UV-C irradiation as an effective tool for sterilization of porcine chimeric VP1-PCV2bCap recombinant vaccine.

Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial difference in the production of VP1-PCV2bCap specific IgG was observed in piglets immunized with VP1-PCV2bCap sterilized by UV-C in comparison with protein sterilized by filtration in combination with the inactivation of baculovirus by binary ethylenimine. UV-C irradiation represents an effective method for vaccine sterilization, where commonly used methods of sterilization are not possible.

Development of modern immunization agent against bovine papillomavirus type 1 infection based on BPV1 L1 recombinant protein.

Bovine papillomavirus type 1 L1 protein was produced in a baculovirus expression system and purified as virus-like particles (VLPs) by affinity chromatography using lectins. The morphological integrity of VLPs was confirmed by electron microscopy. Differences between the two detected variants were deciphered by mass spectrometry of peptides (MALDI-TOF). Mice were immunized with purified VLPs in doses of 10, 25, or 50 µg in combination with 1% saponin and 15% alhydrogel per dose as adjuvants. Analysis of the humoral immune response revealed increased levels of specific antibodies detected 3 weeks after the first immunization in all groups of animals. This was further significantly increased by the booster applied 3 weeks after the first dose, with the best immune response in a group of mice immunized by the largest dose of antigen. BPV1 L1 VLPs purified by affinity chromatography using lectins could be used for prophylactic immunization in veterinary medicine.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

Stromal Vascular Fraction Cell Therapy for a Stroke Patient-Cure without Side Effects.

A 48-year-old male, who suffered from a stroke resulting in cerebellum damage and occlusion of the left vertebral artery, underwent stromal vascular fraction therapy. The clinical status of the patient was monitored by a modified Stroke Specific Quality of Life Scale before therapy and at 3, 9, 12, 18, 24, and 32 months after therapy. Three months after therapy, the patient felt a reduction in pain, vertigo, and fatigue. After 9 months, he was able to walk safely on his own. After 24 months, he was able to ride a bicycle. After 32 months, he felt completely healthy without any limitations or handicaps. Therefore, intravenous application of stromal vascular fraction cells represents a promising strategy for the treatment of patients after a stroke.

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice.

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D-isomeric variant LLIII-D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII-D at concentrations of 11.5 µM and 21 µM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida-inhibitory effect of daily intravaginal administration of 0.7 or 1.4 µg of LLIII was assessed in mice with experimentally-induced vaginal candidiasis. LLIII and LLIII-D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature-induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.

[IgA nephropathy - research-generated questions]. / IgA nefropatie - otázky, které nastolil výzkum.

IgA nephropathy (IgAN) is the most common type of glomerulonephritis. Its etiology involves an increased production of polymeric immunoglobulin A1 with an abnormal composition of some carbohydrate chains. The reaction of these abnormal forms of IgA1 with specific autoantibodies while circulating immune complexes arise and settle in the renal mesangium with subsequent inflammatory activation of mesangial cells which in up to 50% of cases results in end-stage kidney failure. Pathogenesis involves an interplay of genetic predisposition and environmental effects, mainly of microbial nature. Current therapy is not sufficiently effective and lacks the focus on the cause of the disease, therefore more efficient and specific ways of therapy are being sought to target the individual stages of the pathogenetic process of IgAN development. With the accumulation of knowledge, new questions arise, concerning detailed mechanisms of the pathological processes, as discussed in the text.Key words: autoimmunity - glycosylation of IgA hinge region - IgA nephropathy - immunoglobulin IgA - IgA1 hinge region.

N-acetylgalactosaminide α2,6-sialyltransferase II is a candidate enzyme for sialylation of galactose-deficient IgA1, the key autoantigen in IgA nephropathy.

BACKGROUND: Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a ß1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing. METHODS: We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry. RESULTS: ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide. CONCLUSIONS: Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.