Optimized synthesis, polymer conjugation, and proof-of-concept studies of the gd-IgA1 epitope for antibody-scavenging therapies in IgA nephropathy.

IgA nephropathy (IgAN) is the most common glomerular autoimmune disease and has severe long-term consequences for patients, with 40% of the patients eventually progressing to end-stage renal disease. Despite the severity, no causal treatment is currently available. While the pathogenesis of IgAN is complex, disease severity is linked to autoantibodies against the gd-IgA1 epitope, a stretch in the hinge region of IgA1 that lacks O-glycans and is found in the characteristic immune complexes deposited in the kidneys of IgAN patients. One elegant, causal approach would be to remove the anti-gd-IgA1 autoantibodies and consequently reduce the immune complex burden on the kidneys. The administration of synthetic polymers that present autoantigens in a multivalent manner have been established as promising therapeutic strategies in other autoimmune diseases and may be applied to IgAN. We here present an improved protocol for the synthesis of the gd-IgA1 epitope, its successful coupling to a poly-L-lysine polymer and proof-of-concept experiments that the polymer-bound synthetic glycopeptide is able to capture the IgAN autoantibodies, making this approach a promising way forward for developing a targeted treatment option for IgAN patients.

Higher serum galactose-deficient immunoglobulin A1 concentration is associated with stronger mesangial cellular inflammatory response and more severe histologic findings in immunoglobulin A nephropathy.

BACKGROUND: Galactose-deficient immunoglobulin A1 (Gd-IgA1) is known to play a key role in the pathogenesis of IgA nephropathy (IgAN). We aimed to evaluate whether serum Gd-IgA1 is associated with in vitro activation of mesangial cells in individual patients and how this affects the clinical and histologic parameters. METHODS: Serum samples and clinical and histologic data were collected in the University Hospital Basel and Hammersmith Hospital, London. Serum levels of IgA1 and Gd-IgA1 were measured by enzyme-linked immunosorbent assay (ELISA) and lectin-binding assay using lectin Helix aspersa (HA). Primary human mesangial cells were stimulated with IgA1 isolated from serum from individual patients and the concentrations of monocyte chemoattractant protein-1 and interleukin-6 were measured in cell culture supernatant by ELISA. RESULTS: Thirty-three patients were enrolled. A significant correlation was observed between serum Gd-IgA1 levels and the concentration of MCP-1 in the culture supernatant in individual patients (Spearman r = 0.5969, P = 0.0002). There was no significant correlation between serum Gd-IgA1 levels and proteinuria or estimated glomerular filtration rate at diagnosis. However, the serum Gd-IgA1 level was significantly higher in patients with segmental glomerulosclerosis (S0 versus S1, P = 0.0245) and tubular atrophy/interstitial fibrosis (T0 versus T1 and T2, P = 0.0336; T0 versus T2, P = 0.0225). CONCLUSIONS: Higher serum Gd-IgA1 concentration is associated with stronger mesangial cell inflammatory response with production of a greater amount of MCP-1 in vitro. This in turn is associated with severe histologic changes. The disease progression with worse renal outcome in patients with higher serum Gd-IgA1 may be therefore mediated by more pronounced mesangial cell inflammatory response leading to more severe histologic changes.

Sodium Polystyrene Sulfonate and Cytomegalovirus-Associated Hemorrhagic Duodenitis: More than Meets the Eye.

