Proučavanje stvaranja agregata rekombinantnog humanog granulocitnog faktora stimulacije kolonija (rHuG-CSF), lenograstima, pomoću kromatografije isključenjem prema veličini i SDS-PAGE

The stability of proteins is a subject of intense current interest. Aggregation, as a dominant degradation pathway for therapeutic proteins, may cause multiple adverse effects, including loss of efficacy and immunogenicity. In the present study, the formation of aggregates in lenograstim under physiological conditions was monitored. For this purpose, a simple and selective size-exclusion high-performance liquid chromatography method for detection and separation of aggregates from intact protein was developed. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions to determine the nature of aggregate bond formation. Using both techniques, the presence of a low aggregate content attached via disulfide bonds was detected.Stabilnost proteina vrlo je aktualna problematika. Agregacija, kao dominantni put razgradnje terapijskih proteina, može uzrokovati različite nuspojave, koje uključuju i gubitak učinkovitosti imunološkog sustava. U ovom radu praćeno je stvaranje agregata u lenograstimu pod fiziološkim uvjetima. Razvijena je jednostavna i selektivna tekućinska kromatografija visoke djelotvornost s isključenjem prema veličini za detekciju i razdvajanje agregata od intaktnog proteina. Pomoću natrij-dodecil sulfat poliakrilamidne gel elektroforeze u reducirajućim i nereducirajućim uvjetima utvrđen je način stvaranja agregata. Pomoću obje tehnike moguće je detektirati prisutnost malih količina agregata koji su spojeni disulfidnim vezama

(Benzoylamino)methyl 4-[(Benzoylamino)methoxy]benzoate

In this note, two procedures for the synthesis of (benzoylamino)methyl 4-[(benzoylamino)­methoxy]benzoate (3) are presented. The first procedure is carried out in dioxane/water using benzoylaminomethyl-4-hydroxybenzoate, while the second one employs a suspension of 4-hydroxy­benzoic acid in dioxane. In both procedures, benzamidomethyl triethylammonium chloride is used for the benzamidomethylation reaction

Methyl 4-[(Benzoylamino)methoxy]benzoate

Methylparabene (2) was simply benzamidomethylated with (benzamidomethyl)­triethylammonium chloride (1) in aqueous medium to afford methyl 4-(benzamido­methoxy)benzoate (3) in high yield. The title compound was characterized by elemental analysis, FT-IR, 1H-NMR and 13C-NMR spectroscopy

Design, synthesis and determination of physical and chemical characteristics of glycoconjugates as model for oligosaccharide vaccines against vibrio cholerae

Cholera is toxin-mediated enteroinfection, with epidemic character and there are approximately 120000 death cases per year worldwide. Protection against cholera has not been accomplished due to deficiencies in the licensed vaccines. Serum vibriocidal activity mediated by LPS antibodies is the only immune segment correlated with the resistance of cholera. On the basis of literature data (Robbins JB, 1990; Ogawa Y, 1996) we synthesized glucoconjugates, composed of detoxified LPS from Vibrio cholerae and protein carriers. Conjugate vaccines were prepared by binding acetic acid and hydrazine-treated lipopolysaccharide (LPS) from Vibrio cholerae O1, serotype Inaba, to cholera toxin B-subunit (CT-B) and bovine serum albumin (cBSA). Adipic acid dihydrazide was used for derivatization of oligosaccharides and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as conjugating agent. SDS-PAGE, glycoprotein detection and TLC dot-blot were used for physical and chemical analysis of the prepared four types of conjugates. Safe level of endotoxins, measured by LAL assay was detected in all conjugates. The synthesized conjugates can be used for monitoring immunization schemes on experimental animals. It is to be expected that conjugated vaccines are safe and efficient and that will have high immunogenic and T-dependant characteristics with long immune protection against cholera

Chemometric evaluation of the efficacy of locally administered chlorhexidine in patients with periodontal disease

The process of assessment of drug efficacy produces multivariate data which are difficult to interpret. The interpretation and extraction of relevant data requires application of chemometric algorithms for multivariate data analysis. The aim of our study was evaluation of the efficacy of local treatment with chlorhexidine (CHX) in patients suffering from periodontal disease by chemometric algorithms for multivariate data analysis. Several algorithms were used: principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The PCA models identified the examined variables as suitable for monitoring the periodontal disease progression at the same time revealing mutual relationship among them. The developed PLS-DA model successfully distinguished patients treated with CHX from non-treated patients. The OPLS-DA model revealed differences in the mechanism of action of the two widely applied treatments in periodontal disease, local administration of CHX and local administration of doxycycline (DOX). The approach presented in this study opens the possibility of application of chemometric algorithms for multivariate data analysis for assessment of treatment efficacy

