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Intestinal organoids grown from adult stem cells have emerged as prototype 3D organotypic models for studying tissue renewal and homeostasis. Owing to their strict dependence on Wnt signaling, intestinal organoids offer an unprecedented opportunity to examine Wnt pathway regulation in normal physiology and cancer. We review how alterations in growth factor dependency and organoid morphology can be exploited to identify Wnt signaling mechanisms, characterize mutated pathway components, and predict responses of patient-derived tumors to targeted therapy. We discuss current deficits in the understanding of genotype-phenotype relationships that are to be considered when interpreting mutation-induced changes in organoid morphology.

Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors., Enhanced Wnt receptor activity is a major cause of cancer development. Here the authors identify camelid single-domain antibody fragments (VHHs) that bind to the Wnt receptor LRP5/6 ectodomain, determine the crystal structures and show that these VHHs selectively inhibit Wnt3- mediated cellular responses and block the growth of mutant Wnt-hypersensitive intestinal tumor organoids.

The intestinal epithelium harbors a remarkable adaptability to undergo injury-induced repair. A key part of the regenerative response is the transient reprogramming of epithelial cells into a fetal-like state, which drives uniform proliferation, tissue remodeling, and subsequent restoration of the homeostatic state. In this review, we discuss how Wnt and YAP signaling pathways control the intestinal repair response and the transitioning of cell states, in comparison with the process of intestinal development. Furthermore, we highlight how organoid-based applications have contributed to the characterization of the mechanistic principles and key players that guide these developmental and regenerative events.

Mutation-induced activation of WNT-β-catenin signalling is a frequent driver event in human cancer. Sustained WNT-β-catenin pathway activation endows cancer cells with sustained self-renewing growth properties and is associated with therapy resistance. In healthy adult stem cells, WNT pathway activity is carefully controlled by core pathway tumour suppressors as well as negative feedback regulators. Gene inactivation experiments in mouse models unequivocally demonstrated the relevance of WNT tumour suppressor loss-of-function mutations for cancer growth. However, in human cancer, a far more complex picture has emerged in which missense or truncating mutations mediate stable expression of mutant proteins, with distinct functional and phenotypic ramifications. Herein, we review recent advances and challenges in our understanding of how different mutational subsets of WNT tumour suppressor genes link to distinct cancer types, clinical outcomes and treatment strategies.

Pseudomonas aeruginosa is a major health challenge that causes recalcitrant multidrug-resistant infections, especially in immunocompromised and hospitalized patients. P. aeruginosa is an important cause of nosocomial and ventilator-associated pneumonia characterized by high prevalence and fatality rates. P. aeruginosa also causes chronic lung infections in individuals with cystic fibrosis. Multidrug- and totally drug-resistant strains of P. aeruginosa are increasing threats that contribute to high mortality in these patients. The pathogenesis of many P. aeruginosa infections depends on its ability to form biofilms, structured bacterial communities that can coat mucosal surfaces or invasive devices. These biofilms make conditions more favorable for bacterial persistence, as embedded bacteria are inherently more difficult to eradicate than planktonic bacteria. The molecular mechanisms that underlie P. aeruginosa biofilm pathogenesis and the host response to P. aeruginosa biofilms remain to be fully defined. However, it is known that biofilms offer protection from the host immune response and are also extremely recalcitrant to antimicrobial therapy. Therefore, development of novel therapeutic strategies specifically aimed at biofilms is urgently needed. Here, we review the host response, key clinical implications of P. aeruginosa biofilms, and novel therapeutic approaches to treat biofilms relevant to lung infections. Greater understanding of P. aeruginosa biofilms will elucidate novel avenues to improve outcomes for P. aeruginosa pulmonary infections.

The intestinal stem cell (ISC) marker LGR5 is a receptor for R‐spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self‐renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post‐translational regulation of LGR5. Here, we show that the HECT‐domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high‐grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/β‐catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post‐translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression., NEDD4 and NEDD4L E3 ligases control LGR5 receptor turnover in intestinal homeostasis and colorectal cancer.

Mammalian Wnt proteins are believed to act as short-range signals(1-4), yet have not been previously visualized in vivo. Selfrenewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt(5). Wnt3 is produced specifically by Paneth cells(6,7). Here we have generated an epitopetagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and ` plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.

Differential WNT and Notch signaling regulates differentiation of Lgr5+ crypt-based columnar cells (CBCs) into intestinal cell lineages. Recently we showed that mitochondrial activity supports CBCs, while adjacent Paneth cells (PCs) show reduced mitochondrial activity. This implies that CBC differentiation into PCs involves a metabolic transition toward downregulation of mitochondrial dependency. Here we show that Forkhead box O (FoxO) transcription factors and Notch signaling interact in determining CBC fate. In agreement with the organoid data, Foxo1/3/4 deletion in mouse intestine induces secretory cell differentiation. Importantly, we show that FOXO and Notch signaling converge on regulation of mitochondrial fission, which in turn provokes stem cell differentiation into goblet cells and PCs. Finally, scRNA-seq-based reconstruction of CBC differentiation trajectories supports the role of FOXO, Notch, and mitochondria in secretory differentiation. Together, this points at a new signaling-metabolic axis in CBC differentiation and highlights the importance of mitochondria in determining stem cell fate.

