Dimethyl fumarate decreases short-term but not long-term inflammation in a focal EAE model of neuroinflammation.

BACKGROUND: Dimethyl fumarate (DMF) is an oral immunomodulatory drug used in the treatment of autoimmune diseases. Here, we sought to study whether the effect of DMF can be detected using positron emission tomography (PET) targeting the 18-kDa translocator protein (TSPO) in the focal delayed-type hypersensitivity rat model of multiple sclerosis (fDTH-EAE). The rats were treated orally twice daily from lesion activation (day 0) with either vehicle (tap water with 0.08% Methocel, 200 µL; control group n = 4 (3 after week four)) or 15 mg/kg DMF (n = 4) in 0.08% aqueous Methocel (200 µL) for 8 weeks. The animals were imaged by PET using the TSPO tracer [18F]GE-180 in weeks 0, 1, 2, 4, 8, and 18 following lesion activation, and the non-displaceable binding potential (BPND) was calculated. Immunohistochemical staining for Iba1, CD4, and CD8 was performed in week 18, and in separate cohorts of animals, following 2 or 4 weeks of treatment. RESULTS: Using the fDTH-EAE model, DMF reduced the [18F]GE-180 BPND in the DMF-treated animals compared to control animals after 1 week of treatment (two-tailed unpaired t test, p = 0.031), but not in weeks 2, 4, 8, or 18 when imaged in vivo by PET. Immunostaining for Iba1 showed that DMF had no effect on the perilesional volume or the core lesion volume after 2 or 4 weeks of treatment, or at 18 weeks. However, the optical density (OD) measurements of CD4+ staining showed reduced OD in the lesions of the treated rats. CONCLUSIONS: DMF reduced the microglial activation in the fDTH-EAE model after 1 week of treatment, as detected by PET imaging of the TSPO ligand [18F]GE-180. However, over an extended time course, reduced microglial activation was not observed using [18F]GE-180 or by immunohistochemistry for Iba1+ microglia/macrophages. Additionally, DMF did affect the infiltration of CD4+ and CD8+ T-lymphocytes at the fDTH-EAE lesion.

Au nanostar nanoparticle as a bio-imaging agent and its detection and visualization in biosystems.

In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.

Cessation of anti-VLA-4 therapy in a focal rat model of multiple sclerosis causes an increase in neuroinflammation.

BACKGROUND: Positron emission tomography (PET) can be used for in vivo evaluation of the pathology associated with multiple sclerosis. We investigated the use of longitudinal PET imaging and the 18-kDa translocator protein (TSPO) binding radioligand [18F]GE-180 to detect changes in a chronic multiple sclerosis-like focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) rat model during and after anti-VLA-4 monoclonal antibody (mAb) treatment. Thirty days after lesion activation, fDTH-EAE rats were treated with the anti-VLA-4 mAb (n = 4) or a control mAb (n = 4; 5 mg/kg, every third day, subcutaneously) for 31 days. Animals were imaged with [18F]GE-180 on days 30, 44, 65, 86 and 142. Another group of animals (n = 4) was used for visualisation the microglia with Iba-1 at day 44 after a 2-week treatment period. RESULTS: After a 2-week treatment period on day 44, there was a declining trend (p = 0.067) in [18F]GE-180-binding in the anti-VLA-4 mAb-treated animals versus controls. However, cessation of treatment for 4 days after a 31-day treatment period increased [18F]GE-180 binding in animals treated with anti-VLA-4 mAb compared to the control group (p = 0.0003). There was no difference between the groups in TSPO binding by day 142. CONCLUSIONS: These results demonstrated that cessation of anti-VLA-4 mAb treatment for 4 days caused a transient rebound increase in neuroinflammation. This highlights the usefulness of serial TSPO imaging in the fDTH-EAE model to better understand the rebound phenomenon.

A multicentre verification study of the QuantiFERON®-TB Gold Plus assay.

