity of myeloma cells to polyethylene glycol-conjugated L-asparaginase (PEG-L-asparaginase) using the human tumor clonogenic assay. Of the 19 myeloma samples evaluated, 63% were inhibited at 0.075 IU/mL, and 74% were inhibited at 0.75 IU/mL. PEG-L-asparaginase is a form of Escherichia coli-derived L-asparaginase that is bound covalently to polyethylene glycol. Compared with the native form, it has a longer half-life and is less likely to cause allergic reactions., Methods: The authors conducted a Phase I-II trial using PEG-L-asparaginase as a single agent in patients with recurrent and/or refractory multiple myeloma., Results: Twenty-two patients received a median of two doses of PEG-L-asparaginase. In the 17 patients who are evaluable for response, a complete response was observed in one patient after four doses, and stable disease was observed in eight patients. Progression of disease was the reason for termination from study in the remaining eight patients. The median survival was 31.7 months, with four patients who were alive at 72 months after the start of therapy. Grade 3-4 toxicity was noted by the PEG-L-asparaginase 2000 mg/m(2) level. Severe allergic reactions were noted only at the highest dose level., Conclusions: Current data suggest that the maximal tolerated dose for single agent PEG-L-asparaginase in relapse/refractory multiple myeloma patients is 1000 mg/m(2) every 4 weeks. We could not identify any correlation between dose, plasma level and response. In this advanced group of patients we noted stable disease and/or response in 52% of evaluable patients. PEG-L-asparaginase has lower tolerance when used in the standard dosage as a single agent in this group of patients. We therefore recommend further studying of PEG-L-asparaginase dose of 1000 mg/m(2) on alternate weeks with steroids and/or other immune modulators., (Copyright 2003 American Cancer Society.)

Imozymase treatment should be started from the first hospital days in patients with destructive pulmonary tuberculosis concurrent with nonspecific purulent endobronchitis. Two-week ultrasound inhalations with imozymase in a dose of 1 ml (60 PU) may rapidly abolish not only the nonspecific inflammatory bronchial process, but also accelerate the attenuation of symptoms of intoxication, the time of abacillation, cavernous closure, and preparation for surgical interventions.

There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-rehydration method', followed by the 'extrusion technique' has shown to be superior over other procedures. In addition to many investigations in the field of cheese production--there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in the enzyme-replacement therapy or for immunoassays)--including a few in vivo studies. In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous. Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase. Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products. Using physical (e.g. microwave irradiation) or chemical methods (e.g. addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent. A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there. There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates. Using micrometer-sized vesicles (so-called 'giant vesicles') as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules.

The impact of proteolytic enzymes on the humoral immune response, survival rate and mean survival time of mice, infected with S. aureus culture and receiving antibiotics was studied. Infection with staphylococcal suppressed the formation of antibodies to sheep red blood cells. Ampicillin made this immunosuppression even more pronounced, while gentamicin produced practically no effect on the degree of immunosuppression in the infected animals. Proteolytic enzymes terrilytin and terridecase exhibited immunocorrecting properties when used in combination with antibiotics. Terridecase, the immobilized form of the enzyme proved to have the highest activity. In experimental generalized staphylococcal infection all preparations under study produced a protective effect. The maximum effect was noted after the use of ampicillin in combination with terridecase.

L-asparaginase is a very effective anti-tumor agent. To take the most advantage of enzyme treatment method, the natural L-asparaginase could be chemically modified, entrapped or immobilized into/onto other carriers. The extracorporeal shunt system with immobilized enzyme reactor deserves to receive special attention. It provides an entirely new idea for leukemia treatment and is proved to be very prospective in the future leukemia cure.

In vitro experiments on fibrin films using purulent exudate from the abdominal cavity of rats with experimental peritonitis demonstrate the fibrinolytic effect of bacterial proteinases immobilized on a polymeric matrix. The application of Imozimaza in the complex treatment of experimental peritonitis by the way of intraperitoneal lavage resulted in reliable lowering of mortality, due to the lysis of fibrinopurulent abdominal contents and better contact between antibacterial agents and peritonitis pathogens. In the clinic, prolonged abdominal proteolysis was applied to 44 patients with postoperative diffuse purulent peritonitis of >24 h duration. Under the conditions of programmable relaparotomy, intraperitoneal Imozimaza infusion led (as in in vitro tests) to the lysis of fibrinopurulent masses, which contained micro-organisms of an order higher than exudate. It was accompanied by increase in the drainage efficacy, absence of fragmentation of abdominal contents and absence of secondary abscesses. The use of Imozimaza on the background of complex antibacterial treatment and combined homeostatic therapy resulted in lowering of mortality from 65.8% to 27.3%. Complications and contra-indications for Imozimaza use in diffuse purulent peritonitis were not registered.

Joint immobilization of a proteolytic enzyme (terrilytin, protease C, or collytin) and an antimicrobial compound or the bacteriolytic enzyme lysozyme in the structure of polyvinyl alcohol was studied. Changes in the activity of immobilized enzymes upon x-ray sterilization were studied. These materials were highly effective in wound healing.

The influence of immobilized proteases such as catalytin (a complex preparation containing the protease terrilytin and the antiseptic catapol) and streptodecase (an activator of fibrinolysis proteases) on the humoral immune response induced by the thymus-dependent antigen (SRBC) under conditions of burns and Staphylococcus infected burns was studied. The immobilized proteases catalytin and streptodecase had an immunostimulating effect under conditions of extended burns. Catalytin had an immunocorrigating effect under conditions of the infected burns. It was suggested that the complex enzyme-containing preparation catalytin and the immobilized activator of fibrinolysis proteases streptodecase were promising immunopharmacological agents in combined treatment of pathological processes accompanied by the development of immune deficiency as a secondary condition.

A new method for the immobilization of phospholipase A2 (PLA2) has been developed to enhance the activity retention of immobilized PLA2. When PLA2 from the venom of Agkistrodon piscivorus piscivorus was pretreated with 4-nitro-3-octanoyl-oxybenzoic acid to acylate epsilon-amino groups of two lysines (Lys-7 and Lys-10) and the resulting acylated enzyme was covalently coupled onto carbonyldiimidazole-activated cross-linked agarose beads, the immobilized acylated enzyme showed high retention of activity toward various aggregated phospholipids. Toward densely packed phospholipid bilayers, such as large unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the immobilized acylated A. p. piscivorus PLA2 was 25-fold more active than the soluble A. p. piscivorus PLA2. The general applicability of our immobilization protocol was demonstrated by the high retention of activity achieved for the immobilized acylated PLA2 from the venom of Naja naja naja. In particular, full activity retention of the immobilized acylated A. p. piscivorus PLA2 toward phospholipids on the surface of human low density lipoproteins suggests its potential usefulness in a newly developed PLA2-based therapy for hypercholesterolemia.

The paper summarises the experience gained in the use of immozymase in tuberculous patients to advance the disease diagnosis and treatment. Immozymase proved valuable in obtaining sputum enriched with M. tuberculosis in patients who were previously considered noncarriers. M. tuberculosis became detectable in 28.3% of them. Immozymase instillations of the urethra before its massage for stimulation of prostatic secretion led to getting secretion in all the patients (0.35 ml, on the average). Immuzymase inhalations were used in the treatment of purulent bronchitis in patients with destructive pulmonary tuberculosis in conservative and preoperative regimens. Postoperative immozymase inhalations promoted prevention of pleuropulmonary complications. Open treatment of the caverns with immozymase after cavernotomy shortens the treatment duration. Positive results were also reached in the treatment of pleural empyema.

Pretreatment characteristics of 138 dogs with malignant lymphoma were analyzed to determine prognostic factors associated with outcome (ie, complete response rate, time to relapse after complete response, survival time). Dogs were all treated for 10 weeks, using a standard induction chemotherapy protocol, and were then given asparaginase weekly. Once the disease became progressive, second-line chemotherapy was instituted. Age, sex, weight, clinical stage, performance grade, immunophenotype, and malignancy grade assigned according to the National Cancer Institute's Working Formulation were not associated with complete response rate. However, malignancy grade assigned according to the Kiel classification was found to be associated with complete response rate; dogs with high-grade malignancies had a significantly higher complete response rate than did dogs with low-grade malignancies. By means of multivariate analysis, clinical stage and immunophenotype were found to be prognostic factors for time to relapse (among dogs that had had a complete response) and survival time. In addition, malignancy grade assigned according to the Kiel classification was found to be a prognostic factor for time to relapse; whereas, malignancy grade assigned according to the Working Formulation was determined to be a prognostic factor for survival time.