Glycoconjugates as target antigens in peripheral neuropathies

Identification and characterization of antigens present at the human peripheral nerve is a great challenge in the field of neuroimmunology. The latest investigations are focused on the understanding of the biology of glycoconjugates present at the peripheral nerve, and their immunological reactivity. Increased titers of antibodies that recognize carbohydrate determinants of glycoconjugates (glycolipids and glycoproteins) are associated with distinct neuropathic syndromes. There is considerable cross-reactivity among anti-ganglioside antibodies, resulting from shared oligosaccharide epitopes, possibly explaining the overlap in syndromes observed in many affected patients. Sera from patients with neuropathies (GBS, chronic inflammatory demielynating polyneuropathy - CIDP, multifocal motor neuropathy - MMN), cross-react with glycoproteins isolated from human peripheral nerve and from Campylobacter jejuni O:19. The frequency of occurrence of antibodies against these glycoproteins is different, depending of the type of neuropathy. Identification of the cross-reactive glycoproteins and possible additional auto antigens could be useful in laboratory evaluation of peripheral neuropathies and help to develop a more effective therapeutic approach

Antibodies to Glycoproteins Shared by Human Peripheral Nerve and Campylobacter jejuni in Patients with Multifocal Motor Neuropathy

We have tested serum samples from 24 patients with multifocal motor neuropathy (MMN) for reactivity to ganglioside GM1 and to Gal(β1–3)GalNAc-bearing glycoproteins isolated from human peripheral nerve and from Campylobacter jejuni (Cj) serotype O:19. IgM anti-GM1 antibodies were detected by ELISA in 11 patients (45.8%) with MMN and in only one subject (4%) from the control group. Western blots showed positive reactivity of sera from 6 patients (25%) with MMN to several Gal(β1–3)GalNAc-bearing glycoproteins from human peripheral nerve and from Cj O:19 isolates. Sera from three patients (12.5%) with MMN showed positively reactive bands with similar electrophoretic mobility in all isolates (60–62 kDa, 48–51 kDa, 42 kDa, and 38 kDa). All six patients showed positive reactivity to 48–52 kDa protein isolated from human peripheral nerve. Increased titer of IgG antibodies to 60–62 kDa protein isolated from Cj O:19 associated with Guillain-Barré syndrome was detected in three patients, and their serum showed also IgG positive reactivity to peripheral nerve antigen with the same electrophoretic mobility. One of these patients had a previous history of Cj infection which suggests the possibility that Cj may be also involved in the pathogenesis of MMN

Immunoreactivity of glycoproteins isolated from human peripheral nerve and Campylobacter jejuni (O:19)

Objective: Antibodies to ganglioside GM1 are associated with Guillain-Barré Syndrome (GBS) in patients with serologic evidence of a preceding infection with Campylobacter jejuni. Molecular mimicry between C. jejuni Lipopolysaccharide (LPS) and ganglioside GM1 has been proven to be the immunopathogenic mechanism of the disease in the axonal variant of GBS. GM1-positive sera cross-react with several Gal-GalNAc-bearing glycoproteins from the human peripheral nerve and C. jejuni (O:19). This study aimed to examine the immunoreactivity of the digested cross-reactive glycoproteins isolated from the human peripheral nerve and C. jejuni (O:19) with Peanut Agglutinin (PNA) as a marker for the Gal-GalNAc determinant, and with sera from patients with GBS. Materials and Methods: For this purpose, the cross-reactive glycoproteins from peripheral nerve and C. jejuni (O:19) were enzymatically digested with trypsin and the obtained peptides were incubated with PNA and GBS sera. Results: Western blot analysis of the separated peptides revealed several bands showing positive reactivity to PNA and to sera from patients with GBS, present in both digests from peripheral nerve and C. jejuni (O:19). Conclusions: These data indicate the possible molecular mimicry between the cross-reactive glycoproteins present in C. jejuni and human peripheral nerve and its potential role in the development of GBS following infection with C. jejuni (O:19)

Involvement of serum HSP 70 in Guillain-Barré Syndrome: An exploratory study and a review of current literature

The evolutionary conserved family of heat shock proteins (HSP) is responsible for protecting cells against different types of stress. Although the levels of HSP can be readily measured in serum, the levels of HSP 70 in patients Guillain-Barre Syndrome (GBS) have not been studied before. To this aim we investigate whether patients with GBS (n=21) had altered serum HSP 70 levels compared to healthy controls (HC, n=9) and to patients affected by other immune disorders such as multifocal motor neuropathy (MMN, n=4) and chronic inflammatory demyelinating polyneuropathy (CIDP, n=6). The highest HSP 70 value (15.78 ± 1.72 ng/mL) was found in one patient in the GBS group, although we have found that serum HSP70 levels were significantly higher in 2 out of the 21 GBS patients (9.5%). Hence, it is of interest to underline that the patient with the highest HSP70 level, had also the best recovery rate. Моrе extensive research is required in order to support the hypothesis that HSP 70 serum concentration may be a useful biomarker for the prediction of remission outcome for GBS patients