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

The β‐catenin destruction complex is a dynamic cytosolic multiprotein assembly that provides a key node in Wnt signalling regulation. The core components of the destruction complex comprise the scaffold proteins axin and adenomatous polyposis coli and the Ser/Thr kinases casein kinase 1 and glycogen synthase kinase 3. In unstimulated cells, the destruction complex efficiently drives degradation of the transcriptional coactivator β‐catenin, thereby preventing the activation of the Wnt/β‐catenin pathway. Mutational inactivation of the destruction complex is a major pathway in the pathogenesis of cancer. Here, we review recent insights in the regulation of the β‐catenin destruction complex, including newly identified interaction interfaces, regulatory elements and post‐translationally controlled mechanisms. In addition, we discuss how mutations in core destruction complex components deregulate Wnt signalling via distinct mechanisms and how these findings open up potential therapeutic approaches to restore destruction complex activity in cancer cells. Linked Articles This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc

The mechanism by which Wnt receptors transduce signals to activate downstream beta-catenin-mediated target gene transcription remains incompletely understood but involves Frizzled (Fz) receptor-mediated plasma membrane recruitment and activation of the cytoplasmic effector Dishevelled (Dvl). Here, we identify the deubiquitinating enzyme CYLD, the familial cylindromatosis tumor suppressor gene, as a negative regulator of proximal events in Wnt/beta-catenin signaling. Depletion of CYLD from cultured cells markedly enhances Wnt-induced accumulation of beta-catenin and target gene activation. Moreover, we demonstrate hyperactive Wnt signaling in human cylindroma skin tumors that arise from mutations in CYLD. At the molecular level, CYLD interacts with and regulates K63-linked ubiquitination of Dvl. Enhanced ubiquitination of the polymerization-prone DIX domain in CYLD-deficient cells positively links to the signaling activity of Dvl. Together, our results argue that loss of CYLD instigates tumor growth in human cylindromatosis through a mechanism in which hyperubiquitination of polymerized Dvl drives enhancement of Wnt responses.

Summary Organoid technology provides the possibility of culturing patient-derived colon tissue and colorectal cancers (CRCs) while maintaining all functional and phenotypic characteristics. Labeling stem cells, especially in normal and benign tumor organoids of human colon, is challenging and therefore limits maximal exploitation of organoid libraries for human stem cell research. Here, we developed STAR (stem cell Ascl2 reporter), a minimal enhancer/promoter element that reports transcriptional activity of ASCL2, a master regulator of LGR5+ intestinal stem cells. Using lentiviral infection, STAR drives specific expression in stem cells of normal organoids and in multiple engineered and patient-derived CRC organoids of different genetic makeup. STAR reveals that differentiation hierarchies and the potential for cell fate plasticity are present at all stages of human CRC development. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research into human adult stem cell biology., Graphical Abstract, Highlights • ASCL2-responsive minigene (STAR) is specific for intestinal stem cells • STAR is compatible with user-friendly techniques like lentiviral infections • STAR enables stem cell labeling in normal and in cancer organoids of human colon • Cellular plasticity is present at all stages of colorectal cancer development, Oost et al. present an ASCL2-responsive minigene (STAR) that enables stem cell labeling in patient-derived colorectal cancer organoids, as well as in normal and benign colorectal tumor samples. The user-friendly nature of STAR applications in combination with organoid technology will facilitate basic research into human adult stem cell biology.

Two lipids (palmitate and palmitoleic acid) are appended onto Wnt proteins. It has been suggested that palmitate is required for signalling, whereas palmitoleic acid is necessary for progression through the secretory pathway. By mutating the relevant amino acids, we have investigated how these adducts contribute to the secretion and signalling activity of Wingless, the main Drosophila member of the Wnt family. Analysis of Wingless with a Cysteine 93 to Alanine mutation ([C93A]Wingless) shows that palmitoylation is essential for signalling activity in vivo (as well as in cultured cells). Moreover, without palmitate, Wingless fails to reach the surface of imaginal disc cells and, as electron microscopy (EM) analysis suggests, appears to accumulate in the endoplasmic reticulum (ER). Artificial targeting of palmitate-deficient Wingless to the plasma membrane does not rescue signalling activity. Therefore, palmitate at C93 has a dual role: in secretion and signalling. From our analysis of [S239A]Wingless, which lacks a conserved residue shown to be acylated in Wnt3a, we infer that palmitoleic acid is not, as previously suggested, absolutely required for secretion. Nevertheless, this mutant has poor signalling activity, suggesting that palmitoleic acid contributes significantly to signalling. We suggest that the overall level of lipidation affects signalling activity.

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.

