Purpose Various profibrotic and proinflammatory cytokines have been found upregulated in uncomplicated primary retinal detachment (pRD), but without providing a uniform picture. Here, we compare the cyto- and chemokine profiles in pRD with and without proliferative vitreoretinopathy (PVR) in an attempt to unravel relevant differences not in single cytokines, but in the cytokine profiles at diagnosis. Methods Undiluted vitreous fluid (VF) was obtained at the beginning of surgery from 174 eyes with pRD without relevant PVR (maximally grade B; group 1; n = 81) and with moderate or advanced PVR requiring a gas tamponade (group 2; n = 49) or silicon oil filling (group 3; n = 44). VF of eyes undergoing macular hole (MH) surgery served as controls (group 4; n = 26). Forty-three cytokines were quantified in parallel using a multiplex cytokine analysis system (Bioplex). For all comparisons we applied Holm’s correction to control for multiple comparisons. Results 44.9% of group 2 eyes presented grade C1 and 55.1% C2-C3, whereas 86.4% of group 3 eyes exhibited a PVR grade of C2-D. CCL19 was the only cytokine that displayed higher concentrations in the vitreous of eyes with PVR C1 compared to lower PVR grades. Eyes with PVR C2-D showed higher levels of CCL27, CXCL6, IL4, IL16, CXCL10, CCL8, CCL22, MIG/CXCL9, CCL15, CCL19, CCL 23 and CXCL12 compared to controls. Interestingly, no difference of cytokine levels was detected between C1 and C2-D PVR. Conclusions CCL19 may represent a potential biomarker for early PVR progression that holds promise for future diagnostic and therapeutic applications.
Thrombotic microangiopathies are rare disorders characterized by the concomitant occurrence of severe thrombocytopenia, microangiopathic hemolytic anemia, and a variable degree of ischemic end-organ damage. The latter particularly affects the brain, the heart, and the kidneys. The primary forms, thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), although their clinical presentations often overlap, have distinctive pathophysiologies. TTP is the consequence of a severe ADAMTS-13 deficiency, either immune-mediated as a result of circulating autoantibodies, or caused by mutations in ADAMTS-13. HUS develops following an infection with Shiga-toxin producing bacteria, or as the result of excessive activation of the alternative pathway of the complement system because of mutations in genes encoding complement system proteins.
Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form isletlike tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a perwell normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to distinct therapeutic regimes. Treatment response of islet-like tumoroids differs also between patient samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy Molecular mechanisms underlying the development and progression of PanNET are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this.
PurposeTo compare the cyto- and chemokine profiles in the aqueous humor of PEXS eyes in the absence or presence of secondary glaucoma with or without luxation of the intraocular lens (IOL).MethodsSamples of aqueous humor were collected intraoperatively from 20 healthy controls and from 73 eyes with PEX-syndrome, which was manifested in the absence of any other local or systemic desease. The latter group was sub-devided into eyes with an early form of PEX-syndrome in the absence of complications (PEX, n = 33), those with a late form of PEX-syndrome and glaucoma (PEXG, n = 30), and those with a late form of PEX-syndrome with luxation of the IOL that required surgery (PEXL, n = 10). The samples were analyzed in parallel after storage at -80°C. The levels of 40 cytokines were simultaneously quantified using the Bio-Plex® multiplex beads system. The inter-group data were statistically compared using the Kruskal-Wallis test (p ≤ 0.01).ResultsPEX and PEXG were comparable in their cytokine profiles for all 40 cytokines, whereas the cytokine profile in PEXL-eyes revealed higher levels of all but 5 cytokines (CXCL13, CCL27, IL-2, CCL3, CCL20; p ≤ 0.01). This latter finding is indicative of a non-specific inflammatory reaction in the context of IOL-luxation. The concentrations of 6 cytokines lay below the detection limit in all groups.ConclusionsThe local up-regulation of 85% of the detectable cytokines in the aqueous humor of PEXL-eyes may be linked either with a progression of the disease or a breakdown of the antero-posterior barrier in the context of IOL-luxation.
Purpose To compare the cytokine profiles of phakic (p) and pseudophakic (ps) eyes with primary rhegmatogenous retinal detachment ( RD ) to eyes with macular holes ( MH ) and to identify differences in the specific cytokine profiles. Methods Aqueous humour ( AH ) and vitreous fluid ( VF ) were obtained from patients with primary RD without proliferative vitreoretinopathy undergoing vitrectomy. AH and VF of patients with macular holes ( MH ) served as controls. Forty‐three different cytokines were quantified using multiplex cytokine analysis. Intergroup and intragroup comparisons were performed. To control for multiple comparisons, Holm's correction was applied. Results VF and AH samples of 71 eyes with RD ( pRD N = 38; ps RD N = 33) and 26 eyes with MH were included. Cytokine levels in ps RD and pRD were similar (none with >10‐fold difference). The levels of 39 of 43 cytokines in the VF were significantly higher in eyes with RD than in those with MH (>10‐fold: CXLC 5, CCL 26, CCL 1, IL ‐6, CXCL 11, CCL 7, CCL 13, MIG / CXCL 9, CCL 19 and TGF ‐ β 1). In the AH , 23 of 43 cytokines were significantly higher compared to MH (>10‐fold: CXCL 5, IL ‐4, IL ‐6, IL ‐8/ CXCL 8 and CCL 7). Conclusion A complex, but nonspecific cytokine environmental response seems to initiate immunological and profibrotic processes following RD . Relevant differences in the cytokine profiles of eyes with pRD and ps RD were not identified, whereas cytokine differences between AH and VF in RD could be explained by upregulation in the vitreous, a higher turn around in the anterior chamber, or differences in inflammatory cascades in both compartments.
