Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Development of a self-limiting model of methotrexate-induced mucositis reinforces butyrate as a potential therapy.

Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. The protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005-50 µg/mL) folinic acid. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.

[Hypokalemia due to a liquorice-containing thirst-quencher]. / Pseudohyperaldosteronisme door Arabische dorstlesser.

BACKGROUND: Liquorice consumption can cause pseudohyperaldosteronism and potentially lead to life-threatening complications. Besides correcting hypokalemia and hypertension, finding the triggering factor for pseudohyperaldosteronism is essential to prevent recurrence. CASE DESCRIPTION: A 68-year-old Syrian man presented in the Emergency Department with complaints of fatigue, weakness and exercise-related shortness of breath. Blood tests revealed severe hypokalemia for which suppletion and cardiac rhythm surveillance was necessary. Talking to the patient's son, it occurred that our patient drank copious amounts of Erk Sous, a thirst-quenching drink made from liquorice. The diagnosis pseudohyperaldosteronismwas confirmed by a high level of glycyrrhetinic acid in the patient's urine. After correction of the hypokalemia, our patient recovered successfully. CONCLUSION: Erk Sous is a thirst-quenching drink that can cause pseudohyperaldosteronism. The drink is popular in the Middle East during summer and Ramadan. If a patient from the Middle East presents with hypokalemia and/or hypertension, ask for consumption of Erk Sous.

Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition.

Proteins of the Rho family regulate cytoskeletal rearrangements in response to receptor stimulation and are involved in the establishment and maintenance of epithelial cell morphology. We recently showed that Rac is able to downregulate Rho activity and that the reciprocal balance between Rac and Rho activity is a major determinant of cellular morphology and motility in NIH3T3 fibroblasts. Using biochemical pull-down assays, we analyzed the effect of transient and sustained oncogenic Ras signaling on the activation state of Rac and Rho in epithelial MDCK cells. In contrast to the activation of Rac by growth factor-induced Ras signaling, we found that sustained signaling by oncogenic RasV12 permanently downregulates Rac activity, which leads to upregulation of Rho activity and epithelial-mesenchymal transition. Oncogenic Ras decreases Rac activity through sustained Raf/MAP kinase signaling, which causes transcriptional downregulation of Tiam1, an activator of Rac in epithelial cells. Reconstitution of Rac activity by expression of Tiam1 or RacV12 leads to downregulation of Rho activity and restores an epithelial phenotype in mesenchymal RasV12- or RafCAAX-transformed cells. The present data reveal a novel mechanism by which oncogenic Ras is able to interfere with the balance between Rac and Rho activity to achieve morphological transformation of epithelial cells.

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.

Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin-mediated cell-cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin-mediated cell-cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin-mediated cell- cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase- PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin-mediated cell- cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.

Inhibition of invasion of epithelial cells by Tiam1-Rac signaling.

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.

Recommendations for the successful control of a large outbreak of vancomycin-resistant Enterococcus faecium in a non-endemic hospital setting.

BACKGROUND: A large outbreak of three epidemic vancomycin-resistant Enterococcus faecium (VRE) clones affected the study hospital for almost two years. AIM: To describe the strategy to successfully control this outbreak and eradicate VRE from the study hospital. METHODS: Infection control interventions started after detection of VRE in three patients. Hospital-wide surveillance was started after ongoing transmission despite isolation precautions, cleaning and contact tracing. Hygiene education and discipline were enhanced. Despite these interventions, additional measures were required to control the outbreak, such as ward disinfection with hydrogen peroxide vapour and the introduction of a VRE quarantine ward. Ultimately, ciprofloxacin prophylaxis for haematological patients on chemotherapy was abandoned. FINDINGS: Over a 22-month period, 242 VRE carriers were identified. Of these, 128 (53%) patients were detected by hospital-wide surveillance alone. Three epidemic clones were detected: ST494-vanA (N = 160), ST78-vanA (N = 23) and ST117-vanB (N = 32). In total, 5614 possible contacts were identified. VRE transmission occurred on 13 out of 23 wards. VRE was cultured from clinical specimens in 22 patients (seven with bacteraemia). Since January 2014, no further transmission of these VRE clones has been observed. CONCLUSION: Infection control measures according to international guidelines were insufficient to expose the outbreak to its full extent and control it. Its full extent only became apparent after sustained hospital-wide screening. Successful control of this hospital-wide VRE outbreak was feasible, but required great effort. Final containment and eradication of the epidemic clones was achieved by environmental decontamination with hydrogen peroxide vapour, strict isolation precautions, a VRE quarantine ward and antimicrobial stewardship.