We studied the cervico-ocular reflex (COR) alone and in combination with the optokinetic (OKN) reflex in head-fixed pigeons. We analyzed these responses in two behavioral conditions: (1) animals were hung in a harness (“resting” condition); and (2) animals were additionally submitted to a frontal airflow that provoked a flight posture (“flying” condition). In both conditions, cervical stimulation provoked a slow phase of very low gain (around 0.05) in the opposite direction to that of the stimulation and fast phases triggered near the head–body alignment in the same direction as the stimulation. The slow phase showed a phase lag of 20 deg at 0.5 Hz. The gain of the slow phase was not modified by the velocity, amplitude, or frequency of the stimuli. This gain was not changed by the presence of a fixed visual surround.When cervical stimuli (0.05–0.5 Hz) were added to an optokinetic stimulation (30 deg/s) in the “resting” condition, the slow phase velocity (SPV) of the optokinetic reflex was modulated with a time course close to that produced by the cervico-ocular reflex alone. The SPV was alternately increased and decreased round the SPV level corresponding to the steady-state OKN. In the “flying” condition, optokinetic-cervical stimulation provoked an eye beating field and a strong SPV modulation synchronized with the position of the cervical stimulation. The number of nystagmic beats (OKN) and the amplitude and velocity of the fast phases were modulated in correlation with the SPV. Consequently, the optokinetic response was increased or decreased according to whether the cervical stimuli were in the reverse direction or in the same direction as the optokinetic stimulation, respectively.These data are interpreted as an improvement of gaze stabilization by the COR. This mechanism is context dependent, since it is strongly reinforced during the flight.

A novel microscope system is presented for observation of corneal cells in a living mouse. It enables tracking of individual cells in all layers of the cornea at various times, thus allowing the generation of time-lapse recordings. The system consists of three major components: an upright fluorescence microscope for visualization of corneal cells, a mouse-holding unit for immobilization of the animal and the eye, and a set of gimbals which permit observation of a wide area of corneal surface without refocusing. The same cells could be observed at different limes with the help of fiducial marks in the cornea, allowing their changes in position to be determined under natural and experimental conditions. This technique should prove useful in investigation of the cell movement in normal and diseased corneas, including the study of wound healing after an injury or surgery.

Programmed cell death (apoptosis) is crucial for thymocyte development. We analyzed the role of the ubiquitin (Ub)–proteasome pathway in dexamethasone-triggered and TCR-mediated apoptosis in fetal thymic organ culture (FTOC). Proteasome activity was increased in apoptotic thymocytes, as visualized by active-site labeling of proteasomal β subunits. The activity of deubiquitinating enzymes in murine apoptotic thymocytes was likewise examined by active-site labeling. We show that the deubiquitinating enzyme USP7 (HAUSP) is proteolytically processed upon dexamethasone-, γ-irradiation-, and antigen-induced cell death. Such processing of HAUSP does not occur in caspase 3 −/− thymocytes, or upon pretreatment of wild type thymocytes with the general caspase inhibitor ZVAD-fmk. Thus, our results suggest that thymocyte apoptosis leads to modification of deubiquitinating enzymes by caspase activity and may provide an additional link between the ubiquitin–proteasome pathway and the caspase cascade during programmed cell death.

Crosslinking of the T cell receptor has been proposed to be a prerequisite for T cell activation. Although the evidence supports this notion for CD4 T cells, the situation for CD8 T cells is less clear. Soluble class I monomers have been used to determine activation requirementsin vitrowith contradictory results. The possibility of transfer of peptide from soluble class I molecules onto class I molecules present on the surface of CD8 T cells, with ensuing presentation to other CD8 T cells, has been widely ignored. We show that monomeric and tetrameric class I molecules as well as free peptide can stimulate naïve CD8 T cellsin vitro. We generate and characterize CD8 T cells that express the OT-I T cell receptor (for Kb/SIINFEKL) yet lack Kband Dbmolecules, and show that their activation requirements differ from their class I positive counterparts when stimulated with soluble Kbmolecules. By eliminating the confounding effect of peptide transfer, we unmask the true activation requirements for naïve CD8 T cells and show that multivalent engagement of T cell receptors, as well as costimulation, is required for optimal stimulation.

The H-2Ldalloreactive 2C T cell receptor (TCR) is commonly considered as being positively selected on the H-2Kbmolecule. Surprisingly, 2C TCR+CD8+single-positive T cells emerge in massive numbers in fetal thymic organ culture originating from 2C transgenic, H-2KbDb−/−(2C+KbDb−/−) but not in fetal thymic organ culture from β2-microglobulin−/−2C transgenic animals. Mature CD8+T cells are observed in newborn but not in adult 2C+KbDb−/−mice. These CD8+T cells express the α4β7integrin, which allows them to populate the intestine, a pattern of migration visualized by intrathymic injection of FITC and subsequent accrual of FITC-labeled lymphocytes in the gut. We conclude that the 2C TCR is reactive not only with H-2Ldand H-2Kb, but also with nonclassical MHC class I products to enable positive selection of 2C+T cells in the fetal and newborn thymus and to support their maintenance in the intestine.

