Sortagged anti-EGFR immunoliposomes exhibit increased cytotoxicity on target cells.

PURPOSE: Conventional chemotherapy is associated with therapy-limiting side effects, which might be alleviated by targeted chemotherapeutics such as immunoliposomes. The targeting ligands of immunoliposomes are commonly attached by unspecific chemical conjugation, bearing risk of structural heterogeneity and therewith related biological consequences. Chemoenzymatic methods may mitigate such risks through site-specific conjugation. METHODS: The formulation parameters for pentaglycine-modified, doxorubicin-loaded liposomes and the reaction conditions for a site-specific, Sortase-A mediated conjugation with monoclonal antibodies were thoroughly evaluated. The cytotoxicity of such sortagged, epidermal growth factor receptor (EGFR)-specific immunoliposomes was tested on human breast cancer cells. RESULTS: Sortaggable liposomes with a defined size (140 nm, PDI < 0.25) and high encapsulation efficiency (>90%) were obtained after manufacturing optimization. A ratio of 1.0-2.5 µM mAb/100 µM pentaglycine yielded stable dispersions and circumvented carrier precipitation during ligand grafting. The cytotoxicity on EGFR+ MDA-MB-468 was up to threefold higher for EGFR-specific immunoliposomes than for the nontargeted controls. CONCLUSIONS: Sortase-A is suitable to generate immunoliposomes with a site-specific ligand-carrier linkage and hence improves chemical homogeneity of targeted therapeutics. However, the sweet spot for manufacturability utilizing mAbs with two Sortase-A recognition sites is narrow, making mono-reactive binders such as scFvs or Fab's preferable for a further development. Despite this, the immunoliposomes demonstrated a targeted delivery of doxorubicin, indicating the potential to increase the therapeutic window during the treatment of EGFR+ tumors.

Lipoprotein modifications by gingipains of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low-density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro. MATERIAL AND METHODS: Plasma lipoproteins were isolated from whole blood treated with wild-type and gingipain-mutant (lacking either the Rgp- or Kgp gingipains) P. gingivalis by density/gradient-ultracentrifugation and were studied using 2-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid-reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation. RESULTS: Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB-100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high-density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation. CONCLUSION: Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.

Bioavailability of House Dust Mite Allergens in Sublingual Allergy Tablets Is Highly Dependent on the Formulation.

BACKGROUND: In sublingual immunotherapy (SLIT), the immune system is addressed by solubilized allergen that interacts with immunocompetent cells of the oral mucosa, the efficiency of which is governed by 2 main factors of SLIT allergen bioavailability: the allergen concentration and the mucosal contact time. Recently, 3 house dust mite (HDM) SLIT tablets were developed that differ with regard to allergen content, nominal strength (maintenance doses: 6 SQ-HDM/10,000 Japanese Allergen Units [JAU], 12 SQ-HDM/ 20,000 JAU, and 300 IR/57,000 JAU), and formulation (freeze-dried/compressed). Here, the importance of the SLIT tablet formulation for HDM major allergen bioavailability is examined. METHODS: The HDM major allergen content, tablet disintegration times, and allergen release kinetics were determined. Dissolution kinetics (allergen concentration vs. time) of Der f 1, Der p 1, and Der 2 were measured. Area under the curve (AUC) was used as a surrogate parameter for allergen bioavailability. RESULTS: The release of HDM major allergens from the freeze-dried tablets was complete after 30 s, while only partial release was achieved with the compressed tablets, even after prolonged dissolution. At 1 min, i.e., the recommended sublingual holding time for the freeze-dried tablets, the allergen bioavailability (AUC) of the compressed 300 IR/57,000 JAU tablet was 4.7-fold (Der f 1), 10.8-fold (Der p 1), and 23.6-fold (Der 2) lower than that of the freeze-dried 12 SQ-HDM/20,000 JAU tablet and similar to (Der f 1) and 5.3-fold (Der p 1) and 12.5-fold (Der 2) lower than that of the freeze-dried 6 SQ-HDM/10,000 JAU tablet. CONCLUSIONS: SLIT tablet allergen bioavailability depends highly on the tablet formulation. Only the fast-dissolving freeze-dried tablets provide maximal delivery of soluble allergens and achieve allergen concentrations that reflect the nominal tablet strengths within the recommended sublingual holding time.