3118 Background: EB1, a protein located on the plus-ends of microtubules is involved in microtubule function and has been associated with glioblastoma (GBM) stem-cell-ness and more aggressive disease. Lisavanbulin (BAL101553) is a prodrug of the lipophilic small molecule BAL27862, that promotes tumor cell death by modulating the spindle assembly checkpoint and has been shown in rodents to efficiently penetrate the brain. Data from GBM mouse models and recent phase 1 clinical data (Lopez et al. ESMO 2020) suggest that EB1 is a response-predictive marker for lisavanbulin in GBM. A phase 2 study is ongoing to confirm this hypothesis (NCT02490800). A proof-of-concept in GBM would support an expansion of EB1-directed lisavanbulin clinical development in non-GBM tumors, which requires prevalence estimates of EB1-positivity in non-GBM tumor types. Methods: Tissue samples from GBM and other tumor types were stained for EB1 using a CE-marked immunohistochemistry Clinical Trial Assay (Targos Molecular Pathology GmbH, Kassel Germany). EB1-positivity was assessed by a board-certified pathologist based on the percentage of tumor cells showing moderate or strong staining for EB1, using thresholds of ≥50%, ≥60% and ≥70% of tumor cells with EB1 positivity. Whole transcriptome sequencing (WTS) using RNAseq was performed in a subset of tissue samples to develop a potential RNA-based predictive response signature for lisavanbulin. Results: 73 GBM tissue samples and 333 tissue samples from 13 other cancer types were stained for EB1. The strongest overall signal for EB1-positivity was obtained for medulloblastoma, neuroblastoma and GBM. In addition, moderate or strong EB1-staining in ≥50% of tumor cells was observed in samples from colorectal cancer (CRC), non small-cell lung cancer (NSCLC), metastatic melanoma, small-cell lung cancer (SCLC) and triple-negative breast cancer (TNBC). An expanded staining campaign is ongoing in these cancer types. Initial results from the ongoing WTS analyses show marked differences in gene expression profiles between EB1-positive and -negative cases. Conclusions: Strong EB1-positivity is infrequent but occurs in a variety of tumor types, with the strongest signals in medulloblastoma, neuroblastoma and GBM. A phase 2 study is ongoing to assess prospectively whether EB1 is a response-predictive biomarker for lisavanbulin in GBM.[Table: see text]
Introduction: Antibodies are the primary effector molecules in the humoral immune system. To create a diversity of receptors-antibodies B-cells undergo V(D)J gene recombination and somatic hypermutation. This allows the recognition of most of pathogens but also sometimes of self-antigen, which can lead to autoimmune diseases. Autoantibodies (Abs) neutralizing and/or accelerating the clearance of ADAMTS13 are present in nearly all acquired thrombotic thrombocytopenic purpura (aTTP) patients with severe ADAMTS13 activity (<5%). As increasing evidence points at the spleen as a major reservoir for antigen specific memory B-cells, we investigated the splenic B-cell repertoire. We sequenced heavy and corresponding light chains from single antigen-specific memory B-cells and deep sequenced global antibody repertoire among different B-cell populations. Combining both, the single antigen-specific repertoire and the general global repertoire from the same patients specimens allows an investigation of the V(D)J gene usage and abundance of specific V(D)J junction in the B-cell populations within one patient and also among different patients. Analysis of the repertoire enables better understanding of the humoral autoimmune response. Methods: Specimens were analyzed from 8 aTTP patients who were splenectomized due to a refractory course of the autoimmune disease. Splenic mononuclear cells were sorted by flow-cytometry into naïve and transitional (CD27-, IgD+), unswitched memory (CD27+, IgD+) and switched memory (CD27+, IgD-) as well as (CD27+, CD20-/CD19-, CD38+) plasma cells. The frequencies of highly positive anti-ADAMTS13 B-cells in each B-cell population were calculated. Library consisting of antibody cDNA from each B-cell population was deep sequenced on MiSeq Ilumina. Prime analysis were performed in CLC Genomics Workbench. High quality sequences were submitted to IMGT/high V-QUEST web-based analysis tool to determine for V(D)J genes alignments, and V(D)J junction (antigen binding region) and mutations analysis. The IMGT output was parsed into MySQL database for further analysis. In addition, from 4 patients anti-ADAMTS13 specific IgG bearing cells were individually sorted. Gene transcripts of single cells were reverse-transcribed followed by nested PCR of IgG heavy/light chains. V(D)J pairings were visualized with Circos software. In MySQL we compared single cell antigen specific sequence with global repertoire. Results: Anti-ADAMTS13 specific B-cells were detected in the spleen of all patients (average 0.01% of total B-cell population). Deep sequencing of the total B-cell repertoire revealed ~3 million productive sequences, from which ~2 million were unique. Splenic anti-ADAMTS13 specific B-cells of four aTTP patients revealed 80 antibodies with unique V(D)J junction. Among those most frequently used V-genes were IGHV1-69 and IGHV3-30 (15% and 12% respectively) in global repertoire 1.5% of antibodies were encoded with IGHV1-69 gene within respective population. The average identity to germline was lower in ADMTST13 specific B-cells (92% compare to 96%). Four V(D)J junctions were convergent in at least 2 patients (identical amino acid sequence). Conclusion: Anti-ADAMTS13 specific B-cells were found in all aTTP patients in different B-cell populations. We observed enrichment of some variable gene segments when comparing the specific anti-ADAMTS13 to the total splenic repertoire (mainly IGHV1-69). Finding convergent V(D)J junction is very promising and also confirms previous finding of our group where similar V(D)J junction were found among two patients. Currently we clone selected single sorted monoclonal Abs (Schaller et al, Blood 2014;124(23):3469-79). Functional testing will allow the selection of the inhibitory Abs to be used as tools to develop anti-idiotypic specific therapies for aTTP patients. Disclosures No relevant conflicts of interest to declare.