The principles of preoperative management of patients with varicosity complicated by trophic ulcers are discussed. The main type of treatment is surgery including subfascial ligation of the perforating veins of the thigh, leg, and foot+phlebectomy after Troyanov-Trendelenburg-Bebcock-Narath. The author suggests or modifies approaches to the perforating veins. The basis of preoperative management is formed of measures causing a general effect on the organism (lysozymo--and immunoglobulinotherapy, stimulation of regeneration by means of zinc sulphate, transfusion of albumins, etc.), local treatment (cleansing of the ulcer by low-frequency ultrasound, and with immobilized enzymes), and measures for improving microcirculation (magnesium preparations, disaggregants, ultrasound). Complex treatment based on subfascial ligation of perforating veins and removal of varicose superficial veins led to stable healing of ulcers in 98.2% of patients.

Earlier studies have shown that a mixture of glucose oxidase and a peroxidase exerts a tumoricidal effect on rats bearing Novikoff hepatomas when the enzyme mixture is injected intraperitoneally. The enzyme mixture was shown to be nontoxic when injected into healthy animals at levels up to 600 times the therapeutic dose. In the present study, we have evaluated the possibility that the host immune defense system may be involved in the antitumor activity of the peroxidase system, using the murine Ehrlich ascites tumor as the target. The results revealed that the antitumor activity of the peroxidase system is absent in tumor-bearing animals whose immune system has been compromised by whole body gamma-irradiation or by an induced selenium deficiency. The peroxidase system was also found to be inactive in tumor-bearing mice whose immune system was suppressed by the administration of cyclosporin A as well as in athymic (nu/nu) mice. These results indicate that T lymphocytes may directly or indirectly be involved in the in vivo antitumor activity of the peroxidase system. This could explain the observed high selectivity toward tumor cells by the enzyme system in vivo and its lack of toxicity in healthy animals.

L-asparaginase is an enzyme that inhibits protein synthesis by the depletion of sources of L-asparagine, which is necessary for transformed lymphoid cells to proliferate. L-asparaginase is used in the treatment of childhood acute lymphoblastic leukemia. A problem with L-asparaginase therapy is the immunogenicity of the enzyme and the development of anaphylactic reactions. Canine lymphoma is a predominantly B-cell tumor with widespread disease; without treatment, dogs with lymphoma usually survive 1-2 months. Canine lymphoma will respond to L-asparaginase therapy. A randomized double-blind study evaluated a polyethylene glycol (PEG) conjugate L-asparaginase combined with chemotherapy (vincristine, cyclophosphamide, doxorubicin, and prednisone). Thirty-five dogs were randomized to the PEG L-asparaginase group, and 34 dogs were randomized to the native L-asparaginase group. Thirty dogs (85.7%) achieved a complete remission (CR) with a median time to relapse of 217 days, and 32 (94.1%) dogs in the native L-asparaginase group achieved a CR with a median time to relapse of 214 days (P greater than 0.05). The asparaginase was well tolerated in both groups. Two dogs in the native L-asparaginase group had severe allergic reactions, and one dog in the PEG asparaginase group had a generalized urticarial reaction after repeated injections. This study indicates that PEG L-asparaginase has equal therapeutic efficacy to native L-asparaginase.

The article analyses the results of treatment of 150 patients with purulent diseases of the soft tissues of various localization, in which after surgical treatment the purulent focus immobilized trypsin (50 patients), trypsin with lysozyme immobilized on gauze (50 patients), and antiseptics (50 patients) were used for topical treatment of the formed wounds. The results of the treatment showed that topical prolonged enzyme therapy of purulent wounds with the use of trypsin immobilized on gauze and that with lysozyme promotes cleansing of the wounds, reduces microbial dissemination, intensifies reparative processes, and reduces the terms of treatment.