OBJECTIVES: The aim of this verification study was to compare the QuantiFERON®-TB Gold Plus (QFT-Plus) to the QuantiFERON®-TB Gold In Tube (QFT-GIT). The new QFT-Plus test contains an extra antigen tube which, according to the manufacturer additionally elicits a CD8+ T-cell response above the CD4+ T-cell response. We assessed the value of this tube in detecting recent latent tuberculosis infections. METHODS: Between May 2015 and December 2016, 1031 subjects underwent QFT-Plus and QFT-GIT test. Overall agreement between both tests and performance for different test indications and/or immune states was assessed. A difference of >0.6 IU/mL interferon-γ release between the two antigen tubes of the QFT-Plus assay was considered a true difference and used as estimation for CD8+ T-cell response. RESULTS: Analysis of the QuantiFERON tests resulted in an overall agreement between assays of 95%. Subjects considered to be recently exposed to tuberculosis had significantly more often a true difference in interferon-γ release compared to all other subjects (p = 0.029). CONCLUSION: Results of QFT-Plus are highly comparable to QFT-GIT. Although there is an indication that a true difference in interferon-γ release between the antigen tubes is associated with recent latent tuberculosis infection, the QFT-Plus could not be used to exclude recent exposure.

Diagnostic yield of repeat sampling with immunoassay, real-time PCR, and toxigenic culture for the detection of toxigenic Clostridium difficile in an epidemic and a non-epidemic setting.

Current international guidelines lack definite conclusions regarding repeat stool sampling for the detection of toxigenic Clostridium difficile. We assessed the value of repeat sampling and compared the diagnostic yield in an epidemic to a non-epidemic setting. Consecutive fecal samples obtained during two time frames were analyzed using direct stool immunoassay toxin testing (enzyme immunoassay [EIA]), direct stool real-time PCR toxin gene testing, and toxigenic culture. Samples collected within 7 days of the initial sample were considered repeat tests. In the epidemic setting 989 patients were analyzed, and in the non-epidemic setting 1,015. In the epidemic setting 204 patients had two or more specimens included for analysis and in the non-epidemic setting 287 patients. In the epidemic setting 136 samples yielded a positive results, either by EIA or toxigenic culture; of these, 108 were positive according to EIA and 123 according to toxigenic culture. In the first test round 98 (90.7%, 95% CI 85.3 to 96.2), 114 (92.7%, 88.1 to 97.3), and 126 (92.6%, 88.3 to 97.0) positives were detected. Subsequent test rounds yielded 10 (9.3%, 3.8 to 14.7), 9 (7.3%, 2.7 to 11.9), and 10 (7.4%, 3.0 to 11.7) extra positives. In the non-epidemic setting EIA, toxigenic culture and PCR detected 33, 66, and 83 positives. The three tests combined 93 detected positives. In the first test round 30 (90.9%, 81.1 to 100.7), 63 (95.5%, 90.4 to 110.5), 76 (91.6%, 85.6 to 97.5), and 87 (93.5%, 88.6 to 98.5) positives were detected. Subsequent test rounds yielded 3 (9.1%, -0.7 to 18.9), 3 (4.5%, -0.5 to 9.6), 7 (8.4%, 2.5 to 14.4), and 6 (6.5%, 1.5 to 11.4) extra positives. In conclusion, repeat testing resulted in 4.5% to 9.3% extra positives. No significant difference between the settings studied could be demonstrated. Repeat sampling and multimodality testing may be chosen in an outbreak situation to detect all cases, effectively controlling nosocomial spread.

Skin equivalents: skin from reconstructions as models to study skin development and diseases.

While skin is readily available for sampling and direct studies of its constituents, an important intermediate step is to design in vitro and/or in vivo models to address scientific or medical questions in dermatology and skin biology. Pioneered more than 30 years ago, human skin equivalents (HSEs) have been refined with better cell culture techniques and media, together with sophisticated cell biology tools including genetic engineering and cell reprogramming. HSEs mimic key elements of human skin biology and have been instrumental in demonstrating the importance of cell-cell interactions in skin homeostasis and the role of a complex cellular microenvironment to coordinate epidermal proliferation, differentiation and pigmentation. HSEs have a wide field of applications from cell biology to dermocosmetics, modelling diseases, drug development, skin ageing, pathophysiology and regenerative medicine. In this article we critically review the major current approaches used to reconstruct organotypic skin models and their application with a particular emphasis on skin biology and pathophysiology of skin disorders.

