Oxygen supply by photosynthesis to an implantable islet cell device.

Transplantation of islet cells is an effective treatment for type 1 diabetes with critically labile metabolic control. However, during islet isolation, blood supply is disrupted, and the transport of nutrients/metabolites to and from the islet cells occurs entirely by diffusion. Adequate oxygen supply is essential for function/survival of islet cells and is the limiting factor for graft integrity. Recently, we developed an immunoisolated chamber system for transplantation of human islets without immunosuppression. This system depended on daily oxygen supply. To provide independence from this external source, we incorporated a novel approach based on photosynthetically-generated oxygen. The chamber system was packed sandwich-like with a slab of immobilized photosynthetically active microorganisms (Synechococcus lividus) on top of a flat light source (LEDs, red light at 660 nm, intensity of 8 µE/m(2)/s). Islet cells immobilized in an alginate slab (500-1,000 islet equivalents/cm(2)) were mounted on the photosynthetic slab separated by a gas permeable silicone rubber-Teflon membrane, and the complete module was sealed with a microporous polytetrafluorethylene (Teflon) membrane (pore size: 0.4 µm) to protect the contents from the host immune cells. Upon illumination, oxygen produced by photosynthesis diffused via the silicone Teflon membrane into the islet compartment. Oxygen production from implanted encapsulated microorganisms was stable for 1 month. After implantation of the device into diabetic rats, normoglycemia was achieved for 1 week. Upon retrieval of the device, blood glucose levels returned to the diabetic state. Our results demonstrate that an implanted photosynthetic bioreactor can supply oxygen to transplanted islets and thus maintain islet viability/functionality.

A novel device for islet transplantation providing immune protection and oxygen supply.

Islet transplantation as a biological ß-cell replacement therapy has emerged as a promising option for achieving restoration of metabolic control in type 1 diabetes patients. However, partial or complete loss of islet graft function occurs in relatively short time (months to few years) after implantation. The high rate of early transplant dysfunction has been attributed to poorly viable and/or functional islets and is mediated by innate inflammatory response at the intravascular (hepatic) transplant site and critical lack of initial nutrient/oxygen supply prior to islet engraftment. In addition, the diabetogenic effect of mandatory immunosuppressive agents, limited control of alloimmunity, and the recurrence of autoimmunity limit the long-term success of islet transplantation. In order to abrogate instant blood-mediated inflammatory reaction and to provide oxygen supply for the islet graft, we have developed an extravascular (subcutaneous) transplant macrochamber (the 'ßAir' device). This device contains islets immobilized in alginate, protected from the immune system by a thin hydrophilized teflon membrane impregnated with alginate and supplied with oxygen by daily refueling with oxygen-CO (2) mixture. We have demonstrated successful utilization of the oxygen-refueling macrochamber for sustained islet viability and function as well as immunoprotection after allogeneic subcutaneous transplantation in healthy minipigs. Considering the current limitations of intraportal islet engraftment and the restricted indication for islet transplantation mainly due to necessary immunosuppressive therapy, this work could very likely lead to remarkable improvements in the procedure and moreover opens up further strategies for porcine islet cell xenotransplantation.

PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase.

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERalpha expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERalpha expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERalpha mRNA levels, suggesting that PRL signaling to the ERalpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERalpha promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERalpha promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERalpha expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.

Phenotypic expression of tissue mosaicism in a 45,X/46,X,dicY(q11.2) female.

