Molecular underpinnings of enzalutamide resistance

Prostate cancer (PCa) is among the most common adult malignancies, and the second leading cause of cancer-related death in men. As PCa is hormone dependent, blockade of the androgen receptor (AR) signaling is an effective therapeutic strategy for men with advanced metastatic disease. The discovery of enzalutamide, a compound that effectively blocks the AR axis and its clinical application has led to a significant improvement in survival time. However, the effect of enzalutamide is not permanent, and resistance to treatment ultimately leads to development of lethal disease, for which there currently is no cure. This review will focus on the molecular underpinnings of enzalutamide resistance, bridging the gap between the preclinical and clinical research on novel therapeutic strategies for combating this lethal stage of prostate cancer.

Alpha particle spectroscopy using FNTD and SIM super-resolution microscopy.

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry.

Fast-spiking Parvalbumin Interneurons are Frequently Myelinated in the Cerebral Cortex of Mice and Humans.

Myelination, the insulating ensheathment of axons by oligodendrocytes, is thought to both optimize signal propagation and provide metabolic support. Despite the well-established physiological importance of myelination to neuronal function, relatively little is known about the myelination of GABAergic interneurons in the cerebral cortex. Here, we report that a large fraction of myelin in mouse cerebral cortex ensheaths GABAergic interneurons, reaching up to 80% in hippocampal subregions. Moreover, we find that a very high proportion of neocortical and hippocampal parvalbumin (PV) interneurons exhibit axonal myelination. Using a combination of intracellular recordings and biocytin labeling of ex vivo human neocortex, we also confirm that axons of fast-spiking PV interneurons are extensively myelinated in the human brain. PV interneuron myelination in both mice and humans exhibits a stereotyped topography with a bias towards proximal axonal segments and relatively short internodes (~27 µm) interspersed with branch points. Interestingly, myelin-deficient Shiverer mice exhibit an increased density and more proximal location of en passant boutons, suggesting that myelination might function in part to regulate synapse formation along PV interneuron axons. Taken together, fast-spiking interneuron myelination is likely to have broad implications for cerebral cortex function in health and disease.

Automated spherical aberration correction in scanning confocal microscopy.

Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy to compensate for spherical aberrations due to coverslip thickness mismatch. With a motorized coverslip correction collar, the adjustment procedure consists of xz image scans, image processing, correction quality evaluation, the mismatch estimation, and eventually the optimal adjustment of the correction collar. For fast correction with less photodamage, coarse-fine Gaussian fitting algorithms are proposed and evaluated with various specimen for their estimation accuracy. The benefits of the proposed automated correction are demonstrated for various coverslips with biological specimens, showing the optimized resolution of the confocal microscope.

Variable loss of functional activities of androgen receptor mutants in patients with androgen insensitivity syndrome.

Androgen receptor (AR) mutations in androgen insensitivity syndrome (AIS) are associated with a variety of clinical phenotypes. The aim of the present study was to compare the molecular properties and potential pathogenic nature of 8 novel and 3 recurrent AR variants with a broad variety of functional assays. Eleven AR variants (p.Cys177Gly, p.Arg609Met, p.Asp691del, p.Leu701Phe, p.Leu723Phe, p.Ser741Tyr, p.Ala766Ser, p.Arg775Leu, p.Phe814Cys, p.Lys913X, p.Ile915Thr) were analyzed for hormone binding, transcriptional activation, cofactor binding, translocation to the nucleus, nuclear dynamics, and structural conformation. Ligand-binding domain variants with low to intermediate transcriptional activation displayed aberrant Kd values for hormone binding and decreased nuclear translocation. Transcriptional activation data, FxxFF-like peptide binding and DNA binding correlated well for all variants, except for p.Arg609Met, p.Leu723Phe and p.Arg775Leu, which displayed a relatively higher peptide binding activity. Variants p.Cys177Gly, p.Asp691del, p.Ala766Ser, p.Phe814Cys, and p.Ile915Thr had intermediate or wild type values in all assays and showed a predominantly nuclear localization in living cells. All transcriptionally inactive variants (p.Arg609Met, p.Leu701Phe, p.Ser741Tyr, p.Arg775Leu, p.Lys913X) were unable to bind to DNA and were associated with complete AIS. Three variants (p.Asp691del, p.Arg775Leu, p.Ile915Thr) still displayed significant functional activities in in vitro assays, although the clinical phenotype was associated with complete AIS. The data show that molecular phenotyping based on 5 different functional assays matched in most (70%) but not all cases.