Background. Autoantibodies (Abs) neutralizing and/or accelerating clearance of the von Willebrand factor-cleaving protease-ADAMTS13 are the major culprits in acquired Thrombotic Thrombocytopenic Purpura (aTTP). Despite the first-line treatment with daily plasma exchange (PEX), acute aTTP is still today associated with a mortality rate of ~20% leaving survivors at high risk of relapse. To improve patient care and/or treatment of aTTP we explored the capacity of anti-idiotypic small molecules to specifically bind and potentially neutralize pathogenic anti-ADAMTS13 autoantibodies thereby restoring plasma-derived ADAMTS13 activity in acquired TTP patients. Method: As selecting tools we used previously generated spleen-derived inhibitory human monoclonal anti-ADAMTS13 antibodies consisting of the 4 antigen-binding CDR3 motifs shared by the two aTTP patients (Schaller et al, Blood 2014;124(23):3469-79). Either 3 mAbs representative for CDR3 Motif-1 or 3 mAbs for CDR3 Motif-3 or 2 mAbs representing CDR3 Motif-4 were pooled at equimolar concentrations by ribosomal display to screen for binders in two combinatorial small protein libraries (DARPins from Molecular Partners, Schlieren, Switzerland) containing either two (N2C) or three (N3C) randomized ankyrin repeat modules, respectively From each of the 6 libraries single anti-idiotypic DARPins were cloned into a pMPAG6 expression vector and purified by His-tag affinity chromatography. In total 22 unique anti-idiotypic DARPins, 4 N2C-DARPins and 2 N3C-DARPins selected by Motif-1, 9 N2C-DARPins by Motif-3 and 7 N2C-DARPins by Motif-4 mAbs were identified. Inhibitor targeting Strategy: The neutralization potential towards plasma-derived anti-ADAMTS13 Abs, was tested by pre-incubating the DARPins prior measuring the residual activity by functional FRETS assay, in a cohort of 50 acute aTTP patients with pathological inhibitor titers >0.4 BU/ml (diagnosed at our Center 2006-2014). First, equimolar pools (1000 nM) of anti-idiotypic DARPins (Motif-1, Motif-3 or Motif-4) were tested in 11/50 patients, secondly the contribution of the single DARPins from the responsive pools was assessed in 18/50 patients to finally test the entire cohort with a combination of all neutralizing anti-idiotypic DARPins. Results: The pool of all Motif-1 anti-idiotypic DARPins (n=6) neutralized inhibitor titers below the pathological threshold in 9/11 (82 %) aTTP patients tested, whereas only a slight reduction of the inhibitor titers (in average 15%) was observed for Motif-3 and Motif-4 DARPin pools, revealing the Motif-1 DARPins as the most universal neutralizers. To test the contribution of each Motif-1 anti-idiotypic DARPin to the observed inhibitor neutralization, single DARPins were tested in 18/50 aTTP revealing a reduction of the inhibitor of 20-50% (N2C-19) or 20% (N3C-74) in 5/18 patients tested. In contrast 90% (45/50) aTTP patients incubated with the pool of all 6 Motif-1 DARPins caused inhibitor titers to drop either completely or below the pathological threshold. The inhibitor titer was reduced below 1BU/ml in 3/50, but was irresponsive in 2/50 patients. Conclusions: Our data clearly show that anti-idiotypic DARPins can potently and universally neutralize inhibitory anti-ADAMTS13 Abs of aTTP patients. However an effective neutralization, restoring ADAMTS13 activity above 10-15% in most patients was only achieved when pooling all Motif-1 DARPins. If a combination of Motif-3 and/or Motif-4 DARPins with Motif-1 DARPins can additionally increase neutralization capacity also in relapsing patients is currently under investigation. Disclosures No relevant conflicts of interest to declare.
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