The prenatal development of the human orbit.

During the 1970s, as part of his work for a doctor's thesis in which he described the development of the human orbit in great detail, the first author established the largest anatomical collection of embryonic and fetal orbits ever. Unfortunately, he died before the thesis could be finished. The thousands of sections have now been scanned at high resolution and made publicly available on the Internet at www.visible-orbit.org; 3-D reconstruction software is being developed. The Discussion and part of the 'Methods' section of this thesis are published in translation in this article. The conclusions of the first author at the time read as follows: (1) initially, the developing orbit is vaguely indicated by condensations in the mesenchymal connective tissue area; (2) in this connective tissue area, chondral, osseous and muscular structures develop and grow until, in the fully developed stage, the orbital content is surrounded by bony surfaces with a thin layer of connective tissue as periosteum, and by a muscle fragment; (3) the embryonic and early fetal phase, during which one can only speak of a 'regio orbitalis,' is followed by a period in which we can speak of a primordial orbit; (4) the phase of the primordial orbit extends until after birth; (5) the surface area of all orbital walls increases more or less linearly; (6) the 'musculus orbitalis Mülleri' occupies a special place in the orbital wall; (7) the so-called 'regio craniolateralis' is the primordium, which, in the fully developed stage, is occupied by the thick intersection of the frontolateral and the horizontal part of the frontal bone; (8) in the frontal plane, the shape of the primordial orbit, as well as that of the fully developed orbit, is more or less round; (9) the prenatal development of an eye socket is a complex event, characterized by changes in composition, shape and size of the orbital wall; and (10) the orbit can only be denoted by the term "eye socket" when it is fully developed. At the end of the thesis, he also presented the following postulates: (1) in the prenatal orbit, the development of the so-called 'periorbita' is at the forefront; (2) the mutual rotation of the orbital axes and the frontalization of the eyes from approx. 180 degrees in the early prenatal stages to approx. 50 degrees in adulthood do not seem to be caused by mechanical influences of the surrounding tissue; (3) the pterygopalatine fossa and the 'cavum cerebri' are not part of the orbit at any developmental stage; (4) in the prenatal skull, the inferior nasal concha, which forms part of the maxilla in the fully developed skull, is part of the 'capsula nasalis'; and (5) in order to achieve normal development of the eye socket in microphthalmus and anophthalmus, the normal orbital content should be restored.

Substrain and light regime effects on integrated anxiety-related behavioral z-scores in male C57BL/6 mice-Hypomagnesaemia has only a small effect on avoidance behavior.

Magnesium (Mg) has been described to possess an anxiolytic function, but a number of studies present inconsistent results on this matter. In this study the effect of Mg deficiency on anxiety-related behavior, brain and blood plasma Mg in young adult male C57BL/6JOlaHsd and C57BL/6NCrl mice was studied. The animals were put on a control or Mg deficient diet from day 0 and significant hypomagnesaemia was evident from day 12 onwards in the test animals. Housing and test conditions were under either conventional light regime (white light behavioral test conditions) or reverse light regime (red light behavioral test conditions). The animals were tested in three tests for unconditioned anxiety: the modified Hole Board (day 14), the light-dark test (day 21) and the elevated plus maze (day 28). Overall integrated behavioral z-scores were calculated over these three behavioral tests. Mg showed a structure dependent distribution at the level of the brain, that differed between C57BL/6 substrain and light regime (conventional versus reverse), respectively. Likewise, total brain Mg did differ between substrain and light regime, but was not affected by the diet. Animals on the Mg deficient diet housed under conventional light regime had a higher final (day 28) blood plasma corticosterone level as compared to controls. Animals housed under reverse light regime exhibited no diet effect of plasma corticosterone levels. The significant hypomagnesaemia at blood plasma level resulted in an effect of Mg deficiency on avoidance, but not overall anxiety-related behavior. Significant differences regarding avoidance behavior were found between the two substrains and light regimes, respectively.