BACKGROUND Hemorrhagic duodenitis is an exceptionally rare adverse event of sodium polystyrene sulfonate (SPS) treatment and is a common manifestation of cytomegalovirus (CMV) reactivation. SPS is known to cause marked inflammation in the lower gastrointestinal tract, including colonic necrosis, whereas involvement of the small bowel is uncommon. Although its effectiveness and safety has been disputed since its introduction, SPS remains widely used due to lack of alternatives. CMV infection and reactivation are well-known complications after solid-organ transplantation, particularly in seronegative recipients receiving organs from seropositive donors, and is associated with significant morbidity and mortality. The lower gastrointestinal tract is more commonly involved, but infections of all parts of the intestine are observed. CASE REPORT Here, we report the case of a 56-year-old man who presented with severe upper-gastrointestinal bleeding. Hemorrhagic duodenitis was initially attributed to the use of SPS, as abundant SPS crystals were detected in the duodenal mucosa but we found only 2 CMV-infected endothelial cells. Two weeks later, gastrointestinal bleeding recurred. However, this time, abundant CMV-infected cells were demonstrated in the duodenal biopsies. CONCLUSIONS Our case report highlights an uncommon adverse event after SPS use with a simultaneous CMV reactivation. The main difficulty was to differentiate between CMV reactivation and CMV as an "innocent bystander". This demonstrates the challenge of decision-making in patients with complex underlying diseases.

Bile Cast Nephropathy: The Unknown Dangers of Online Shopping.

Renal dysfunction in the setting of cholestatic liver disease is multifactorial but most often due to decreased kidney perfusion from intravascular volume depletion, acute tubular injury/necrosis, and hepatorenal syndrome. Drug-induced hepatotoxicity may be associated with a cholestatic injury pattern. We report a case of a 56-year-old man with a diagnosis of bile cast nephropathy, as a complication of drug-induced severe hyperbilirubinemia due to the abuse of intramuscular anabolic steroids bought on the internet to increase muscular mass for bodybuilding training. Kidney biopsy showed the histological pattern of diffuse potentially reversible tubular damage with intratubular bile casts obstructing the renal tubules. The patient developed acute kidney injury and needed dialysis treatment for 4 weeks until renal function recovered. The severity of the kidney injury and the requirement of hemodialysis observed in our patient are unique and have not been previously described. As full recovery of renal function is suspected with decreasing bilirubin levels, early recognition and treatment of the underlying disease is essential.

Cytokine patterns in nasal secretion of non-atopic patients distinguish between chronic rhinosinusitis with or without nasal polys.

BACKGROUND: Being one of the most common nasal diseases, chronic rhinosinusitis (CRS) is subdivided into CRS with nasal polyps (NP) and CRS without nasal polyps (CRSsNP). CRSsNP presents itself with a TH1 milieu and neutrophil infiltration, while NP is characterised by a mixed TH1/TH2 profile and an influx of predominantly eosinophils, plasma cells and mast cells. For the purpose of discovering disease-specific cytokine profiles, the present study compares levels of mediators and cytokines in nasal secretions between CRSsNP, NP, and healthy controls. METHODS: The study included 45 participants suffering from NP, 48 suffering from CRSsNP and 48 healthy controls. Allergic rhinitis constituted an exclusion criterion. Nasal secretions, sampled using the cotton wool method, were analysed for IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, IL-8, GM-CSF, G-CSF, IFN-γ, MCP-1, MIP-1α, MIP-1ß, eotaxin, and RANTES, and for ECP and tryptase, using Bio-Plex Cytokine assay or ELISA, respectively. RESULTS: Elevated levels of IL-5, IL-17, G-CSF, MCP-1, MIP-1α, MIP-1ß, ECP, and tryptase, as well as decreased levels of IL-10, IL-12, IL-13, and IFN-γ were detected in NP. CRSsNP presented increased levels of RANTES and MIP-1ß while IL-13 was decreased. No differences between the three groups were found for IL-4, IL-8, GM-CSF, and eotaxin. CONCLUSIONS: The present work suggests a disequilibrium of TH1 and TH2, together with a down-regulation of regulatory T lymphocytes and up-regulated TH17 in NP. Moreover, elevated levels of diverse mediators represent the activation of various inflammatory cells in this disease entity. The inflammation in CRSsNP, however, is only weakly depicted in nasal secretions. Therefore, cytokines in nasal secretions may provide helpful information for differential diagnosis.

Cytokine profiles in nasal fluid of patients with seasonal or persistent allergic rhinitis.