Functional analysis of novel androgen receptor mutations in a unique cohort of Indonesian patients with a disorder of sex development.

Mutations in the androgen receptor (AR) gene, rendering the AR protein partially or completely inactive, cause androgen insensitivity syndrome, which is a form of a 46,XY disorder of sex development (DSD). We present 3 novel AR variants found in a cohort of Indonesian DSD patients: p.I603N, p.P671S, and p.Q738R. The aim of this study was to determine the possible pathogenic nature of these newly found unclassified variants. To investigate the effect of these variants on AR function, we studied their impact on transcription activation, AR ligand-binding domain interaction with an FxxLF motif containing peptide, AR subcellular localization, and AR nuclear dynamics and DNA-binding. AR-I603N had completely lost its transcriptional activity due to disturbed DNA-binding capacity and did not show the 114-kDa hyperphosphorylated AR protein band normally detectable after hormone binding. The patient with AR-I603N displays a partial androgen insensitivity syndrome phenotype, which is explained by somatic mosaicism. A strongly reduced transcriptional activity was observed for AR-Q738R, together with diminished interaction with an FxxLF motif containing peptide. AR-P671S also showed reduced transactivation ability, but no change in DNA- or FxxLF-binding capacity and interferes with transcriptional activity for as yet unclear reasons.

Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching.

To study protein-protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor-acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein-protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5' endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.

Multiple object tracking in molecular bioimaging by Rao-Blackwellized marginal particle filtering.

Time-lapse fluorescence microscopy imaging has rapidly evolved in the past decade and has opened new avenues for studying intracellular processes in vivo. Such studies generate vast amounts of noisy image data that cannot be analyzed efficiently and reliably by means of manual processing. Many popular tracking techniques exist but often fail to yield satisfactory results in the case of high object densities, high noise levels, and complex motion patterns. Probabilistic tracking algorithms, based on Bayesian estimation, have recently been shown to offer several improvements over classical approaches, by better integration of spatial and temporal information, and the possibility to more effectively incorporate prior knowledge about object dynamics and image formation. In this paper, we extend our previous work in this area and propose an improved, fully automated particle filtering algorithm for the tracking of many subresolution objects in fluorescence microscopy image sequences. It involves a new track management procedure and allows the use of multiple dynamics models. The accuracy and reliability of the algorithm are further improved by applying marginalization concepts. Experiments on synthetic as well as real image data from three different biological applications clearly demonstrate the superiority of the algorithm compared to previous particle filtering solutions.

Cytokine production induced by binding and processing of calcium oxalate crystals in cultured macrophages.

Deposition of calcium oxalate (CaOx) crystals in the renal interstitium is common in humans with primary oxalosis and secondary hyperoxaluria, as well as in kidneys of rats with CaOx nephrolithiasis. In vivo, macrophages and multinucleated giant cells mostly encapsulate these crystals. To investigate whether macrophages are able to dispose of CaOx crystals after phagocytosis, we used a nontransformed macrophage cell line derived from mouse spleen progenitors. Cytokine assays showed that in response to crystal binding and phagocytosis, these macrophages release tumor necrosis factor-alpha. This release was evident at 8 hours, maximal at 24 hours, and decreased to control values after 48 hours of incubation with crystals. A very low but significant release of interleukin-6 into the culture medium was only noticed after 32 hours. Radiochemical experiments showed that these cells bind 38.8% of the CaOx crystals added. After 4 days, all internalized crystals had been dissolved and their molecular constituents released into the extracellular environment. Confocal laser scanning microscopy followed by morphometrical analyses confirmed these results. Long-term (survival) analyses showed that in the interval under study and at the crystal doses used, cell viability was not significantly affected. These findings support the view that properly functioning macrophages are able to remove CaOx deposits from the renal interstitium and that these cells produce inflammatory cytokines before crystal dissolution.

Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors. METHODS AND RESULTS: Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake. CONCLUSIONS: Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells.

Antibody-targeted chemotherapy is a promising therapy in patients with acute myeloid leukemia (AML). In a phase II study of Mylotarg (CMA-676, gemtuzumab ozogamicin), which consists of a CD33 antibody linked to calicheamicin, saturation and internalization by leukemic and normal myeloid cells were analyzed in 122 patients with relapsed AML. Peripheral blood samples were obtained just before and 3 and 6 hours after the start of the first and second Mylotarg treatment cycles. Within 3 to 6 hours after infusion, near complete saturation of CD33 antigenic sites by Mylotarg was reached for AML blasts, monocytes, and granulocytes, whereas Mylotarg did not bind to lymphocytes. Saturation levels prior to the start of the second Mylotarg treatment cycle were significantly increased compared with background levels before the start of the first cycle. This apparently was caused by remaining circulating Mylotarg from the first treatment cycle (approximately 2 weeks earlier). On binding of Mylotarg to the CD33 antigen, Mylotarg was rapidly internalized, as determined by the decrease in maximal surface membrane Mylotarg binding. Internalization of Mylotarg was also demonstrated in myeloid cells in vitro and was confirmed by confocal laser microscopy. In vitro studies using pulse labeling with Mylotarg showed a continuous renewed membrane expression of CD33 antigens, which can significantly increase the internalization process and thereby the intracellular accumulation of the drug. Finally, Mylotarg induced dose-dependent apoptosis in myeloid cells in vitro. These data indicate that Mylotarg is rapidly and specifically targeted to CD33(+) cells, followed by internalization and subsequent induction of cell death.

Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching.

Regulation and structural requirements of vital nuclear processes such as DNA replication, transcription, RNA processing and DNA repair inside the eukaryote nucleus are as yet poorly understood. Although a wealth of evidence exists pointing to a considerable degree of spatial organisation of chromatin and nuclear processes, there are still questions concerning the dynamics and interaction of nuclear proteins that remain unanswered. The cloning of the gene encoding the green fluorescent protein (GFP) has revolutionised the study of proteins in living cells. The expression of recombinant cDNA fusion plasmids of GFP and proteins of interest currently enables the investigation of those proteins in living cells. Time-lapse confocal microscopy as well as quantitative fluorescence methods such as fluorescence redistribution after photobleaching (FRAP) and fluorescence resonance energy transfer are widely applied to living cells expressing GFP fusion proteins. This review gives an overview of the current state of knowledge of nuclear structure and function. In particular, the different applications of FRAP technology to study the dynamics of GFP-tagged nuclear proteins will be summarised.

Role of macrophages in nephrolithiasis in rats: an analysis of the renal interstitium.

Interstitial calcium oxalate (CaOx) crystals can be found in primary oxalosis and in secondary hyperoxaluria. In a rat model for nephrolithiasis, we investigated whether such crystals can be removed by the surrounding interstitial cells. CaOx crystals were induced by a crystal-inducing diet based on ethylene glycol (EG) and ammonium chloride (CID). Both lithogenic compounds were added to the drinking water. After 9 days, the animals received normal drinking water for 2 days. Using this CID, only the interstitial crystals are retained. Subsequently, half of the population remained on normal drinking water (normo-oxaluria), whereas the other half received a low dose of EG alone (chronic hyperoxaluria). The rats were killed at regular times thereafter. The results showed that the kidney-associated oxalate significantly declined during normo-oxaluria, but remained high during chronic hyperoxaluria. Interstitial cells positive for the leukocyte common antigen (CD45; which identifies all types of leukocytes), the ED1 antigen (which is specific for monocytes and macrophages), and the major histocompatibility class II antigen (MCHII), respectively, had increased in number, with minor differences between both rat populations. The cells around the interstitial crystals were mostly positive for ED1. Multinucleate giant cells were regularly observed. These cells were positive for CD45 and ED1 and sometimes also for MCHII. The crystals in these cells were moderately positive for acid phosphatase and carbonic anhydrase II. It is concluded that interstitial CaOx crystals can be removed under normo-oxaluric conditions and that, in all likelihood, macrophages and multinucleate giant cells are involved in that process.

Apoptosis is present in the primate macula at all ages.

BACKGROUND: It has become increasingly clear that apoptosis is a main event in photoreceptor cell death in a variety of retinal degenerations. We investigated the role of apoptosis in the physiologically aging primate macula. METHODS: Twenty maculae of rhesus monkeys, aged 6-34 years, were investigated. Apoptosis was determined in formalin-fixed, paraffin-embedded eyes using the TUNEL (TdT-mediated dUTP-biotin nick end labeling) method and quantitatively analyzed. Morphology of TUNEL-positive cells was studied by confocal laser microscopy and transmission electron microscopy. The thickness of the outer nuclear layer (ONL) was determined by image analysis. Furthermore, expression of apoptosis-regulating proteins Bcl-x, Fas and Fas Ligand was studied by immunohistochemistry. RESULTS: TUNEL-positive nuclei showed apoptotic features on confocal laser microscopy. They were scattered and sparsely found in the macula, most frequently in the ONL. The thickness of the ONL decreased with increasing age. Apoptosis was found equally distributed at all ages, although in the two oldest maculae up to 13 times more apoptosis was found. Expression of Bcl-x, Fas and Fas Ligand was equal at all ages. CONCLUSION: Our findings indicate that apoptosis in the primate macula occurs at all ages at similar rates, possibly increasing in the oldest age group, and may account for the decreasing thickness of the primate macula with age.

Chromosome no. 1 of Crepis capillaris shows defined 3D-shapes in mitotic prophase.

The shape of mitotic prophase chromosomes has been studied in root tip nuclei by confocal microscopy and 3D-image analysis. Crepis capillaris chromosome no. 1 was used as a test object. Chromosome conformation was studied in early, mid- and in late prophase. In mid- and late prophase, individual chromosomes could be distinguished on the basis of their length. Early prophase chromosomes could not be distinguished as individuals. The central axes of prophase chromosomes were traced with an automated computer procedure and then represented as a string of 3D coordinates. This representation facilitated measurement along the chromosome axis of shape parameters such as curvature (amount of bending), torsion (helical winding) and torsion sign (helical handedness). Stretches of early prophase chromosomes showed full helical turns, which could be left- or right-handed. In the later prophase stages curvature and torsion were statistically analysed. Our data on 40 midprophase chromosomes no. 1 show that they are still highly curved, but full helical turns were no longer found. Instead, an overall meandering pattern was observed. In late prophase, one central loop persisted, flanked by two preferential regions of high curvature.

The 5S rRNA gene clusters have a defined orientation toward the nucleolus in Petunia hybrida and Crepis capillaris.

The 3D localization of the 5S ribosomal RNA genes was studied in cells of the cortex zone of roots in the plant species Petunia hybrida inbred line V26 and in Crepis capillaris. The analysis was carried out on interphase nuclei (both species) and on prophase nuclei (C. capillaris). The 5S ribosomal RNA genes were detected by fluorescence in-situ hybridization and 3D images were obtained by confocal scanning laser microscopy. In both plant species, the 5S ribosomal genes were localized at the short arm of chromosome 2, which, in both plants, also possesses a satellite at its end. Statistical and visual analysis of interphase nuclei showed that: (1) there is a preference for an association of the 5S rRNA gene clusters of the two homologous chromosomes, and (2) the 5S rRNA gene clusters in both species had a preserved spatial position within the interphase nucleus and they tended to be polarized with respect to their neighbouring cells (i.e. a relic telophase orientation). Moreover, tracing of the chromosomal segment between the 5S loci and the active NOR revealed that the homologous chromosomes during early/mid prophase were aligned and that they entered the nucleolus side by side, at least for these chromosome segments. We interpret our data to mean that location of 5S rRNA near the nucleolus favours their functioning in ribosome biogenesis.

Cytogenetic clonality analysis of megakaryocytes in myelodysplastic syndrome by dual-color fluorescence in situ hybridization and confocal laser scanning microscopy.