Alcohol-induced premature permeability in mouse placenta-yolk sac barriers in vivo.

OBJECTIVE: Acute alcohol exposure induces malformation and malfunction of placenta-yolk sac tissues in rodents, reducing the labyrinth zone in the placenta and altering the permeability and fluidity of the cell membrane. During normal mouse placentation the cells line up in an optimal way to form a hemotrichorial placenta where layers II and III are connected through gap junctions. These act as molecular sieves that limit the passage of large molecules. PlGF is a developmentally regulated protein that controls the passage of molecules in the vasculosyncytial membranes and media of large blood vessels in the placental villi. In addition to the chorioallontoic placenta, rodents also have another type of placenta that consists of Reichert's membrane within the trophoblast cell layer on the maternal side and the parietal endodermal cells on the embryonic site. This forms a separate materno-fetal transport system. We study here whether alcohol affects these two placental barriers, leading to placental malfunction that in turn diminishes the nutrient supply to the embryo. STUDY DESIGN: CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals at 8.75 days post coitum (dpc). The placentas were collected on 9.5, 11.5 and 14.5 dpc and used for histopathological protein studies. Hemotrichorial cell layer structure interactions through connective tissue and gap junction were analyzed by electron microscopy. The permeability of the feto-maternal barrier was visualized with Evans Blue. RESULTS: VEGF, a permeability inducer, was found to be up-regulated in the mouse placenta after acute alcohol exposure, and permeability was also affected by altered structures in the barriers that separate the feto-maternal blood circulation which destroyed the gap junctions in the hemotrichorial cell layer, reduced the thickness of Reichert's membrane and interfered with with Reichert's trophoblast/Reichert's parietal interaction. These defects together could have caused the permeability malfunction of the placenta-yolk sac tissues as visualized and quantified here by Evans Blue leakage. CONCLUSIONS: An altered PlGF/VEGF ratio together with barrier malformation may contribute to placental malfunction by altering the permeability of the feto-maternal barriers. Further studies are needed in order to show whether premature permeability is involved in the intrauterine growth restriction observed in human FAS embryos.

Expression of Sprouty genes 1, 2 and 4 during mouse organogenesis.

We demonstrate that Sprouty genes 1, 2 and 4 are expressed in several developing organs of the craniofacial area and trunk, including the brain, cochlea, nasal organs, teeth, salivary gland, lungs, digestive tract, kidneys and limb buds. In organs such as the semicircular canal, Rathke's pouch, nasal organs, the follicle of vibrissae and teeth, Sprouty1 and Sprouty2 are expressed in the epithelium and Sprouty4 in the mesenchyme or neuronal tissue, while in the lung Sprouties1, 2 and 4 are all expressed mainly in the epithelial tissue. In the kidney, Sprouty1 is prominent in the ureteric bud whereas Sprouty2 and 4 are expressed in both the ureteric bud and the kidney mesenchyme and glomeruli deriving from it. The expression profiles suggest roles for these Sprouties in the epithelial-mesenchymal interactions that govern organogenesis.

Wnts and the female reproductive system.

Wnts are intercellular growth and differentiation factors that regulate several key developmental steps, such as gastrulation, neurulation, and organogenesis, including the development of the midbrain, central nervous system, kidney, and limbs. Wnts are also needed for a normal development of the reproductive system. Deficiency of Wnt-4, -5a, and -7a, for example, results in sex reversal, infertility, and/or malformation of the internal and external genitals. Here we focus on the importance of Wnts in the female reproductive system.

Induction of ureter branching as a response to Wnt-2b signaling during early kidney organogenesis.