Mapping of avirulence genes in Phytophthora infestans with amplified fragment length polymorphism markers selected by bulked segregant analysis.

In this study we investigated the genetic control of avirulence in the diploid oomycete pathogen Phytophthora infestans, the causal agent of late blight on potato. The dominant avirulence (Avr) genes matched six race-specific resistance genes introgressed in potato from a wild Solanum species. AFLP markers linked to Avr genes were selected by bulked segregant analysis and used to construct two high-density linkage maps, one containing Avr4 (located on linkage group A2-a) and the other containing a cluster of three tightly linked genes, Avr3, Avr10, and Avr11 (located on linkage group VIII). Bulked segregant analysis also resulted in a marker linked to Avr1 and this allowed positioning of Avr1 on linkage group IV. No bulked segregant analysis was performed for Avr2, but linkage to a set of random markers placed Avr2 on linkage group VI. Of the six Avr genes, five were located on the most distal part of the linkage group, possibly close to the telomere. The high-density mapping was initiated to facilitate future positional cloning of P. infestans Avr genes.

The involvement of glutamate-gated channels in negative feedback from horizontal cells to cones.

Photoreceptors are the light sensitive cells in the retina. They project to horizontal cells and bipolar cells via a glutamatergic feed forward pathway. Horizontal cells are strongly electrically coupled and integrate in that way the input from the photoreceptors. Horizontal cells feedback to cones negatively. The combined signal from the photoreceptors and the horizontal cells is sent to the bipolar cells. The feedback pathway from horizontal cells to cones is thought to form the basis for the center/surround organization of bipolar cells. The nature of the feedback pathway is an issue of intense debate. It was thought for a long time that this feedback pathway was GABAergic, because cones have GABA-receptors and horizontal cells release GABA via a GABA-transporter working in the reversed direction. However, recently we showed in goldfish that horizontal cells feed back to cones via an alternative mechanism. In goldfish, negative feedback from horizontal cells to cones shifts the calcium current of the cone to more negative potentials. This feedback pathway is independent of GABA, since feedback cannot be blocked by either saturating concentrations of PTX, the GABA-transporter blocker SKF89976A, or application of GABA. The mechanism of negative feedback from horizontal cells to cones involves hemichannels located at the tips of the invaginating horizontal cells, just opposite to the calcium channels of the cones. Current flowing through these hemichannels changes the extracellular potential deep in the synaptic cleft and in that way modulates the calcium current of the cones. Such a modulation of the extracellular potential is called ephaptic. If negative feedback from horizontal cells to cones is indeed ephaptic, other channels present in the synapse should also be able to act as a current source, i.e., should also be able to change the output of the cone. We showed that glutamate-gated channels present at the tips of the horizontal cell dendrites can also mediate feedback responses. Surprisingly, although the glutamate-gated conductance of the horizontal cells is eight times the hemichannel conductance, glutamate-gated channels are not the major current source in negative feedback from horizontal cells to cones. In this chapter we present evidence that this is due to the more focal localization of the hemichannels, compared to a diffuse and extrasynaptic localization of the glutamate-gated channels.

Severe hypokalaemic paralysis and rhabdomyolysis due to ingestion of liquorice.

