The diagnostic significance of C4d deposits, as an immunohistochemical proof of complement activation, in kidney glomerular pathologies and kidney transplantation.

C4d, a split product of C4 activation in classical and lectin pathways of the complement system activation, has been regarded as a footprint of tissue damage in antibody-mediated rejection in transplantology. The introduction of C4d staining into daily clinical practice aroused an ever-increasing interest in the role of antibody-mediated mechanisms in kidney allograft rejection. However, this marker of complement activation is also important in other various kidney glomerular pathologies such as immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, lupus nephritis, and others. In routine histopathological practice, C4d staining can be done by two histological methods, specifically by immunofluorescence on frozen tissue using monoclonal antibody to C4d (with the downside of unsteady availability of frozen tissue) or by immunohistochemistry using C4d antibodies on formalin-fixed and paraffin-embedded renal tissue. The aim of this narrative review is to summarize recent knowledge about the complement fragment C4d and its significance in different kidney pathologies, focusing on its immunohistochemical detection in renal tissue biopsies. We have supplemented this review with our experience with our proprietary methodology of preparation and practical use of antibodies such as anti-C4d, on a small national level. Immunohistochemical staining for C4d has revolutionized the field of renal histopathology. Despite being a simple diagnostic test, its utility can be of utmost importance, especially in a resource-poor setting where immunofluorescence and frozen tissue may not be available (Fig. 2, Ref. 53). Keywords: C4d deposition, immunohistochemistry, kidney glomerular diseases, kidney transplant, renal tubular damage.

Identification of new phosphorylation sites of CD23 in B-cells of patients with chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common lymphoproliferative disorder in adults. Patients with B-CLL strongly express the CD23 - C type of lectin (low affinity IgE receptor, Fc epsilon RII), which is linked to B cell activation and proliferation. Phosphorylation in lymphocytes is tightly associated with regulation of protein activities, functional regulation and cell signaling, and may thus affect initiation and/or progression of the disease. Here we report changes in the phosphorylation of CD23 on threonine (pThr314) and two serine residues (pSer254, pSer265) in B lymphocytes of B-CLL patients, using a flow cytometry approach. The majority of tested patients with active forms of B-CLL presented a notable overexpression of CD23 along with pThr314, pSer254, and pSer265 CD23 phosphorylation positivity. Moreover, we have experimentally stimulated the CD23 phosphorylations in a subset of peripheral blood lymphocytes of healthy controls by phorbol-12-myristate-13-acetate treatment. This affects the activation of competent phosphorylation mediating kinases, resulting in the enhanced phosphorylation pattern. Together, these data confirm that CD23 protein is phosphorylated in B cells of B-CLL patients, report the identification of new CD23 phosphorylation sites, and suggest a possible role(s) of such phosphorylations in the activation of CD23 during the process of lymphocytic activation in B-CLL.

Deciphering the interface between a CD40 receptor and borrelial ligand OspA.

Neuroborreliosis is serious sequelae of Lyme borreliosis. Neuroinvasion is largely relied on successful translocation of Borrelia across the blood-brain barrier. Adherence of Borrelia to brain microvascular endothelial cell (BMEC) seems to be critical for translocation. Here we unfold the interface between OspA and CD40 molecules, major ligand and receptor, that are involved in adhesion of Borrelia to BMECs. We found that a region between Asn127 and Asp205 of OspA forms the CD40-receptor binding site. This region encompasses human umbilical vein endothelial cell (HUVEC) binding domain and contains a potential ligand-binding pocket lined by three amino acid residues: Arg139, Glu160 and Lys189. Disruption of this pocket (by truncation of the HUVEC binding domain) caused complete abrogation of its ability to bind CD40. To identify the amino acid residues within the HUVEC binding domain involved in the CD40 binding, site-directed mutagenesis and binding assays were performed. Results showed that Asp149, Phe165, Ala172, Val186 and Leu192 might form interface with CD40 molecule. Other side of the interface was also identified with the help of a ligand-binding assay with OspA and truncated CD40 fragments. Results exposed that cysteine rich domain 2 (CRD2) of CD40 might be the site for OspA binding. Precise knowledge of the molecular basis of the ligand-receptor interactions is essential in order to understand mechanisms of pathogenesis and could help in the development of novel therapeutics and vaccines.

Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design.

Several protein expression systems for construction of protein arrays have been established in recent years. However, current protocols for protein synthesis are still time consuming, laborious and expensive. This study has established an alternative workflow that covers rapid construction of expression cassettes, in-tube and on-membrane synthesis of recombinant proteins, and straightforward screening of synthesized proteins. Eighteen membrane associated eukaryotic proteins and two secretory complement regulators (C1 inhibitor and vitronectin) were included in the study. To generate hybrid genes, double-overlap extension PCR was employed to fuse the 5' fragment (consisting of a T7 promoter and a species independent translation sequence), ORFs of the target proteins, and the 3' fragment (encompassing GFP fusion, Myc-tag and stop codon). OE-PCR generated fragments were directly mixed with the Leishmania torentolae lysate (translation mix) for protein synthesis. In order to establish a cheap and user-friendly alternative to existing cell-free protein array techniques, PCR products were spotted on the hydrophobic substrate (PVDF membrane), air-dried and covered with only 2 µL of translation mix. All synthesized proteins were spontaneously immobilized on the membrane due to the hydrophobic interaction between C-terminally fused GFP and PVDF. Synthesis and immobilization of proteins were confirmed simply by assessing the GFP chromophore under a laser scanner or a fluorescent microscope.

Prevalence of Francisella tularensis and Francisella-like endosymbionts in the tick population of Hungary and the genetic variability of Francisella-like agents.

Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.

Variation in the coding region of the prion protein gene in Slovak cattle.

This study was conducted to investigate the presence of single nucleotide polymorphisms (SNPs) in the coding region of the bovine prion protein (PrP) gene among healthy and bovine spongiform encephalopathy (BSE-) affected cattle in Slovakia. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) followed by DNA sequencing were used to identify SNPs and variations in octapeptide repeats. Altogether three single nucleotide polymorphisms (g234a, c339t and c576t) and variations in the number of octapeptide repeat units (5 or 6) were found in the analysed part of the prion protein gene. All single nucleotide polymorphisms were silent, causing no amino acid changes. Significant differences (P < 0.05) in the genotype distribution of g234a polymorphism were observed when the homozygous genotype with a mutated allele (caa/caa) was compared to the heterozygous genotype -/cag among healthy and BSE-affected cattle. The homozygous genotype caa/caa was characteristic of the group of BSE-affected cattle. Additionally, the homozygous genotype caa/caa was significant for the group of Simmental crossbreeds among healthy cattle. The allele and genotype distribution of the other polymorphisms was not significantly different among groups of healthy and BSE-affected cattle. The possible influence of a silent mutation on expression of the gene is not clearly determined and needs further investigations.

Variable regions in the sushi domains 6-7 and 19-20 of factor H in animals and human lead to change in the affinity to factor H binding protein of Borrelia.

Borrelia binds host's complement regulatory factor H (fH) to evade complement attack. However, binding affinities between fH-binding-proteins (FHBPs) of Borrelia and fH from various hosts are disparate. Experiments performed to unfold the underlying molecular basis of this disparity revealed that recombinant BbCRASP-1 (major FHBP of Borrelia burgdorferi) neither interacted with sushi 6-7, nor with sushi 19-20 domains of fH in cattle and pig, however, showed binding affinity to both sushi domains of human fH, sushi 6-7 of mouse and sushi 19-20 of sheep. Further, peptide-spot assay revealed three major binding sites (sushi 6:(335-346), sushi 7:(399-410) and sushi 20:(1205-1227)) in human fH that can form BbCRASP-1:fH interface, while (337)HENMR(341) residues in sushi 6 are crucial for rigid BbCRASP-1:fH complex formation. Amino acid stretches DTIEFTCRYGYRPRTALHTFRTT in ovine sushi 19-20 and SAYWEKVYVQGQ in mouse sushi 7 were important sites for fH:BbCRASP-1 interaction. Comparative analysis of the amino acid sequences of sushi 6 of cattle, pig and human revealed that bovine and porcine fH lack methionine and arginine in HENMR pocket, that may impede formation of fH:BbCRASP-1 interface. Increasing numbers of FHBPs from animal and human pathogens are being discovered, thus results presented here can be important benchmark for study of other FHBPs:FH interactions.

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application.

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.