BACKGROUND: New therapeutic approaches with biologic agents such as anti-cytokine antibodies are currently on trial for the treatment of asthma, rhinosinusitis or allergic diseases necessitating patient selection by biomarkers. Allergic rhinitis (AR), affecting about 20 % of the Canadian population, is an inflammatory disease characterised by a disequilibrium of T-lymphocytes and tissue eosinophilia. Aim of the present study was to describe distinct cytokine patterns in nasal secretion between seasonal and perennial AR (SAR/PAR), and healthy controls by comparing cytokines regulating T-cells or stimulating inflammatory cells, and chemokines. METHODS: Nasal secretions of 44 participants suffering from SAR, 45 participants with PAR and 48 healthy controls were gained using the cotton wool method, and analysed for IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, GM-CSF, G-CSF, IFN-γ, MCP-1, MIP-1α, MIP-1ß, eotaxin, and RANTES by Bio-Plex Cytokine Assay as well as for ECP and tryptase by UniCAP-FEIA. RESULTS: Participants with SAR or PAR presented elevated levels of tryptase, ECP, MCP-1, and MIP-1ß, while values of GM-CSF, G-CSF, IL-1ß, and IL-6 did not differ from the controls. Increased levels of IL-5, eotaxin, MIP-1α, and IL-17 and decreased levels of IFN-γ, IL-12 and IL-10 were found in SAR only. RANTES was elevated in SAR in comparison to PAR. Interestingly, we found reduced levels of IL-4 in PAR and of IL-13 in SAR. CONCLUSIONS: Elevated levels of proinflammatory cytokines were seen in both disease entities. They were, however, more pronounced in SAR, indicating a higher degree of inflammation. This study suggests a downregulation of TH1 and Treg-lymphocytes and an upregulation of TH17 in SAR. Moreover, the results display a prominent role of eosinophils and mast cells in AR. The observed distinct cytokine profiles in nasal secretion may prove useful as a diagnostic tool helping to match patients to antibody therapies.

Quantification of vancomycin in human serum by LC-MS/MS.

BACKGROUND: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. METHODS: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. RESULTS: In a bi-center validation total CVs of <4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was ±4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement. CONCLUSIONS: Employing a closely related homologue molecule for internal standardization and the use of MS/MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.

Deproteination of serum samples for LC-MS/MS analyses by applying magnetic micro-particles.

OBJECTIVES: Investigation of the practicability and performance of a magnetic micro-particle based method for protein depletion of serum samples, preceding the quantitative analysis of small molecules by LC-MS/MS. DESIGN AND METHODS: A commercially available kit including a protein denaturation reagent and functionalized magnetic particles together with a magnetic separator device was tested by addressing the quantification of amiodarone in serum as an exemplary analyte by LC-MS/MS with on-line SPE. A standard method validation protocol was applied. RESULTS: The sample preparation protocol was found to be convenient, straightforward and robust. Validation data characterized the entire analytical method - combining particle-based protein depletion and two-dimensional chromatography - as compatible with the analytical needs regarding selectivity, accuracy (102-106%), linearity (r(2)≥0.99), reproducibility (CV<7%), and control of ion suppression. CONCLUSIONS: Since this novel approach of sample preparation does neither require centrifugation nor the technically demanding application of positive or negative pressure, as in conventional solid phase extraction protocols, it seems highly attractive for developing fully automatized preparation systems for LC-MS/MS analyzers.

Sample preparation for measurement of plasma mycophenolic acid concentrations using chromatographically functionalized magnetic micro-particles.

Utilizing chromatographically modified magnetic micro-particles is an innovative principle of sample preparation for quantitative analysis of small molecules in complex biomedical samples by liquid chromatography tandem mass spectrometry. Since no vacuum or pressure has to be applied-in contrast to cartridge based solid phase extraction protocols-the principle's main characteristics are potentially straightforward automation and a high extraction performance (in terms of µg of extraction material per µL of sample). Following first descriptions of the approach, this article reports, the validation of a magnetic particle-based, analytical method for the quantification of the immunosuppressant mycophenolic acid in plasma. This sample preparation technology has shown a good performance for this clinically relevant analyte. As a result, we conclude that further work towards the implementation of this technology in a multi- analyte approach on robotic systems, aiming towards a fully automated process, is justified.