Split–Combine Click-SELEX Reveals Ligands Recognizing the Transplant Rejection Biomarker CXCL9

Renal rejection is a major incidence in patients after kidney transplantation and associated with allograft scarring and function loss, especially in antibody-mediated rejection. Regular clinical monitoring of kidney-transplanted patients is thus necessary, but measuring donor-specific antibodies is not always predictive, and graft biopsies are time-consuming and costly and may come up with a histological result unsuspicious for rejection. Therefore, a noninvasive diagnostic approach to estimate an increased probability of kidney graft rejection by measuring specific biomarkers is highly desired. The chemokine CXCL9 is described as an early indicator of rejection. In this work, we identified clickmers and an aptamer by split–combine click-SELEX (systematic evolution of ligands by exponential enrichment) that bind CXLC9 with high affinity. The aptamers recognize native CXCL9 and maintain binding properties under urine conditions. These features render the molecules as potential binding and detector probes for developing point-of-care devices, e.g., lateral flow assays, enabling the noninvasive monitoring of CXCL9 in renal allograft patients

The levels of IL-18, VEGF, MIP-2 and TNFα is increased in the serum but not in the kidney tissue of CD73^−/−mice.

<p>The levels of IL-18, VEGF, MIP-2 and TNFα were analyzed in serum and kidney tissue of CD73^−/− and WT mice using BioPlex analysis. 7 animals from each mice group were analyzed. Data are presented as mean l ± SEM. Level of significance as indicated according to according to Mann-Whitney-U-test (p<0.005).</p

Total proteinuria in CD73^−/−mice is a low molecule proteinuria.

<p>(A) Profiling the urinary protein content, 10 µg of total urinary protein from WT and CD73^−/−mice were subjected per lane to a 12.5% nonreducing SDS-PAGE (silver staining). Mice groups are shown as indicated, Alb  =  bovine serum albumin, M  =  rainbowmolecule weight marker with characteristic proteins as indicated (CA  =  carbonic anhydrase, GD  =  glutamatic dehydrogenase, Myo  =  myoglobin red). Proteins bands identified by size are indicated: α1-M  =  α 1-microglobulin, RBP  =  retinol binding protein. (B) Urinary albumin excretion was not different as compared to WT mice. 9 to 24 mice per group were used. Data are presented as means SEM.</p

Presentation_2_Immunomodulatory regulator blockade in a viral exacerbation model of severe asthma.pptx

Asthmatics are more susceptible to viral infections than healthy individuals and are known to have impaired innate anti-viral defences. Influenza A virus causes significant morbidity and mortality in this population. Immuno-modulatory regulators (IMRs) such as PD-1 are activated on T cells following viral infection as part of normal T cell activation responses, and then subside, but remain elevated in cases of chronic exposure to virus, indicative of T cell exhaustion rather than activation. There is evidence that checkpoint inhibition can enhance anti-viral responses during acute exposure to virus through enhancement of CD8+T cell function. Although elevated PD-1 expression has been described in pulmonary tissues in other chronic lung diseases, the role of IMRs in asthma has been relatively unexplored as the basis for immune dysfunction. We first assessed IMR expression in the peripheral circulation and then quantified changes in IMR expression in lung tissue in response to ex-vivo influenza infection. We found that the PD-1 family members are not significantly altered in the peripheral circulation in individuals with severe asthma but are elevated in pulmonary tissues following ex-vivo influenza infection. We then applied PD-1 Mab inhibitor treatment to bronchial biopsy tissues infected with influenza virus and found that PD-1 inhibition was ineffective in asthmatics, but actually increased infection rates in healthy controls. This study, therefore, suggests that PD-1 therapy would not produce harmful side-effects when applied in people with severe asthma, but could have important, as yet undescribed, negative effects on anti-viral responses in healthy individuals that warrant further investigation.</p

Humoral inflammation in glomeruli and cellular infiltrates in the interstitium of CD73^−/−mice.

<p>The renal cortex of CD73^−/−mice showed glomerular deposits of IgG and C3 as well as increased presence of CD 11b- a<b>n</b>d CD8- positive cells as well as CD25- and GR-1–positive cells in the interstitium of CD73^−/− mutants which were absent in WT-mice. (A) IgG staining (green, FITC conjugated secondary antibodies) and C3 staining (red, rhodamin red λ conjugated secondary antibodies) was carried out in cryo-conserved, 7-µm sections of the kidneys of WT and CD73^−/−mice. Images are representative of >3 independent experiments. (B) CD11b and CD 8 staining (green, FITC conjugated secondary antibodies) and CD25 and GR-1 staining (red, Cy 3) was performed in cryo-conserved, 6–7-µm sections of the mice kidneys. Images are representative of 5 to 7 animals with more than 2 sections per kidney.</p

Proinflammatory pattern of angiogenetic factors, chemokines and cytokines observed in the serum but not in the renal tissue of CD73^−/−mice.