We found that the magnesium salt of ilimaquinone, named 201-F, specifically disassembled dynamically unstable microtubules in fibroblasts and various epithelial cell lines. Unlike classical tubulin- interacting drugs such as nocodazole or colchicine which affect all classes of microtubules, 201-F did not depolymerize stable microtubules. In WIF-B–polarized hepatic cells, 201-F disrupted the Golgi complex and inhibited albumin and alpha1-antitrypsin secretion to the same extent as nocodazole. By contrast, 201-F did not impair the transport of membrane proteins to the basolateral surface, which was only affected by the total disassembly of cellular microtubules. Transcytosis of two apical membrane proteins—the alkaline phosphodiesterase B10 and dipeptidyl peptidase IV—was affected to the same extent by 201-F and nocodazole. Taken together, these results indicate that only dynamically unstable microtubules are involved in the transport of secretory proteins to the plasma membrane, and in the transcytosis of membrane proteins to the apical surface. By contrast, stable microtubules, which are not functionally affected by 201-F treatment, are involved in the transport of membrane proteins to the basolateral surface. By specifically disassembling highly dynamic microtubules, 201-F is an invaluable tool with which to study the functional specialization of stable and dynamic microtubules in living cells.

There have been reports of the clearing of sclera that has been grafted into the cornea. This clearing was studied in the rabbit by transplanting full-thickness scleral buttons or by implanting slivers of sclera, both autografts and allografts, into the cornea. Some clearing around the edges of the implanted scleral tissues was observed to take place slowly over the following months. Electron microscopic observation of the borders of either type of graft showed that the collagen fibrils of the sclera were breaking down either by unraveling into smaller fibrils similar in diameter to those of the cornea by fraying into microfibrils. Attracting blood vessels over the scleral slivers did not appear to accelerate the changes. Both active macrophages and fibroblasts could be found in the transitional area.

The study reports a reconstruction of the sacrum in STS 14 based on extrapolation from the measurements of the first two sacral vertebrae of STS 14 and of the angle formed by the anterior surfaces of their vertebral bodies. Reconstruction is based on comparisons of, and extrapolation from, sacra of Pan troglodytes, Homo sapiens, and Australopithecus afarensis. The reconstructed sacrum has an anterior sacral curvature of 39°. The two ossa coxae were also completed by mirror imaging of one side by the other. With the pelvis completely reconstructed, the pelvic dimensions for the antero-posterior (AP) diameters of the pelvic inlet, midpelvis, and pelvic outlet are 85, 68, and 69 mm and the corresponding transverse (TR) diameters are 109, 88, and 103 mm, respectively. The posterior sagittal diameters in the three pelvic planes are small compared to the anterior sagittal diameters. This analysis indicates that the STS 14 pelvis is platypelloid in the three pelvic planes; i.e., all the AP diameters are smaller than the corresponding TR diameters. This makes the STS 14 pelvis similar to that to Al 288-1, save for a less pronounced degree of platypelloidy at the inlet in the former. © 1995 Wiley-Liss, Inc.

The low rate of blinking in the rabbit as compared to the human suggests that there may be a greater contact time for drugs applied topically in the animal. However, there have been no experiments performed to examine this belief directly.A small drop containing either fluorescein or FITC Dextran was applied to the eye of a rabbit without the use of tranquilizer or anesthetic. Subsequently, the change in the fluorescence of the precorneal tear film was followed with a noninvasive fluorometer.The animal did not blink for 20-30 min. after the instillation. With either fluorophore, the tear film fluorescence remained constant after the first few minutes and fell precipitously at the first blink. Comparison with the loss kinetics in the human eye, an exponential decline with an average rate constant of 0.16 min-1, indicates that the AUC will be 3 times greater in the rabbit, and the penetration of a drug can be overestimated to the same degree from the low blink rate alone. There was a rise in the corneal fluorescence, attributable to penetration of the fluorophore, after the eye was washed at the end of the experiment. This was used to estimate the epithelial permeability which averaged 1.5 x 10(-4) cm hr for fluorescein and 1.9 x 10(-5) cm hr-1 for FITC Dextran. For most drugs, however, the epithelial permeability is sufficiently high that all the material present in the tear film will partition into the cornea in a few minutes. Considerations of contact time become irrelevant under these circumstances.

SummaryStable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.

A bench specular microscope, modified to perform as a scanning microfluorometer, was used to make simple and precise measurements of the absorption of visible light by the isolated rabbit cornea. The tissue was scanned while identical FITC dextran solutions were perfused over both the surfaces, and the measured fluorescence levels in the solutions on the two sides were compared. The alteration in level corresponds to the difference between the absorption of the cornea and that of of an equal thickness of the solution, which was determined in a separate experiment. The absorption of light at 500 nm ranged from 0-8% with a mean of 3.3% (n = 20; S.E. = 0.52). Pigmented and non-pigmented rabbits did not show any clear difference in absorption. These results suggests that light absorption is not a major source of error in anterior segment fluorophotometry.

