Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization

Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization.BackgroundAnaphylatoxin C5a mediates inflammatory responses through interaction with a specific C5a receptor (C5aR), the expression of which is thought to be restricted to peripheral blood leukocytes. Although the presence of C5aR on cultured mesangial cells and tubular epithelial cells has recently been documented, the tissue distribution of C5aR in diseased kidney has not yet been determined.MethodsImmunohistochemistry and nonradioactive in situ hybridization for C5aR were performed in 34 tissue samples of kidneys from patients with various renal diseases, including 4 with minimal change nephrotic syndrome (MCNS), 5 with membranous nephropathy (MN), and 25 with mesangial proliferative glomerulonephritis (mesGN; 15 patients with IgA nephropathy, 5 with non-IgA mesGN, and 5 with lupus nephritis). Normal portions of surgically resected kidney served as the control.ResultsIn normal kidneys, C5aR protein was detected in tubular epithelial cells, while C5aR mRNA was detected in a few glomerular cells, tubular epithelial cells, and vascular endothelial and smooth muscle cells. In MCNS, the distribution of C5aR protein and mRNA was similar to that in normal kidneys. In MN and mesGN, C5aR protein and mRNA were detected in mesangial cells, glomerular epithelial and endothelial cells, Bowman's capsule cells, tubular cells, infiltrating cells, and vascular endothelial and smooth muscle cells. The glomerular expression of C5aR mRNA and protein correlated positively with the degree of mesangial hypercellularity and mesangial matrix expansion in mesGN. In the tubulointerstitium, interstitial expression of C5aR mRNA correlated positively with the degree of tubular atrophy and interstitial broadening in mesGN. Furthermore, the interstitial expression of C5aR mRNA correlated positively with the level of serum creatinine.ConclusionsOur results indicate that renal cells produce C5aR and that activation of C5a/C5aR pathway on renal cells may be involved in tissue injury in mesGN

Co-existence of acute myeloid leukemia with multilineage dysplasia and Epstein-Barr virus-associated T-cell lymphoproliferative disorder in a patient with rheumatoid arthritis: a case report

Rheumatoid arthritis (RA) is an autoimmune disease mediated by inflammatory processes mainly at the joints. Recently, awareness of Epstein-Barr virus (EBV)-associated T-cell lymphoproliferative disorder (T-LPD) has been heightened for its association with methotraxate usage in RA patients. In the contrary, acute myeloid leukemia with multilineage dysplasia (AML-MLD) has never been documented to be present concomitantly with the above two conditions. In this report we present a case of an autopsy-proven co-existence of AML-MLD and EBV-associated T-LPD in a patient with RA

Synthesis of Peptide-Immobilized Magnetic Beads, and Peptide Reactivity Assay for Assessing Skin Sensitization Utilizing Chromophore

DPRA (direct peptide reactivity assay) and ADRA (amino acid derivative reactivity assay), which are based on the biological events of skin sensitization, were developed as alternatives to the controversial animal experiments. These assays are described in the OECD (Organization for Economic Co-operation and Development) guideline, Test No. 442C. Although these assays have been endorsed by the industries and internationally accepted as promising and effective tests for in vitro skin sensitization, they suffer from several drawbacks, such as incompatibility with hydrophobic chemicals and complicated sample processing. Here, we demonstrated a chromophore-based solid phase peptide reaction assay in vitro using peptides immobilized on magnetic beads (C-SPRA-MB). We successfully synthesized lysine (Lys) and cysteine (Cys) immobilized on magnetic microbeads. However, Cys immobilized magnetic microbeads showed gradual decomposition of the magnetic beads due to SH oxidation. Using Lys immobilized magnetic microbeads, we demonstrated the capacity of C-SPRA-MB to predict skin sensitization by measuring free amino groups of the Lys after reaction with test chemicals. First, the free amines on the microbeads were reacted with bromophenol blue (BB). Then, by treatment with a saturated solution of Lys, the bound BBs were released and quantified. C-SPRA-MB provides high-throughput and accurate assays for assessments of chemicals, including with low-potency as skin sensitizers and poor water solubility. C-SPRA-MB may be useful for effective prediction of their skin sensitization potential in the process of compound screening, especially in the case of misclassified by DPRA and ADRA. Thus, C-SPRA-MB can be applied to assessing the sensitization potential of medical, pharmaceutical, cosmetics, and industrial compounds

Mass Spectrometry-Based Solid Phase Peptide Reaction Assay for Detecting Allergenicity Using an Immobilized Peptide-Conjugating Photo-Cleavable Linker

The biological process of skin sensitization depends on the ability of a sensitizer to modify endogenous proteins. A direct peptide reactivity assay (DPRA), based on the biological process of skin sensitization, was developed as an alternative to controversial animal experiments. Although DPRA has been endorsed by industries and is internationally accepted as promising, it has several drawbacks, such as incompatibility with hydrophobic chemicals, inability to perform detailed reaction analysis, and ability to evaluate only single components. Here, we demonstrated that sensitizers and peptide adducts can be easily identified using a mass spectrometry-based solid-phase peptide reaction assay (M-SPRA). We synthesized peptides with a photo-cleavable linker immobilized on resins. We showed the potential of M-SPRA in predicting skin sensitization by measuring the peptide adducts that were selectively eluted from the resin after cleaving the linker post-reaction. M-SPRA provides more detailed information regarding chemical reactivity and accurate assessment of test samples, including mixtures. M-SPRA may be helpful for understanding the binding mechanism of sensitizers (toxicology), which may assist in further refining reactivity assays and aiding in the interpretation of reactivity data

Hill–Sachs lesion measurement with tridimensional models in anterior shoulder instability

ABSTRACT Objective To evaluate the reproducibility and repeatability of Hill–Sachs lesion measurement from computed tomography images, with computer software and tridimensional prototype. Methods Three-dimensional models were made from computed tomography images from 14 patients with anterior shoulder instability, using InVesalius 3.0® software. Hill–Sachs lesions were measured with Rhinocerus 5.0® software with pre-determined position. Mid-lateral distance, perpendicular to humeral shaft, cranial-caudal distance, parallel to humeral shaft, and the longitudinal distance of the lesion were measured. Using the Printer-ZP 310 three-dimensional printer, plaster models were made. To measure the Hill–Sachs lesion, a calibrated universal digital caliper was used in the same way as the software. Results There was intra-observer and inter-observer variability for measurement of the same model. Observers did not perform the measurements in a similar way, showing difficulty to use the method (p < 0.05). Using the software to measure the mid-lateral distance, as well as in the measurement with the caliper, the model type influenced the measurements for each of the observers, rendering the method invalid (p < 0.05). Conclusion There was no reproducibility and repeatability for Hill–Sachs lesion measurement between plaster models and software models

Semi-Quantitative Non-Radioactive In Situ Hybridization and Its Clinical Application.

In situ hybridization (ISH) is widely used to determine the expression of specific mRNA in tissues. However, the results of ISH are influenced by the amount of retained RNA in the tissue. Estimation of retained RNA in the tissue is very important and the results of ISH should be interpreted taking into consideration the amount of retained RNA in samples. In the present study, we determined the amount of retained RNA by performing ISH for 28S rRNA. We also analyzed the results of ISH semi−quantitatively by computer image analysis. The method was applied by calculating the ratio of specific mRNA to 28S rRNA in order to standardize the results of ISH among different tissue samples. Our method is useful for comparing the expression of specific mRNA in different tissue samples with variable levels of retained RNA. Since the conditions of fixation and storage of tissues differ among samples, especially in clinical samples, our semi−quantitative method allows a more precise evaluation of gene expression in different samples