Epithelial-mesenchymal tissue interactions play a central role in vertebrate organogenesis, but the molecular mediators and mechanisms of these morphogenetic interactions are still not well characterized. We report here on the expression pattern of Wnt-2b during mouse organogenesis and on tests of its function in epithelial- mesenchymal interactions during kidney development. Wnt-2b is expressed in numerous developing organs in the mouse embryo, including the kidney, lung, salivary gland, gut, pancreas, adrenal gland, and genital tubercle. Additional sites of expression include the branchial arches and craniofacial placodes such as the eye and ear. The data suggest that the expression of Wnt-2b is associated with organs regulated by epithelial-mesenchymal interactions. It is typically localized in the capsular epithelium or peripheral mesenchymal cells of organ rudiments, e.g., the perinephric mesenchymal cells in the region of the presumptive renal stroma in the developing kidney at E11.5. Functional studies of the kidney demonstrate that cells expressing Wnt-2b are not capable of inducing tubule formation but instead stimulate ureter development. Incubation of isolated ureteric buds on such cells supports bud growth and branching. In addition, recombination of Wnt-2b-pretreated ureteric bud tissue with isolated nephrogenic mesenchyme results in a recovery of organogenesis and the expression of epithelial genes within the reconstituted organ explant. Lithium, a known activator of Wnt signaling (Hedgepeth et al. [1997] Dev Biol 185:82-91), is also sufficient to promote ureter branching in the reconstituted kidney in a comparable manner to Wnt-2b signaling, whereas Wnt-4, which induces tubules, neither supports the growth of a ureteric bud nor leads to reconstitution of the ureteric bud with the kidney mesenchyme. We conclude that Wnt-2b may act in the mouse kidney as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and that the Wnt pathway may regulate ureter branching directly. In addition, Wnt signals in the kidney differ qualitatively and are specific to either the epithelial ureteric bud or the kidney mesenchyme.

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C.

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.

Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription.

T cell factor / lymphocyte enhancer factor (Tcf/Lef) transcription factors complex with the transcriptional co-activator beta-catenin to transduce Wnt signals in a variety of developmental systems. The prototypic family member Tcf-1 is highly expressed in T lineage cells. Tcf1-/- mice are defective in cell cycling of early thymocyte stages. Here, we show that the interaction of beta-catenin with Tcf-1 is required for full thymocyte development. This interaction may be established by signals mediated by Wnt1 and Wnt4, leading to increased Tcf-dependent transcriptional activity in thymocytes, as demonstrated in Tcf-LacZ reporter mice. Transduction of fetal thymocytes with Wnt1 and Wnt4 results in increased survival in an in vitro cell culture system. Retroviral expression of soluble Wnt receptor mutants that block Wnt signaling inhibits thymocyte development. These results imply an important role for the Wnt cascade in thymocyte development.

Associations of FGF-3 and FGF-10 with signaling networks regulating tooth morphogenesis.

The morphogenesis and cell differentiation in developing teeth is governed by interactions between the oral epithelium and neural crest-derived ectomesenchyme. The fibroblast growth factors FGF-4, -8, and -9 have been implicated as epithelial signals regulating mesenchymal gene expression and cell proliferation during tooth initiation and later during epithelial folding morphogenesis and the establishment of tooth shape. To further evaluate the roles of FGFs in tooth development, we analyzed the roles of FGF-3, FGF-7, and FGF-10 in developing mouse teeth. In situ hybridization analysis showed developmentally regulated expression during tooth formation for Fgf-3 and Fgf-10 that was mainly restricted to the dental papilla mesenchymal cells. Fgf-7 transcripts were restricted to the developing bone surrounding the developing tooth germ. Fgf-10 expression was observed in the presumptive dental epithelium and mesenchyme during tooth initiation, whereas Fgf-3 expression appeared in the dental mesenchyme at the late bud stage. During the cap and bell stage, both Fgf-3 and Fgf-10 were intensely expressed in the dental papilla mesenchymal cells both in incisors and molars. It is of interest that Fgf-3 expression was also observed in the primary enamel knot, a putative signaling center of the tooth, whereas no transcripts were seen in the secondary enamel knots that appear in the tips of future cusps of the bell stage tooth germs. Down-regulation of Fgf-3 and Fgf-10 expression in postmitotic odontoblasts correlated with the terminal differentiation of the odontoblasts and the neighboring ameloblasts. In the incisors, mesenchymal cells of the cervical loop area showed partially overlapping expression patterns for all studied Fgfs. In vitro analyses showed that expression of Fgf-3 and Fgf-10 in the dental mesenchyme was dependent on dental epithelium and that epithelially expressed FGFs, FGF-4 and -8 induced Fgf-3 but not Fgf-10 expression in the isolated dental mesenchyme. Beads soaked in Shh, BMP-2, and TGF-beta 1 protein did not induce either Fgf-3 or Fgf-10 expression. Cells expressing Wnt-6 did not induce Fgf-10 expression. Furthermore, FGF-10 protein stimulated cell proliferation in the dental epithelium but not in the mesenchyme. These results suggest that FGF-3 and FGF-10 have redundant functions as mesenchymal signals regulating epithelial morphogenesis of the tooth and that their expressions appear to be differentially regulated. In addition, FGF-3 may participate in signaling functions of the primary enamel knot. The dynamic expression patterns of different Fgfs in dental epithelium and mesenchyme and their interactions suggest existence of regulatory signaling cascades between epithelial and mesenchymal FGFs during tooth development.