The adverse effects of local anesthetics (LAs) on wound healing at surgical sites have been suggested, and may be related to their cytotoxicity. This study was aimed to compare the cellular toxicity of bupivacaine and lidocaine (two well-known LAs), and to explore the molecular mechanism(s).Toxicity of bupivacaine and lidocaine was assessed in cultured mouse C2C12 myoblasts by cell viability and apoptosis assays. Effects of LAs on extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) activation, which are essential for cell proliferation and survival, were evaluated by immunoblotting.Both LAs, especially bupivacaine, prevented cell growth and caused cell death in a dose-dependent manner. The half maximal inhibitory concentrations (IC(50)) for bupivacaine and lidocaine were 0.49+/-0.04 and 3.37+/-0.53 mmol/L, respectively. When applied at the same dilutions of commercially available preparations, the apoptotic effect induced by bupivacaine was more severe than that of lidocaine in C2C12 cells. Furthermore, bupivacaine significantly diminished the ERK activation, which may underlie its anti-proliferative actions. Both LAs suppressed Akt activation, which correlated with their effects on apoptosis.Our study demonstrated that, when used at the same dilutions from clinically relevant concentrations, bupivacaine is more cytotoxic than lidocaine in vitro. Anti-proliferation and cell death with concomitant apoptosis mediated by bupivacaine may offer an explanation for its adverse effects in vivo (eg slowing wound healing at the surgical sites). A less toxic, long-acting anesthetic may be needed.

A chronic sheep model for Doppler umbilical vascular analysis with indwelling probes was used for the investigation of umbilical vein velocity waveforms. Maternal aortic occlusions produced a delayed drop but never eliminated umbilical vein velocity. With umbilical cord occlusion, there was immediate umbilical vein waveform response. Sudden cord occlusion and release showed reactive venous overflow velocity at the beginning and end of occlusion. Total cord occlusion produced zero velocity, but within seconds, the flow velocity was seen despite persistent occlusion in 65% of the cases. A characteristic undulating venous waveform pattern synchronous with fetal heart rate in over half of the cases was often observed. Reduction of blood flow by uteroplacental insufficiency may be differentiated from umbilical cord occlusion.

Severe fetal distress was produced in 16 fetal dogs by successive and/or prolonged occlusion of the maternal abdominal aorta in an acute surgical preparation, and was characterized in fetal arterial blood of 7.06 for pH, 10 mm Hg for O2, 127 mm Hg for CO2, and fetal heart rate decelerations. Five fetuses recovered spontaneously in utero and delivered normally; 3 needed maternal oxygenation before delivery; 3 suddenly died in utero; 5 fetuses were delivered while still alive in utero when the tpH reached 6.85, and eventually expired. Intrauterine recovery and resuscitation was effective in some fetuses.

Antibodies against cytochrome P-450 are found in some children with autoimmune hepatitis (antiliver/kidney microsome 1) and in patients with ticrynafen hepatitis (antiliver/kidney microsome 2). For an immune reaction against cytochrome P-450 to possibly destroy the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of hepatocytes. In a first series of experiments, plasma membranes were prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. After vortexing, beads were coated with a very pure plasma membrane fraction. Microsomal contamination, judged from the specific activities of glucose-6-phosphatase or NADH-cytochrome c reductase, was less than 1%. Nevertheless, the specific content (per milligram of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25%, and ethoxycoumarin deethylase 11%. Immunoblots showed the presence of cytochromes P-450 UT-A, UT-H, PB-B, ISF-G and PCN-E, the last three isoenzymes being inducible by, respectively, phenobarbital, 3-methylcholanthrene and dexamethasone. In a second series of experiments, nonpermeabilized isolated hepatocytes from untreated rats were incubated with anticytochrome P-450 antibodies. Immunofluorescence and immunoperoxidase staining confirmed the presence of cytochromes P-450 UT-A, PB-B and ISF-G on the membrane. In a last series of experiments, human antiliver-kidney microsomal 1 antibodies were found to react specifically with rat liver plasma membrane cytochrome P-450 UT-H (IID subfamily). We conclude that several cytochrome P-450 isoenzymes are present, active and inducible on the plasma membrane surface of hepatocytes. It is therefore conceivable that immunization against plasma membrane cytochrome P-450 might lead to the immunological destruction of hepatocytes in some patients.

Modification of proteins by ubiquitin plays a major role in a broad array of biological processes. Reversal of this process through deubiquitylation likely represents an important regulatory step in the maintenance of cellular homeostasis. However, the biological functions of deubiquitylating enzymes still remain poorly characterized. To investigate the biological role of the herpes virus-associated ubiquitin-specific protease HAUSP/USP7, we have generated stably transfected cells carrying inducible shRNA expression plasmids. USP7 mRNA and protein were strongly down-regulated 48-72 h after shRNA induction. We used a selected clone to compare whole-cell proteomes by 2D-SDS-PAGE before and after knockdown of USP7. Alterations in 36 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of the replication machinery, DNA/RNA binding proteins, enzymes involved in apoptosis and metabolism were found to be down-regulated upon USP7 removal, representing proteins that are either more rapidly turned over or synthesized less efficiently in the absence of USP7-mediated deubiquitylation. Alix/HP95, a protein implicated in endosomal organization and virus budding, was confirmed by immunoblotting to become down-regulated when USP7 levels were reduced. Our results extend the current list of USP7-dependent biological processes and suggest a role for this enzyme not only in transcriptional regulation but also in DNA replication, apoptosis, and possibly endosomal organization.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.

