Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

Monoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these anti-Id antibodies as Ab2 gamma or Ab2 beta. By inhibiting experiments it was shown that these anti-Id antibodies did not recognize interspecies cross-

A human CD5+ B cell clone that secretes an idiotype-specific high affinity IgM monoclonal antibody.

We previously demonstrated the occurrence of a naturally arisen human anti-idiotypic B cell clone, that we transformed with EBV (EBV383). We show evidence that EBV383 not only expresses the CD5 surface Ag, but also contains the 2.7-kb mRNA transcript encoding this protein. In addition, we show the presence of the 3.6-kb mRNA precursor. Most Ig produced by CD5+ B cells are polyreactive natural IgM antibodies encoded by unmutated copies of germline VH genes. However, in this study we present data demonstrating the monoreactive high affinity character of the anti-idiotypic antibody (mAb383) produced by EBV383. These data are in agreement with our previous observations, showing that the VH chain of mAb383 is encoded by an extensive somatically mutated VHV gene in a way that is consistent with an Ag-driven immune response. A possible role for this remarkable anti-idiotypic antibody in the maintenance of B cell memory is discussed

Human conjunctiva organoids to study ocular surface homeostasis and disease

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.</p

Human conjunctiva organoids to study ocular surface homeostasis and disease

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.</p

Urine-derived bladder cancer organoids (urinoids) as a tool for cancer longitudinal response monitoring and therapy adaptation

BACKGROUND: Bladder cancer is one of the most common cancer types worldwide. Generally, research relies on invasive sampling strategies. METHODS: Here, we generate bladder cancer organoids directly from urine (urinoids). In this project, we establish 12 urinoid lines from 22 patients with non-muscle and muscle-invasive bladder tumours, with an efficiency of 55%. RESULTS: The histopathological features of the urinoids accurately resemble those of the original bladder tumours. Genetically, there is a high concordance of single nucleotide polymorphisms (92.56%) and insertions & deletions (91.54%) between urinoids and original tumours from patient 4. Furthermore, these urinoids show sensitivity to bladder cancer drugs, similar to their tissue-derived organoid counterparts. Genetic analysis of longitudinally generated tumoroids and urinoids from one patient receiving systemic immunotherapy, identify alterations that may guide the choice for second-line therapy. Successful treatment adaptation was subsequently demonstrated in the urinoid setting. CONCLUSION: Therefore, urinoids can advance precision medicine in bladder cancer as a non-invasive platform for tumour pathogenesis, longitudinal drug-response monitoring, and therapy adaptation

Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.This work was supported by NIRM/ Clevers and Stichting Vrienden van het Hubrecht (O.B.), EU/232814-StemCellMark and Skolkovo 077 MPA (J.H.v.E.), NIH/MIT Subaward 5710002735 (to D.E.S.), KWF/PF-HUBR 2007-3956 and Stichting Vrienden van het Hubrecht (M.v.d.W.), European Research Council Advanced Grant ERC-AdG 294325-GeneNoiseControl (to K.W. and A.v.O.), and Wellcome Trust Grant 098357/Z/12/Z (to B.D.S.)

Canonical Wnt signaling negatively modulates regulatory T cell function

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that Tcell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both invitro and invivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector Tcells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity

Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche

Identification of a region required for TSC1 stability by functional analysis of TSC1 missense mutations found in individuals with tuberous sclerosis complex

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). Recently it has been shown that missense mutations to the TSC1 gene can cause TSC. Methods: We have used in vitro biochemical assays to investigate the effects on TSC1 function of TSC1 missense variants submitted to the Leiden Open Variation Database. Results: We identified specific substitutions between amino acids 50 and 190 in the N-terminal region of TSC1 that result in reduced steady state levels of the protein and lead to increased mTOR signalling. Conclusion: Our results suggest that amino acid residues within the N-terminal region of TSC1 are important for TSC1 function and for maintaining the activity of the TSC1-TSC2 complex

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs