In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN, SIZE, DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. STUDY FUNDING AND COMPETING INTEREST(S): Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Télevie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

Hearing preservation and tumour control after radiosurgery for NF2-related vestibular schwannomas.

OBJECTIVES: We analyzed the effects of stereotactic radiosurgery on tumour control and cranial nerve function in patients with vestibular schwannomas (VS) secondary to neurofibromatosis type 2 (NF2). Irradiation was performed with a Gamma Knife, model C equipped with a high-precision, robotized positioning system (APS). METHODOLOGY: This study included 18 patients with 25 VSs secondary to NF2 that were treated from 2001 to 2010 with radiosurgery at our Gamma Knife Center. The radiosurgical procedure included high-resolution conformational dose-planning with multiple, small-diameter isocenters, a single-fraction, low-dose irradiation prescription, and highly accurate gamma rays delivery to the target with the APS. RESULTS: The median follow-up time was 4.4 y. For 16 tumours in 12 patients with available follow-up data, we observed an actuarial tumour control of 87.5% at 2 y and 80.2% at 5 y, based on the Kaplan-Meier method. No patient developed facial weakness. Serviceable hearing was preserved in 78% of cases. Patients treated for bilateral and unilateral tumours had similar outcomes. CONCLUSIONS: Radiosurgery could control tumour growth and preserve hearing function and facial weakness in patients with VS secondary to NF2. The enhanced techniques of radiosurgical irradiation provided with the Gamma Knife model C have improved the results of this treatment alternative to microsurgery.

Comparison of plasma and saliva concentrations of levetiracetam following administration orally as a tablet and as a solution in healthy adult volunteers.

OBJECTIVE: Concentrations in saliva, as an alternative to concentrations in blood, can be advantageous for the monitoring of antiepileptic agents. This study assesses the relationship between saliva and plasma concentrations of levetiracetam after administration orally as a solution and as a tablet. The possibility that saliva concentrations of the drug are altered by contamination in the buccal cavity was also examined. METHODS: 4 healthy male subjects received a single 750 mg oral dose of levetiracetam as a 10% solution and 4 subjects received three 250 mg tablets (750 mg). Levetiracetam concentrations in plasma and saliva were monitored for 24 hours post dose. RESULTS: In subjects receiving the levetiracetam solution, maximum saliva concentrations were observed at the first collection point (15 min) after administration and these were 19-74 times higher than corresponding plasma levels. The mean saliva/plasma ratio rapidly decreased thereafter, becoming stable after 4 hours. In subjects receiving tablets, levetiracetam concentration profiles for saliva paralleled the plasma concentration profiles with a fairly constant saliva/plasma concentration ratio throughout the 24-hour sampling period. A significant linear correlation between levetiracetam saliva and plasma concentrations was demonstrated (Pearson r = 0.88; p < 0.001 for tablet (n = 35) and r = 0.87; p < 0.001 for solution at times > or = 4 hours post-dose (n = 20)). The saliva to plasma concentration ratio was 1.11 (95% confidence interval: 0.99 - 1.22) following tablet intake, and 1.55 (95% CI: 1.34 1.77) following oral solution (> or = 4 hours post dose). CONCLUSIONS: Using saliva to monitor therapeutic exposure to levetiracetam is feasible beginning 15 minutes after tablet intake but beginning 4 hours after intake of an oral solution.

Application of WetSpa model for assessing land use impacts on floods in the Margecany-Hornad watershed, Slovakia.

The spatially distributed hydrologic model WetSpa combines elevation, soil and land use data within GIS, to predict flood hydrographs and spatial distribution of hydrologic characteristics in a watershed. The model is applied to the Margecany-Hornad river basin (1131 km2) in Slovakia. Daily hydrometeorological data from 1991-2000, including precipitation data from nine stations, temperature data from four stations and evaporation data measured at one station are used as input to the model. Three base maps, i.e. DEM, land use and soil type are prepared in GIS form, using 100 x 100 m cell size. Results of the simulations show good agreement between calculated and measured hydrographs. The model predicts the daily/hourly hydrographs with 75-80% accuracy according to the Nash-Sutcliff criteria. For assessing the impact of land use changes on floods, the calibrated model is applied for a reforestation scenario, which considers a 50% increase of forest areas. The model results show that the reforestation scenario decreases the peak discharge by 12%. Investigation of peak discharges from the whole simulation period, shows that the scenario results are reduced by 18% on average, while for small discharges the reduction is even about 34%. The time to peak of the simulated hydrograph of the reforestation scenario is 20 hours longer than for the present land use.

Theoretical and experimental study of the product branching in the reaction of acetic acid with OH radicals.