The effects of the behavioural context on the properties of slow and fast phases of the horizontal optokinetic nystagmus (OKN) and on the electromyographic neck response (EMG) were investigated in head-fixed pigeons. Responses in two situations were compared: (i) animals were hung in a harness (‘resting' condition); (ii) animals in harness were subjected to a frontal airflow that provoked a flight posture (‘flying' condition). During optokinetic stimuli the neck muscles responded in synchrony and in synergy with the eye nystagmus in both the ‘resting’ and the ‘flying’ conditions. In the ‘resting’ condition the neck activity was essentially correlated to the slow phase velocity of the eyes (eye SPV) whereas in the ‘flying’ condition, the neck response was also correlated to the eye position. The neck response was independent of the retinal slip velocity during the OKN. The velocity of the slow and fast phases of the OKN was not modified by flight. However, the ‘flying’ condition provoked an increase of the neck response by augmenting both its velocity gain (neck EMG/eye SPV) and its position gain (neck EMG/eye position). These results show that although an optokinetic stimulation results in synchronised eye and head motor commands in head-fixed pigeons, only the head motor command is modified by the behavioural context (‘flying' vs. ‘resting’). This strategy could help pigeons in reorienting their gaze during the flight.

Thymic selection is controlled by the interaction between TCR and MHC/peptide. Strength and quality of the signal determine whether thymocytes are selected or deleted. The factors that contribute to this signal remain poorly defined. Here we show that fetal thymic organ cultures (FTOCs) derived from OT-I transgenic mice (the OT-I TCR is restricted by Kb-SIINFEKL) on a KbDb−/− background support positive selection, but only when provided with soluble H-2Kb-SIINFEKL complexes. Selection of CD8 T cells is independent of the valency of the ligand or its capability to coengage CD8 molecules. Both CD8αα and CD8αβ T cells are selected by H-2Kb-SIINFEKL, but only CD8αβ cells are capable of releasing IFN-γ in response to the same ligand. The α4β7 integrin is up-regulated on postselection thymocytes from FTOCs. After adoptive transfer, FTOC-derived OT-I CD8 T cells divide in response to the agonist peptide SIINFEKL. These results establish that CD8 T cells responsive to their nominal peptide-Ag can be generated in FTOC supplemented with soluble MHC class I molecules equipped with the same peptide.

Arthritis in the K/BxN mouse model results from pathogenic immunoglobulins (Igs) that recognize the ubiquitous cytoplasmic enzyme glucose-6-phosphate isomerase (GPI). But how is a joint-specific disease of autoimmune and inflammatory nature induced by systemic self-reactivity? No unusual amounts or sequence, splice or modification variants of GPI expression were found in joints. Instead, immunohistological examination revealed the accumulation of extracellular GPI on the lining of the normal articular cavity, most visibly along the cartilage surface. In arthritic mice, these GPI deposits were amplified and localized with IgG and C3 complement. Similar deposits were found in human arthritic joints. We propose that GPI-anti-GPI complexes on articular surfaces initiate an inflammatory cascade via the alternative complement pathway, which is unbridled because the cartilage surface lacks the usual cellular inhibitors. This may constitute a generic scenario of arthritogenesis, in which extra-articular proteins coat the cartilage or joint extracellular matrix.

We showed in the Sprague Dawley rat that the principal ascending cardiac lymphatic is similar in location to that of man and dog and can be readily visualized by injection of a small amount of T1824 blue dye (Evans) into the apex of the left ventricle. We also showed that it is possible to ligate and thereby obstruct this principal cardiac lymphatic near its entry beneath the left atrial appendage. This latter technique may be useful for studying in a small relatively inexpensive laboratory animal the effects of blocking cardiac lymph drainage on inflammatory and infectious processes implicated in myocardial and coronary artery disease.

To study the role of tears in the death of keratocytes after epithelium removal in the mouse cornea.In anesthetized mice, an approximately 1-mm circle of epithelium was removed from the center of the cornea, exposing the underlying stroma. In one group of animals, access of tears to the bare stroma was allowed-in vivo, by closing the eyelids, or ex vivo, by dropping tears from another animal onto the denuded stroma of an enucleated eyeball. In another group, tear access was denied-in vivo, by bathing the cornea continuously in saline or by keeping the lids open, or ex vivo, by rinsing the denuded cornea before incubating the enucleated eyeball. In a separate group, corneal epithelial debris from another mouse was placed on the bare stroma of an enucleated eyeball. The corneas were isolated, stained with a fluorescent nuclear dye, and observed en face in a wholemount preparation under a fluorescence microscope, to evaluate the distribution of intact nuclei across the entire depth of the stroma.Between 1.5 and 2 hours after exposure to tears, the nuclei of the anterior keratocytes under the area of epithelial debridement invariably degenerated. When they had been protected from the tears, however, no degeneration was observed. Epithelial debris applied on the bare stroma had no effect on the underlying keratocytes.Factors in tear fluid trigger keratocyte loss after removal of the epithelium in the mouse cornea.