<p>The levels of IL1-β, IL-2, IL-12p40, IL-15, IL-17, IFN-γ, GM-CSF, G-CSF, IL-13, MCP-1, interleukins 3, 4, 5, 10, 12p70, 13, M-CSF, MIG and RANTES were screened in the serum and renal tissue of CD73^−/− and WT mice using BioPlex analysis. The serum levels of most measured cytokines and chemokines were slightly but not significantly elevated in the mutants as compared to the wild type controls, suggesting a proinflammatory pattern. The renal tissue levels were unaltered or even reduced in the CD73^−/−mice compared to WT.</p

Collagen deposition in the renal cortex, peritubular and surrounding the glomeruli in CD73^−/−mice.

<p>(A) Representative images of fibrillar collagen deposition shown by Sirius Red staining (in CD73^−/−as compared to WT controlc mice, ×200 magnification). (B) Image analysis demonstrates that CD73^−/−mice exhibit increased levels of fibrillary collagen deposition within the cortex compared with WT controls (significance as indicated, p<0.002 CD73^−/− vs WT mice, Sirius red staining, 3–5 images per animal, n = 7 animals/group, Student t-test).</p

Glomerular endotheliosis and injury in CD73^−/−mice.

<p>Electrone microscopic analysis was performed n = 7 CD73^−/−mice vs. WT, original magnification x 10000 or 5000. (A) Ultrastructural analysis of the glomerular filtration barrier from a 3 months old WT mouse with regular appearance of foot processes and slit membranes and fenestration of the endothelium. (B) Analysis of the glomerular filtration barrier from a 3 months old CD73^−/− mouse shows cytoplasmic swelling apparent in endothelial cells (cap. Lumen  =  capillary lumen, ery  =  erythrocyte), endothelial fenestrations and single foot effacements were reduced (arrow). No subepithelial depositis were detectable. (C) Overview of a glomerular segment with regular appearance and without glomerulitis (3 months old WT-mouse). (D) Overview of a glomerular segment in a 3 months old CD73^−/−mouse (mes  =  mesangial cell, ly  =  lymphocytes) shows lymphocytic glomerulitis.</p

Increase of total proteinuria and decrease of renal function in mice lacking CD73.

<p>(A) Using immunohistochemistry with a CD73-specific antibody and a rhodamin red λ conjugated secondary antibody, confocal microscopy at a magnitude of 10× revealed staining of glomerular cells in the mesangium as well as peritubular cells in renal tissue of WT mice which is absent in the mutant. Images are representative of 3 of 5 animals with more than 2 sections per kidney. (B) Renal function estimated by the measurement of serum creatinine, auto normalized, in WT and CD73^−/−mice. 8 to19 mice per group were used. Data are presented as means ± SEM, level of significance as indicated according to Student t-test. (C) Total proteinuria was elevated and increased with age in CD73^−/−mice as compared to the wild type controls. We detected proteinuria/creatinuria in [g/g], left panel, and total urine protein in [mg/24h], right panel. 8 to 19 mice per group were used. Data are presented as means ± SEM, level of significance as indicated according to Student t-test.</p

Presentation_3_Immunomodulatory regulator blockade in a viral exacerbation model of severe asthma.pptx

Asthmatics are more susceptible to viral infections than healthy individuals and are known to have impaired innate anti-viral defences. Influenza A virus causes significant morbidity and mortality in this population. Immuno-modulatory regulators (IMRs) such as PD-1 are activated on T cells following viral infection as part of normal T cell activation responses, and then subside, but remain elevated in cases of chronic exposure to virus, indicative of T cell exhaustion rather than activation. There is evidence that checkpoint inhibition can enhance anti-viral responses during acute exposure to virus through enhancement of CD8+T cell function. Although elevated PD-1 expression has been described in pulmonary tissues in other chronic lung diseases, the role of IMRs in asthma has been relatively unexplored as the basis for immune dysfunction. We first assessed IMR expression in the peripheral circulation and then quantified changes in IMR expression in lung tissue in response to ex-vivo influenza infection. We found that the PD-1 family members are not significantly altered in the peripheral circulation in individuals with severe asthma but are elevated in pulmonary tissues following ex-vivo influenza infection. We then applied PD-1 Mab inhibitor treatment to bronchial biopsy tissues infected with influenza virus and found that PD-1 inhibition was ineffective in asthmatics, but actually increased infection rates in healthy controls. This study, therefore, suggests that PD-1 therapy would not produce harmful side-effects when applied in people with severe asthma, but could have important, as yet undescribed, negative effects on anti-viral responses in healthy individuals that warrant further investigation.</p