2D QSAR and similarity studies on cruzain inhibitors aimed at improving selectivity over cathepsin L.

Hologram quantitative structure-activity relationships (HQSAR) were applied to a data set of 41 cruzain inhibitors. The best HQSAR model (Q(2)=0.77; R(2)=0.90) employing Surflex-Sim, as training and test sets generator, was obtained using atoms, bonds, and connections as fragment distinctions and 4-7 as fragment size. This model was then used to predict the potencies of 12 test set compounds, giving satisfactory predictive R(2) value of 0.88. The contribution maps obtained from the best HQSAR model are in agreement with the biological activities of the study compounds. The Trypanosoma cruzi cruzain shares high similarity with the mammalian homolog cathepsin L. The selectivity toward cruzain was checked by a database of 123 compounds, which corresponds to the 41 cruzain inhibitors used in the HQSAR model development plus 82 cathepsin L inhibitors. We screened these compounds by ROCS (Rapid Overlay of Chemical Structures), a Gaussian-shape volume overlap filter that can rapidly identify shapes that match the query molecule. Remarkably, ROCS was able to rank the first 37 hits as being only cruzain inhibitors. In addition, the area under the curve (AUC) obtained with ROCS was 0.96, indicating that the method was very efficient to distinguishing between cruzain and cathepsin L inhibitors.

Enzymatic gluten detoxification: the proof of the pudding is in the eating!

Celiac disease is caused by an immune response to the dietary protein gluten. The only available treatment is the strict exclusion of gluten from the diet; however, this is marred by the virtual omnipresence of this protein. The enzymatic degradation of gluten might become an alternative to the gluten-free diet, and recent work indicates that such approaches are getting close to being tested in clinical trials.

Diferenciación genética por patrones de restricción entre Entamoeba histolytica monoxénica patógena y no patógena / Genetic differentiation of pathogenic from non-pathogenic Entamoeba histolytica monoxenic by restriction patterns

Las cisteín-proteasas de Entamoeba histolytica son consideradas factores de virulencia importantes en la patogénesis de la amibiasis. Con base en la diferencia que presenta el gene enhhic que codifica para una proteasa de 30 kDa, en este trabajo se validó una estrategia para diferenciar Entamoeba histolytica patógena de la no patógena por medio del patrón de restricción. Material y métodos. Se utilizaron 13 muestras de materia fecal con quistes de Entamoeba histolytica aislados de cuatro individuos asintomáticos y nueve sintomáticos de diferentes edades, sexos y se cultivaron en medio de Robinson. A partir del DNA extraído de los trofozoítos se amplificó el gene enhhic por PCR y se cortó con las enzimas de restricción Taq I y Hinf I. Resultados. De todos los aislados cultivados en medio de Robinson, se obtuvo un fragmento amplificado de 530 pb que hibridó con una sonda para Entamoeba histolytica. Sin embargo la evaluación por digestión enzimática con Taq I y Hinf I indicó que sólo dos de las cuatro muestras que pertenecían a individuos asintomáticos correspondían a poblaciones patógenas de acuerdo al patrón de restricción de la cepa control HM-I:IMSS, las nueve restantes correspondientes a pacientes sintomáticos presentaron un patrón de restricción semejante a la cepa patógena. Conclusiones. Estos resultados muestran que la amplificación del gene enhhic por la reacción en cadena de la polimerasa no es suficiente para establecer un diagnóstico diferencial. Para considerar que una cepa es patógena es necesario realizar digestión enzimática.

[Effect of blood plasma inhibitors on activity of serine and cysteine proteinases]. / Vliianie ingibitorov plazmy krovi na aktivnost' serinovykh i tsisteinovykh proteinaz.