The mammalian corneal stroma has some intriguing features related to its lamellar structure. OPTICAL LAMINATION: Stromal sections under the polarizing microscope show birefringent bands that are about 5 times less numerous than the collagenous lamellae seen in transmission electron microscopy (TEM). Scattered light reveals similar laminae in the intact fresh tissue. COHESION: Very thin sections of stroma do not fall apart under water, indicating the presence of a structural framework that holds the collagen fibrils together. SHEAR DISTORTION: The stroma shears very readily in its plane; the shear amplitude increases and then decreases as the tissue hydrates. This suggests that the structural framework is coiled at physiological hydration and can expand about 10 times. FOLDING: When the stroma swells, the lamellae are thrown into waves that lie both in the plane of and perpendicular to its surface. It is difficult to understand how the latter are packed.

Type I phosphodiesterases are differently expressed by different cell types. Three members have been identified, PC-1, B10 and autotaxin. They are between 40 and 50% identical at the amino acid sequence level. Hepatocytes express both B10 and PC-1 at their plasma membrane. However, B10 is exclusively expressed at the apical pole whereas PC-1 is located at the basolateral pole. Studies of the biosynthetic route of B10 in hepatocytes shows that B10 is first transported to the basolateral surface and secondarily reaches the basolateral surface. The transcytotic step between the basolateral and apical surface occurs through a tubular endosomal compartment identical to the transcytotic compartment of the polymeric IgA receptor. Transfected in the polarized cell lines MDCK and Caco-2 of renal and intestinal origin, B10 and PC-1 are expressed at the apical and basolateral poles respectively, as in hepatocytes. However, the biosynthetic transport of B10 occurs directly in MDCK cells and both directly and by transcytosis in Caco-2 cells. Truncation of the cytoplasmic domain of PC-1 generates an apical protein indicating that the basolateral signal of PC-1 is likely to be in the cytoplasmic domain. The nature of the apical targeting signal of B10 is under investigation.

Protein sorting in epithelial cells is the major event that drive the onset and the maintenance of the functional cell polarity. A lot of interdependent steps are involved in protein sorting and targeting. Recent data describing the last results obtained in this field will be reviewed in the first part of this article. Molecular signals harbored by proteins to specify their destination are thought to be the driven force to sort given protein in a given pathway. The basolateral targeting signals so far identified are known for several years and are of the same nature, whereas apical targeting signals are still discussed and are of diverse molecular nature. Dipeptidyl peptidase IV (DPP IV/CD26) targeting signals have not been described so far and it will be interesting to study these signals, since the protein reach the apical membrane of epithelial cells through different pathways that strongly depend on the cell type considered. These different pathways result in DPP IV membrane localizations that may explain the multifunctional properties of DPP IV such as enzymatic digestion, interaction with extracellular matrix proteins, capture and transport of circulating proteins. We have undertaken the study of DPP IV molecular targeting signals and we will described here how the transmembrane domain and the glycosylation of the ectodomain may be involved in DPP IV apical targeting, with a special reference to the cell type specificity.

We previously reported that retroviral vectors displaying epidermal growth factor (EGF) as part of a chimeric envelope glycoprotein are sequestered upon binding to EGF receptor (EGFR)-positive target cells, leading to loss of infectivity. In the current study, we have displayed stem cell factor (SCF) on beta-galactosidase-transducing ecotropic and amphotropic retroviral vector particles as a factor Xa protease-cleavable N-terminal extension of the envelope glycoprotein. Viral incorporation of the SCF chimeric envelopes was demonstrated by immunoblotting of pelleted virions and their specific attachment to Kit receptors was demonstrated by flow cytometry. Gene transfer studies showed that when SCF was displayed on an amphotropic envelope, the infectivity of the SCF-displaying vectors was selectively inhibited on Kit-expressing cells, but could be restored by adding soluble SCF to block the Kit receptors or by cleaving the displayed SCF domain from the vector particles with factor Xa protease. The host range properties of EGF-displaying and SCF-displaying vectors were then compared in cell mixing experiments. When EGFR-positive cancer cells and Kit-positive hematopoietic cells were mixed and exposed to the different engineered vector particles, the cancer cells were selectively transduced by the SCF-displaying vector and the hematopoietic cells were selectively transduced by the EGF-displaying vector. Retroviral display of polypeptide growth factors can therefore provide the basis for a novel inverse targeting strategy with potential use for selective transduction of hematopoietic or nonhematopoietic cells (eg, cancer cells) in a mixed cell population.