The product distribution of the reaction of acetic acid, CH(3)COOH, with hydroxyl radicals, OH, was studied experimentally and theoretically. Mass-spectrometric measurements at 290 K and 2 Torr of He of the CO(2) yield versus the loss of acetic acid yielded a branching fraction of 64 +/- 14% for the abstraction of the acidic hydrogen as follows: CH(3)COOH + OH --> CH(3)COO + H(2)O --> CH(3) + CO(2) + H(2)O. A quantum chemical and theoretical kinetic analysis showed that the abstraction of the acidic hydrogen is enhanced relative to the abstraction of -CH(3) hydrogens because of the formation of a strong pre-reactive H-bonded complex, where the H-bonds are retained in the H-abstraction transition state. The potential energy surface of the reaction is explored in detail, and the reaction products of the individual channels are identified. The theoretical product branching is found to be critically dependent on the energetic and rovibrational differences between the H-abstraction transition states.

Application of a Bayesian approach to stochastic delineation of capture zones.

This paper presents a Bayesian Monte Carlo method for evaluating the uncertainty in the delineation of well capture zones and its application to a wellfield in a heterogeneous, multiaquifer system. In the method presented, Bayes' rule is used to update prior distributions for the unknown parameters of the stochastic model for the hydraulic conductivity, and to calculate probability-based weights for parameter realizations using head residuals. These weights are then assigned to the corresponding capture zones obtained using forward particle tracking. Statistical analysis of the set of weighted protection zones results in a probability distribution for the capture zones. The suitability of the Bayesian stochastic method for a multilayered system is investigated, using the wellfield Het Rot at Nieuwrode, Belgium, located in a three-layered aquifer system, as an example. The hydraulic conductivity of the production aquifer is modeled as a spatially correlated random function with uncertain parameters. The aquitard and overlying unconfined aquifer are assigned random, homogeneous conductivities. The stochastic results are compared with deterministic capture zones obtained with a calibrated model for the area. The predictions of the stochastic approach are more conservative and indicate that parameter uncertainty should be taken into account in the delineation of well capture zones.

SEEPAGE, a new MODFLOW DRAIN package.

The prediction of the location of ground water discharge areas is a key aspect for the protection and (re)development of ground water-dependent wetlands. Ground water discharge areas can be simulated with MODFLOW using the DRAIN package by setting the drain level equal to the topography, while the conductance is mostly set to an arbitrary high value. However, conceptual and practical problems arise in the calculation of the ground water discharge by the DRAIN package as calculated water tables above the land surface, difficult parameterization of the conductance, and large water balance errors. To overcome these problems, a new SEEPAGE package for MODFLOW is proposed. The basic idea of this package is an adaptable constant head cell. It has a variable head, unless the ground water rises above the seepage level, in which case it has a constant head cell. The estimation of the ground water discharge location along a homogeneous, isotropic, linear sloping profile is used to verify the model and to compare it to the DRAIN solution. In an application to three basins in Belgium, it is shown that the SEEPAGE package can be used in combination with the DRAIN package in situations where an upper boundary for a free water table and additional resistance for drainage is required. It is clearly demonstrated that the identification and delineation of regional ground water discharge areas is more accurate using the SEEPAGE package.

The SH2 domain containing inositol 5-phosphatase SHIP2 controls phosphatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin.

The lipid phosphatase SHIP2 (SH2 domain containing inositol 5-phosphatase 2) has recently been shown to be a potent negative regulator of insulin signaling and insulin sensitivity in vivo. We show here that SHIP2 is expressed in Chinese hamster ovary cells overexpressing the insulin receptor (CHO-IR cells) and tyrosine phosphorylated upon insulin stimulation. We show that SHIP2, which is recruited in anti-phosphotyrosine immunoprecipitates in insulin-stimulated cells, accounts for the insulin sensitivity or apparent increase in activity reported by Guilherme et al. (J. Biol. Chem. 271, 29533-29536, 1996). Overexpression of SHIP2 led to a decrease of the insulin-dependent PIP3 production as well as Akt/PKB activation and MAPK stimulation.

The Src homology 2 domain containing inositol 5-phosphatase SHIP2 is recruited to the epidermal growth factor (EGF) receptor and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF-stimulated COS-7 cells.

The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged full-length enzyme was expressed in COS-7 cells and stained with anti-His6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc.

Identification of a second SH2-domain-containing protein closely related to the phosphatidylinositol polyphosphate 5-phosphatase SHIP.

Distinct inositol and phosphatidylinositol polyphosphates 5-phosphatases have recently been cloned. Primers have been designed coding for highly conserved amino acid regions that are shared between sequences of 5-phosphatases. One of the PCR fragment referred to as 51 C, shows 99% identity to a previously reported sequence (INPPL-1) present in the database. We report here the identification of cDNAs for a new SH2-domain-containing protein showing homology to the inositol 5-phosphatase SHIP and therefore referred to as SHIP2. SHIP2 differs at both N- and C-terminal ends with the sequence of INPPL-1. The translated sequence of SHIP2 encodes a 1258 amino acid protein with a predicted molecular mass of 142 kDa. Particularly high levels of SHIP2 were found in human heart, skeletal muscle and placenta as shown by Northern blot analysis. SHIP2 was also expressed in dog thyroid cells in primary culture where the expression was enhanced in TSH and EGF-stimulated cells.

Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells.

D-myo-Inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP3 and Ca2+ signaling. In brain and many other cells, type I InsP3 5-phosphatase is the major phosphatase that dephosphorylates InsP3 and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-phosphatase appears to be associated with the particulate fraction of cell homogenates. Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity. We have now compared the cytosolic Ca2+ ([Ca2+]i) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP3 5-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca2+]i was also measured in cells transfected with an InsP3 3-kinase construct encoding the A isoform. The Ca2+ oscillations detected in the presence of 1 microM ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP3 5-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells. All cells overexpressing the InsP3 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca2+]i returned to base-line levels in between a couple of oscillations. The [Ca2+]i responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry. In permeabilized cells, no differences in extent of InsP3-induced Ca2+ release nor in the threshold for InsP3 action were observed among the four clones and no differences in the expression levels of the various InsP3 receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP3 concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP3 5-phosphatase. This enzyme directly controls the [Ca2+]i response and the Ca2+ oscillations in intact cells.

Cloning and expression of a human placenta inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase.

Distinct inositol and phosphatidylinositol polyphosphate 5-phosphatases have recently been cloned. Primers were designated coding for highly conserved amino acid regions that are shared between sequences of 5-phosphatases. We used degenerate primers to amplify polymerase chain reaction products from rat brain cDNA. A product with a novel sequence was identified and used to clone a 4.9 kb cDNA from human placenta cDNA libraries (hp51CN). COS-7 cells transfected with a C-terminal truncated form of this cDNA showed an increase in Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 hydrolyzing activity, but not in Ins(1,4,5)P3 5-phosphatase. Enzymatic activity was inhibited in the presence of 2,3-bisphosphoglycerate and p-hydroxymercuribenzoate. The presence of an SH2 domain and proline-rich sequence motifs within hp51CN suggests that this 5-phosphatase interacts with various proteins in signal transduction.

Post-translational modification of human brain type I inositol-1,4,5-trisphosphate 5-phosphatase by farnesylation.

In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase (InsP3 5-phosphatase) is the major isoenzyme hydrolyzing the calcium-mobilizing second messenger InsP3. Activity of this enzyme could be measured in both soluble and particulate fractions of tissue homogenates. The protein sequence showed a putative C-terminal isoprenylation site (CVVQ). In this study, two mutants have been generated. The first mutant (C409S) has a serine replacing a cysteine at position 409 of the wild-type enzyme. The second mutant (K407D1) is a deletion mutant that lacks the last five C-terminal amino acids. These constructs were individually expressed by transfection in COS-7 cells. Western blot analysis of wild-type transfected cells indicated that both soluble and particulate fractions had a 43-kDa immunoreactive band, with a higher proportion of the original homogenate associated with the particulate part. On the contrary, when the two mutated constructs were transfected in COS-7 cells, the phosphatase was predominantly soluble. Confocal immunofluorescence studies showed the wild-type enzyme to be present on the cell surface of transfected COS-7 cells and in subcellular compartments around the nucleus. This was not observed for the two mutants, where uniform immunofluorescence labeling was observed throughout the cytosol. Recombinant type I InsP3 5-phosphatase expressed in Escherichia coli was a substrate of purified farnesyltransferase. Altogether, the data therefore suggest a direct participation of Cys-409 in a C-terminally anchored InsP3 5-phosphatase by farnesylation.

Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli. Lack of phosphorylation by protein kinase C.

The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase. The coding region of human brain type I InsP3 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The relative molecular mass of the purified fusion protein (MBP-InsP3-5-phosphatase) was approximately M(r) 85,000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mumol.mg-1.min-1. The molecular mass of purified protein by SDS/PAGE was M(r) 43,000. The activity was inactivated by p-hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP3 5-phosphatase was tested on the purified 43,000 M(r) protein. In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C.

Cloning and expression of human brain type I inositol 1,4,5-trisphosphate 5-phosphatase. High levels of mRNA in cerebellar Purkinje cells.

In brain and many other tissues, Type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isozyme hydrolysing the calcium-mobilizing second messenger InsP3. We recently reported the cloning and expression of dog thyroid InsP3 5-phosphatase. During the course of this cloning, screening of a human brain cDNA library allowed us to isolate a cDNA clone D1 with 91% sequence identity with the thyroid sequence. When clone D1 was expressed in Escherichia coli, the fusion protein had InsP3 5-phosphatase activity. M(r) estimates of the recombinant enzyme made by immunodetection, activity assay after SDS/PAGE or silver staining were consistent with the calculated molecular mass. In situ hybridization on human cerebellum sections localised the mRNA for this enzyme to the Purkinje cells.

Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase.

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.