Data on study of action plasma inhibitors on activity of pancreatic proteolytic enzymes (trypsin, chymotrypsin) and plant proteinases (papain, bromelain), included in composition of enzyme mixes, used for orally application are submitted. It is established, that serine proteases are more sensitive to inactivation of plasma inhibitors, than cysteine enzymes. Main inhibitor of the papain and bromelain is alpha-2-macroglobulin in complex with which they preserve significant part of initial activity. A high-sensitivity method of determination of activity enzyme combinations, enabling to detect nanograms of them in presence of plasma inhibitors is offered. It can be used for study pharmacokinetic and optimization of enzyme mixes application in clinical practice.

Permeabilization of mammalian cells to proteins: poliovirus 2A(pro) as a probe to analyze entry of proteins into cells.

Two hybrid protein molecules containing the poliovirus protease 2A (MBP-2A(pro)) (maltose-binding protein-2A(pro) and MBP-Pseudomonas exotoxin A-2A(pro)) have been constructed and purified. Both hybrid proteins efficiently cleave the translation initiation factor eIF-4G when they are co-internalized into cells with adenovirus particles. Almost no intact eIF-4G can be detected in cells incubated with these proteins following this method. Reovirus infectious subviral particles also promote the delivery of MBP-2A(pro) into cells, although less efficiently than adenovirus particles. None of the other methods employed to permeabilize cells to MBP-2A(pro) achieves the degree of eIF-4G cleavage observed with adenovirus particles. By comparison about 30% of cells electroporated with MBP-2A(pro) still contain intact eIF-4G. More drastic electroporation conditions lead to a significant decrease of cell survival. Osmotic lysis of pinocytic vesicles resulted in 30% of the eIF-4G being cleaved in cells treated in suspension. Delivery of MBP-2A(pro) by pH-sensitive liposomes leads to poor hydrolysis of eIF-4G. Taken together our results indicate that permeabilization of cells with adenovirus particles is the most efficient method for introducing MBP-2A(pro) into cells.

Skin proteasomes (high-molecular-weight protease): purification, enzymologic properties, gross structure, and tissue distribution.

Proteasomes (high-molecular-weight protease) were purified from rat skin, and their enzymologic properties, gross structure, and tissue distribution were investigated. Skin proteasomes were purified by successive (NH4)2SO4 fractionation and by phenyl Sepharose CL-4B and HPLC gel filtration chromatography. The molecular weights of the proteasomes were estimated from gel filtration to be 750 kD. On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the purified enzymes dissociated into several bands, the majority falling into the range of 36-20 kD. Two-dimensional electrophoretic analysis demonstrated approximately 10-15 separate protein spots with pl values varying between 3 and 10. As analyzed by electron microscopy, the gross structure of the enzymes showed an almost symmetrical ring-shaped particle with a small hole in the center. Succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide, a fluorogenic substrate for serine proteinases, demonstrated the highest activity in terms of substrate specificity. Sodium dodecylsulfate, Ca++, and some free fatty acids activated enzyme activity. Activity was inhibited by diisopropylfluorophosphate, leupeptin, N-ethylmaleimide, iodoacetamide, and chymostatin. These results show that both serine and cysteine residues are related to the enzyme activity of proteasomes. Total and specific enzyme activities in the epidermis were, respectively, 10 and 20 times higher than in the dermis. Immunohistochemical studies utilizing the avidin-biotin complex method with monoclonal antibody revealed that the enzyme is distributed throughout the epidermis. These findings indicate the epidermal localization of proteasomes.

The potential of carboxypeptidase G2: antibody conjugates as anti-tumour agents. II. In vivo localising and clearance properties in a choriocarcinoma model.

The in vivo localising and clearance properties of conjugates of the folate-degrading enzyme carboxypeptidase G2 (CPG2) with anti-human chorionic gonadotrophin (W14A) were measured in nude mice bearing CC3 choriocarcinoma xenografts. Conjugates of W14A-F (ab')2 fragment coupled to CPG2 localised in tumour as effectively as native antibody alone but showed lower uptake in other major tissues. The clearance rates of conjugates prepared with intact antibody or F (ab')2 fragment were shown to be up to five-fold faster than for native antibody and two-fold compared to F (ab')2 fragment. Molecular weight analysis of residual conjugate in the blood showed that no degradation of conjugate to its component molecules occurred during circulation. It was concluded that F (ab')2: CPG2 conjugates offered the greatest potential for targeting applications.