Chronic ingestion of liquorice induces a syndrome with findings similar to those in primary hyperaldosteronism. We describe a patient who, with a plasma K+ of 1.8 mmol/l, showed a paralysis and severe rhabdomyolysis after the habitual consumption of natural liquorice. Liquorice has become widely available as a flavouring agent in foods and drugs. It is important for physicians to keep liquorice consumption in mind as a cause for hypokalaemic paralysis and rhabdomyolysis.

Chromosomal deletion in isolates of Phytophthora infestans correlates with virulence on R3, R10, and R11 potato lines.

In Phytophthora infestans, a cluster of three dominant avirulence genes is located on the distal part of linkage group VIII. In a mapping population from a cross between two Dutch field isolates, probe M5.1, derived from an amplified fragment length polymorphism (AFLP) marker linked to the Avr3-Avr10-Avr11 cluster, hybridized only to DNA from the parent and F1 progeny that is avirulent on potato lines carrying the R3, R10, and R11 resistance gene. In the virulent parent and the virulent progeny, no M5.1 homologue was detected, demonstrating a deletion on that part of linkage group VIII. P. infestans is diploid, so the avirulent strains must be hemizygous for the region concerned. A similar situation was found in another mapping population from two Mexican strains. The deletion was also found to occur in many field isolates. In a large set of unique isolates collected in The Netherlands from 1980 to 1991, 37% had no M5.1 homologue and the deletion correlated strongly with gain of virulence on potato lines carrying R3, R10, and R11. Also, in some old isolates that belong to a single clonal lineage (US-1) and are thus highly homogenous, deletions at the M5.1 locus were detected, indicating that this region is unstable.

Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato.

The plant pathogen Botrytis cinerea can infect undamaged plant tissue directly by penetration of the cuticle. This penetration has been suggested to be enzyme-mediated, and an important role for cutinase in the infection process has been proposed. In this study the expression of the cutinase encoding gene cutA of B. cinerea was analyzed using a cutA promoter-GUS reporter gene fusion. Transformants containing the fusion construct were examined for GUS expression on gerbera flowers and tomato fruits. High GUS activity was detected from the onset of conidial germination and during penetration into epidermal cells, indicating that cutA is expressed during the early stages of infection. To determine the biological relevance of cutinase A for successful penetration, cutinase A-deficient mutants were constructed by gene disruption. Pathogenicity of two transformants lacking a functional cutA gene was studied on gerbera flowers and tomato fruits. Their ability to penetrate and cause symptoms was unaltered compared to the wild-type strain. These results exclude an important role for cutinase A during direct penetration of host tissue by B. cinerea.

tef1, a Phytophthora infestans gene encoding translation elongation factor 1alpha.

From a set of Phytophthora infestans cDNA clones randomly selected from a potato-P. infestans interaction cDNA library, three out of 22 appeared to correspond to a gene encoding translation elongation factor 1alpha. The gene, called tef1, is a single copy gene in P. infestans. During the life cycle of P. infestans, tef1 is expressed in all developmental stages. Alignment and phylogeny analysis based on EF-1alpha proteins from several taxonomic groups, including fungi, slime molds, algae, higher plants and archeabacteria, support the view that oomycetes evolved completely independently from the true fungi. In the phylogenetic tree, P. infestans EF-1alpha forms one branch with EF-1alpha from the unicellular alga Cyanophora paradoxa, an organism belonging to a taxonomic group that occupies a key position in the evolution of plastids.

Localization of the AMPA subunit GluR2 in the outer plexiform layer of goldfish retina.