A road to kidney tubules via the Wnt pathway.

Classical in vitro studies indicate that tubule induction in the kidney mesenchyme is mediated by cell-cell contacts between the inducer tissue and the metanephric mesenchyme. Induction is completed within the first 24 h, after which tubules will form because of stimulated cell proliferation, migration, and cell adhesion. Recent evidence has revealed an essential role for the secreted signals from the Wnt gene family. Of these, Wnt-4 is expressed in developing tubules and knocking out its function perturbed kidney development. More detailed studies demonstrated normal condensation, but tubules were missing. Subsequent experiments indicated that Wnt-4 is also a sufficient signal to trigger tubulogenesis. Cells that were engineered to express Wnt-4 not only induced tubulogenesis in the kidney mesenchyme of Wnt-4 mutant embryos, but also induced tubules in the wild type mesenchyme. With the transfilter induction assay, Wnt-4-mediated induction was completed within the first 24 h, depending on the presence of proteoglycans and cell-cell contacts between the interactants. In addition, Wnt-4 autoinduced expression of its own gene and a panel of other components of the Wnt signalling pathway, such as frizzleds and a candidate Wnt antagonist from the secreted frizzled-related protein family. Taken together, the data provide evidence of an essential role for Wnt signal transmission and transduction pathways in the induction of kidney tubules, and the findings have paved the way for detailed molecular studies.

Intestinal fatty acid binding protein polymorphism at codon 54 is not associated with postprandial responses to fat and glucose tolerance tests in healthy young Europeans. Results from EARS II participants.

UNLABELLED: Polymorphism Ala54Thr of the intestinal fatty acid-binding protein 2 (FABP2) has been reported to have an effect on the protein's affinity for long chain fatty acids and to be associated with serum lipid and insulin levels in fasting and especially postprandial states. We wanted to test whether this genetic variation is associated with fasting and postprandial glucose, insulin or lipid levels in 666 male university students participating in the second European Atherosclerosis Study (EARS II). We also studied whether the subgroup of 330 students with paternal history of myocardial infarction (MI) before the age of 55 have different genotype distribution than 336 matched controls. RESULTS: No difference in genotype distribution was observed between offspring with and without paternal history of MI or between populations from 11 European countries. The frequency of the threonine encoding allele was 0.276 in cases and 0.266 in controls. There were no differences in fasting or postprandial serum lipid, glucose or insulin levels between subjects having different genotypes. CONCLUSIONS: In this study FABP2 Ala54Thr polymorphism was not associated with lipid or glucose metabolism. In addition to environmental and genetic factors, selection of study population also may explain the difference between this and earlier studies.

Localization of 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase isoform expression in the developing mouse testis--androstenedione is the major androgen secreted by fetal/neonatal leydig cells.