In the myelodysplastic syndrome (MDS), cytogenetic abnormalities are often present and can be used as markers in studies for cell lineage involvement. Little is known of the involvement of the megakaryocytic lineage due to the variable ploidy of these cells. We applied dual-color fluorescence in situ hybridization (FISH) to routinely prepared bone marrow (BM) smears of cytogenetically normal patients and seven patients with MDS and monosomy 7 or trisomy 8. Probes specific for the centromeric regions of chromosomes 7 and 8 were detected with fluorescein isothiocyanate (FITC) and Texas Red, respectively. This enabled us to assess the ratio between the numbers of chromosomes 7 and 8 in the polyploid cells. We utilized confocal laser scanning microscopy to count the FITC and Texas Red FISH signals in the different focal layers of the megakaryocytes. Fifty-six megakaryocytes in six normal BM smears were analyzed, giving a mean ratio of 1.0, a standard deviation (SD) of 0.12, and a range of 0.8-1.33. This ratio was applied to evaluation of clonal involvement of individual megakaryocytes in the patients with MDS. In two patients with monosomy 7, the majority of the megakaryocytes were monosomic. In the five patients with trisomy 8, all or a majority of the analyzed megakaryocytes were trisomic. These results add direct evidence that in MDS megakaryocytes are involved in the malignant clone. Genes Chromosomes Cancer 25:332-338, 1999.

Action of DNA repair endonuclease ERCC1/XPF in living cells.

To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus, with a diffusion coefficient (15 +/- 5 square micrometers per second) consistent with its molecular size. Ultraviolet light-induced DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.

Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture.

BACKGROUND: Adherence of crystals to the surface of renal tubule epithelial cells is considered an important step in the development of nephrolithiasis. Previously, we demonstrated that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection against the adherence of calcium oxalate monohydrate crystals. We now examined whether this property is cell type specific. The susceptibility of the cells to crystal binding was further studied under different culture conditions. METHODS: Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohydrate crystal binding to LLC-PK1 cells and to two MDCK strains cultured on either permeable or impermeable supports. These cell lines are representative for the renal proximal tubule (LLC-PK1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and present, respectively. RESULTS: Whereas relatively large amounts of crystals adhered to subconfluent MDCK cultures, the level of crystal binding to confluent monolayers was reduced for both MDCK strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired a higher degree of protection than on impermeable surfaces. Crystals avidly adhered to LLC-PK1 cells, irrespective of their developmental stage or growth substrate used. CONCLUSIONS: These results show that the prevention of crystal binding is cell type specific and expressed only by differentiated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of its in situ equivalent, the renal distal tubule/collecting ducts.

Kinetics, localization, and mechanism of 5-aminolevulinic acid-induced porphyrin accumulation in normal and Barrett's-like rat esophagus.

BACKGROUND AND OBJECTIVES: Photodynamic therapy may selectively destroy Barrett's epithelium in the esophagus. To optimize photosensitizer administration, the kinetics of 5-aminolevulinic acid (ALA)-induced porphyrin accumulation in the normal and Barrett's-like esophagus were studied in the rat. STUDY DESIGN/MATERIALS AND METHODS: Animals received 200 mg/kg ALA intravenously (n = 21) or orally (n = 21). Six rats served as controls. At t = 1, 2, 3, 4, 6, 12, and 24 hr, porphyrin concentration in the esophagus was measured by using chemical extraction, and porphyrin localization was determined by laser scanning microscopy (LSM). In addition, in 20 animals, porphobilinogen deaminase, ferrochelatase, and iron concentration were determined. In a second group (n = 24), an esophagojejunostomy was performed to induce a Barrett's-like esophagus. After 18 weeks, animals received ALA, and LSM was performed at t = 1, 2, 3, 4, 6, 8, and 12 hr. RESULTS: Porphyrin accumulation in normal mucosa was 3.5-fold higher than in muscularis, with a maximum at 3 hr after ALA administration. With LSM, strong homogeneous fluorescence of the squamous epithelium was shown, with minor fluorescence of submucosa and muscularis. In Barrett's-like epithelium, fluorescence was heterogeneous but was also restricted to epithelial cells. There was no difference in fluorescence intensity between Barrett's-like and adjacent squamous epithelium. Porphobilinogen deaminase activity was higher and iron concentration was lower in the mucosa than in the muscularis (P < 0.001). CONCLUSION: ALA-induced porphyrin accumulation selectively occurs in esophageal mucosa, whether normal or Barrett's-like, compared with the muscularis, with a maximum at 3 hr after ALA administration. Selectivity may be caused by a different activity of heme-synthetic enzymes or relative iron deficiency in the mucosa.