L-glutamate, the photoreceptor neurotransmitter, depolarizes horizontal cells and OFF bipolar cells by ionotropic AMPA-glutamate receptors. The AMPA-receptor subunit (GluR4) is localized to dendrites of OFF bipolar cells in goldfish retina. Here, we used immunohistochemical techniques to identify AMPA-receptor subunits on horizontal cell dendrites. A monoclonal antibody against rat GluR2, with high sequence homology to the recently cloned goldfish GluR2a receptor, was used for light- and electron-microscopical immunocytochemistry. Light- and dark-adapted retinas were analyzed, with no major difference in results. GluR2-immunoreactivity (IR) was restricted to a narrow band in the outer plexiform layer, in which it appeared as bright dome-shaped structures amidst numerous puncta. At the ultrastructural level, GluR2-IR was found in horizontal cell dendrites that invaginated cones and rods. Dendrites of OFF bipolar cells were not labeled. GluR2-IR was present mostly in horizontal cell dendrites that were the lateral elements of the triad, rather than in dendrites that were the central elements. In light-adapted retinas, GluR2-IR was found in many horizontal cell spinules. GluR2-IR was observed, on occasion, in a mixed rod/cone (Mb) ON bipolar cell process that innervated rod spherules. Verification of the Mb ON bipolar cell was made by protein kinase C and metabotropic mGluR1alpha immunolabeling. The presence of GluR2-IR in lateral elements suggests that lateral horizontal cell dendrites are postsynaptic to cones rather than only sites of feedback inhibition. All horizontal cell types express the GluR2 subunit, uniquely differentiating themselves from OFF bipolar cells that express the GluR4 subunit. This differentiation most likely has a major influence on the glutamate pharmacology and response kinetics of these cell types to glutamate.

Rho family proteins in cell adhesion and cell migration.

Cell migration and the regulation of cadherin-mediated homotypic cell-cell interactions are critical events during development, morphogenesis and wound healing. Aberrations in signalling pathways involved in the regulation of cell migration and cadherin-mediated cell-cell adhesion contribute to tumour invasion and metastasis. The rho family proteins, including cdc42, rac1 and rhoA, regulate signalling pathways that mediate the distinct actin cytoskeleton changes required for both cellular motility and cell-cell adhesion. Recent studies indicate that rac directly influences rho activity at the GTPase level and that the reciprocal balance between rac and rho activity can determine epithelial or mesenchymal cell morphology and migratory behaviour of epithelial (tumour) cells.

Potassium ferricyanide: a stabilizer for horseradish peroxidase.

Sodium nitroprusside has been used as a stabilizing agent for HRP histochemistry since 1919. However, it is known to have toxic effects orally, intravenously, and subcutaneously. In order to avoid the use of sodium nitroprusside as a stabilizer in HRP histochemistry, we have tested other chemically related compounds to stabilize the reaction product equally well. We will show that potassium ferricyanide is an excellent stabilizer of the chromogen reaction product. In addition, the reaction product remains stable without noticeable changes over a period of several months. As it is far less toxic than sodium nitroprusside, it should be the stabilizer of choice, especially in those laboratories where the histochemical HRP reaction is used frequently.

Ultrastructural identification of trigeminal nerve endings in the rat cornea and iris.

Trigeminal nerve terminals in the rat cornea and iris were ultrastructurally identified using anterograde tracing with Phaseolus vulgaris-leukoagglutinin (PHA-L). Electron microscopic immunohistochemistry was used to demonstrate the presence and localization of calcitonin gene-related peptide (CGRP) in cornea and iris. In the cornea and iris, nerve fibers were labelled with PHA-L throughout the stroma. Labelling was most obvious within varicosities, densely packed with mainly clear and a few granular vesicles and containing dark mitochondria. Numerous fibers in the stroma of cornea and iris were CGRP-positive. CGRP-positive staining was most intense within varicosities, containing mainly clear and incidentally granular vesicles and dark mitochondria, similar to the structures labelled with PHA-L. CGRP-positive varicosities packed with mainly clear and few granular vesicles also were demonstrated in fibers adjacent to the sphincter and dilator muscles of the iris. In the corneal epithelium, small terminals containing vesicles were CGRP-positive. Trigeminal nerve fibers innervating the rat cornea and iris contained numerous varicosities packed with vesicles. These areas are CGRP-positive, so it can be implied that CGRP is released from these varicosities as a response to triggering impulses. This agrees with the hypothesis that in addition to their afferent function, sensory fibers also exert an efferent modulating function.