The final step in the biosynthesis of testosterone is reduction of androstenedione by the enzyme 17beta-hydroxysteroid dehydrogenase/ 17-ketosteroid reductase (17betaHSD/17KSR). In this study, we have examined expression of the four known reductive isoforms of 17betaHSD/ 17KSR (types 1, 3, 5, and 7) in the developing mouse testis and have determined changes in the localization of isoform expression and testosterone secretion during development. Using RT-PCR isoforms 1, 3, and 7 were shown to be expressed in the seminiferous tubules of neonatal testis, whereas isoforms 3 and 7 were expressed in the interstitial tissue of the adult testis. The type 7 isoform is unlikely to be involved in androgen synthesis and further study concentrated on the type 3 isoform. Developmentally, isoform type 3 was expressed in the seminiferous tubules up to day 10, showed little or no expression on day 20 and from day 30 was confined to the interstitial tissue. In situ hybridization confirmed that the type 3 isoform was expressed only in the seminiferous tubules in fetal testes and in the interstitial tissue in adult testes. In accordance with the localization of enzyme messenger RNA expression 17-ketosteroid reductase enzyme activity was very low in isolated interstitial tissue from neonatal testes while interstitial tissue from adult testes showed high activity. Seminiferous tubules from both neonatal and adult testes showed high levels of enzyme activity. The major androgen secreted by the interstitial tissue of prepubertal animals was androstenedione up to day 20 while 5alpha-androstanediol and/or testosterone were the major androgens secreted from day 30 onwards. These results show that fetal Leydig cells do not express significant levels of a reductive isoform of 17betaHSD/ 17KSR and that androstenedione is the major androgen secreted by these cells. Production of testosterone up until puberty is dependent upon 17betaHSD/17KSR activity in the seminiferous tubules--a "two cell" requirement for testosterone synthesis. Expression of the 17betaHSD/17KSR type 3 isoform (the main reductive isoform in the testis) declines in the seminiferous tubules before puberty but then reappears in the developing adult Leydig cell population.

Differential expression of mouse laminin gamma2 and gamma2* chain transcripts.

Laminins are large heterotrimeric basement membrane proteins that consist of alpha, beta, and gamma chains. We have previously shown that the human gamma2 and gamma2* transcripts result from the alternative use of the LAMC2 gene 3'-end exons. To explore the biological significance of the alternative gamma2 transcripts, we isolated the cDNA coding for the mouse laminin gamma2* transcript, characterized the 3'-end of the murine LAMC2 gene, and studied the expression of alternative gamma2 transcripts in several mouse tissues. The sequence reported here is the first one containing a full-length gamma2* 3'-UTR from any species. The mouse gamma2* transcript is 4110 bases and encodes a putative polypeptide of 1110 amino acids. This polypeptide lacks the C-terminal cysteine residue thought to be important for heterotrimer formation. The mouse gamma2* transcript was found to be expressed in several tissues by polymerase chain reaction (PCR), but at very low levels. The clearest signals were obtained on embryonic day 7, and in heart and testis of adult tissues. When the laminin gamma2* transcript expression pattern was compared with that of the gamma2 chain, a similar tissue distribution was observed. There was, however, a significant difference in expression levels. The longer gamma2 transcript was found to be much more abundant than the shorter gamma2* variant. Moreover, by whole-mount in situ hybridization, the shorter gamma2* form was localized in the mesenchyme of the developing kidney whereas the longer gamma2 form was exclusively present in the epithelium of the Wolffian (nephric) duct and ureteric bud. The results indicate different functions for the gamma2 variants.

Kidney morphogenesis: cellular and molecular regulation.

Development of an organ is directed by cell and tissue interactions and these also occur during the formation of functional kidney. During vertebrate development inductive signalling between mesenchyme and epithelium controls the organogenesis of all three kinds of kidneys: pronephros, mesonephros and metanephros. In higher animals the metanephros differentiates into the permanent kidney and in this review we will mainly concentrate on its development. Molecular interactions currently known to function during nephrogenesis have primarily been based on the use of knockout techniques. These studies have highlighted the role for transcription factors, signalling molecules, growth factors and their receptors and also for extracellular matrix components in kidney development. Finally in this review we will represent our own model for kidney development according to the knowledge of the genes involved in the development of the functional excretory organ, kidney.