The same quantity of fluorescein labelled dextran was injected into the vitreous humor of 5 rabbits; in one eye, dissolved in 10 microliters solution and in the other, 100 microliters. The level of fluorescence in the two eyes was compared a few days later and found to average only 12% less with the larger volume. It is concluded that regurgitation through the needle hole can be ignored in spite of the very high intraocular pressure created by the 100 microliter injection.

SummaryFoxp3 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 T cell 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 in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells 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.

OBJECTIVE: To design and evaluate an instrument for the objective measurement of haze in rabbits after myopic PRK. METHODS: A circular fluorescent lamp bulb projected light onto the cornea through a circular collimating aperture covered with an orange filter. The image was collected on a centrally mounted CCD camera and the profile of the haze circle along its horizontal diameter determined. RESULTS: Haze circles were observed and quantitated. In the first weeks, they were donut-shaped, frequently with a central bright area, but later they became square-topped. The instrument reproduced measurements with an error of a few percentage points and showed good long-term stability. [J Refract Surg. 1996;12:412-416.]

Hepatocytoma (HPCT) hybrid cells were obtained by fusion of cultured rat hepatocytes with Fao Reuber hepatoma cells H35 by polyethylene glycol treatment. Surviving cells were cloned in HAT (hypoxanthine-aminopterine-thymidine)/ouabain medium and propagated in cell lines over 80 passages. Morphological criteria were chosen to allow differentiation of the clones into two types of cells: 1) Type I cells which formed irregular cell layers, lacked contact inhibition and resembled the parental Fao hepatoma cells and 2) type II cells, which proliferated in monolayer cultures, exhibited contact inhibition during growth in culture plates and formed bile canaliculi thereby resembling cultured hepatocytes by phenotype. Bile canaliculi were absent in type I clones and Fao cells. One particular type II clone 1E3 was studied in detail. These cells formed bile canaliculi sealed by tight junctions and were comparably polarized as cultured hepatocytes. They expressed canalicular antigen B10, canalicular aminopeptidase N, and even secreted the fluorescent bile acid derivative NBD-cholate into the canalicular lumen. This type of HPCT cells lacked malignancy by tests in vivo and in vitro, and contained 110 +/- 5 chromosomes. The cells were considered to represent an immortalized hepatocyte-like cell line.

Goblet cell impressions free from epithelial cells could be collected more consistently from the rabbit conjunctiva by creating a space between the applied cellulose acetate filter and the tissue surface. Successive impressions taken over the same region of the conjunctiva could not be matched. When tight control of the area being sampled was established, the impressions left by cells on successive filters varied widely in intensity.

The ratio pelvic/abdominal cavity is 6.9% in samples of nonhuman mammals and nonhuman primates, and rises to approximately 30% in humans. This relative reduction of the abdomen and increase of the pelvis is associated with a partial or total shift of some organs from the abdomen to the pelvis: rectosigmoid colon, bladder, and genital organs, which are mostly abdominal in quadrupeds and are mostly pelvic in humans. Pregnancy, always abdominal in nonhumans, is pelvic during the first trimester and becomes abdominal later on in humans. Near term the pregnancy expands easily in nonhumans in view of relatively small fetus and relatively large abdominal cavity. But, for the opposite reasons (large fetus, small abdomen), the human pregnancy is limited space-wise during its abdominal expansion. Unlike that of nonhumans, human pregnancy is faced with multiple problems. These include: 1) "squeezing" between the anterior abdominal wall and the lordosis of the lumbar spine; 2) compression of the aortocaval vessels; and 3) forward expansion of the abdomen resulting in reorientation of the trunk during erect posture as the pregnant woman approaches term. All these conditions are responsible for numerous pathological entities that occur during human pregnancy and are almost unknown in nonhuman mammals.

In hepatocytes, all newly synthesized plasma membrane (PM) proteins so far studied arrive first at the basolateral domain; apically destined proteins are subsequently endocytosed and sorted to the apical domain via transcytosis. A mechanism for the sorting of newly synthesized glycophosphatidylinositol (GPI)-linked proteins has been proposed whereby they associate in lipid microdomains in the trans-Golgi network and then arrive at the apical domain directly. Such a mechanism poses a potential exception to the hepatocyte rule. We have used pulse-chase techniques in conjunction with subcellular fractionation to compare the trafficking of 5' nucleotidase (5NT), an endogenous GPI-anchored protein of hepatocytes, with two transmembrane proteins. Using a one- step fractionation technique to separate a highly enriched fraction of Golgi-derived membranes from ER and PM, we find that both 5NT and the polymeric IgA receptor (pIgAR) traverse the ER and Golgi apparatus with high efficiency. Using a method that resolves PM vesicles derived from the apical and basolateral domains, we find that 5NT first appears at the basolateral domain as early as 30 min of chase. However the subsequent redistribution to the apical domain requires > 3.5 h of chase to reach steady state. This rate of transcytosis is much slower than that observed for dipeptidylpeptidase IV, an apical protein anchored via a single transmembrane domain. We propose that the slow rate of transcytosis is related to the fact that GPI-linked proteins are excluded from clathrin-coated pits/vesicles, and instead must be endocytosed via a slower nonclathrin pathway.