We describe a girl who presented at the age of 11 years with short stature. She had female external genitalia and some clinical features of Turner syndrome. At laparotomy a uterus and Fallopian tubes and small gonad-like tissue masses in the region of the Fallopian fimbria were found. The tissue masses were removed and histological examination revealed no organized testicular or ovarian morphology. Remnants of Fallopian tubes, epididymis, and clusters of Leydig cells were seen but no Sertoli cells were found. Endocrine studies showed levels of sex hormones consistent with primary gonadal failure. G-banding analysis of 16 blood lymphocytes revealed the karyotype 46,X,dicY(q11.2) in all cells. Varying proportions of X and Y centromeres in blood lymphocytes, skin fibroblasts, and in the incompletely formed Wolffian and Müllerian duct derivatives were demonstrated by FISH. Molecular studies confirmed the absence of most of the long arm of the Y chromosome and an intact short arm. The SRY gene was shown to be present, but we presume that due to the mosaicism the dose was insufficient to allow normal testicular development.

Characterization of a rat uterine cell line, U(III) cells: prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor beta, alpha(2)-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway.

Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (U(III)) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, U(III) cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor beta (ER beta) and alpha(2)-macroglobulin (alpha(2)-MG), which are known to be PRL-regulated genes. However, when U(III) cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 and the transcription factor Stat5 were endogenously phosphorylated and their phosphorylation states were not enhanced in the presence of exogenous PRL. To examine whether the endogenously secreted PRL affects the expression of PRL-regulated genes, U(III) cells were treated with either an anti-PRL receptor antibody or a Jak2 inhibitor, AG490. The anti-PRL receptor antibody decreased alpha(2)-MG expression. AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding to its DNA consensus sequence, and also caused a dose-dependent down-regulation of alpha(2)-MG and ER beta expression. In contrast, AG490 enhanced PRL mRNA levels. In summary, we have established that the U(III) stromal cells of uterine origin produce PRL. Furthermore, we have shown for the first time that decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-regulate important genes involved in decidual growth and placentation.

Involvement of SOCS-1, the suppressor of cytokine signaling, in the prevention of prolactin-responsive gene expression in decidual cells.

The cells forming the rat decidua produce PRL and PRL-related proteins and express both the long and short forms of the PRL receptor. Yet, only a defined subpopulation, the mesometrial cells, express the PRL-dependent alpha2-macroglobulin gene. This gene is silenced in vivo in the antimesometrial cells and in the GG-AD cell line, derived from antimesometrial cells. To examine whether the lack of alpha2-macroglobulin expression is due to defective components in the PRL signaling pathway, we compared the relative expression of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 a and b (Stat5 a and b), suppressor of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2 mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of pseudopregnancy, the time of maximal alpha2-macroglobulin expression. We found no significant differences in the relative expression of either Jak2, Stat5 (a and b), or SHP-2 in the two cell populations. However, we discovered a profound difference in the expression of SOCS-1, an inhibitor of the Jak/Stat pathway. This gene was highly expressed in the antimesometrial cells and in the GG-AD cells, which do not produce alpha2-macroglobulin. Immunoprecipitation experiments with GG-AD cells revealed that although Jak2 and Stat5 coprecipitate in response to PRL stimulation, no phosphorylation of Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a role in silencing the alpha2-macroglobulin gene, we cultured GG-AD cells in the presence of either a SOCS-1 antisense oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28 h. Cells were also treated with PRL. Within 4 h of SOCS-1 antisense treatment, alpha2-macroglobulin mRNA expression was initiated. After 28 h, only cells treated with PRL and SOCS-1 antisense oligonucleotide retained the ability to express the alpha2-macroglobulin gene. In summary, results of this study reveal that constitutive expression of SOCS-1 can prevent PRL signaling and that the lack of PRL-induced expression of alpha2-macroglobulin in a defined decidual cell population is largely due to SOCS-1 expression in these cells.

The expression of interleukin-6 (IL-6), IL-6 receptor, and gp130-kilodalton glycoprotein in the rat decidua and a decidual cell line: regulation by 17beta-estradiol and prolactin.

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-beta (ERbeta). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0-100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-beta.

Molecular cloning and characterization of the rat ovarian 20 alpha-hydroxysteroid dehydrogenase gene.

The rat 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is an enzyme responsible for the catabolism of progesterone to the inactive 20 alpha-hydroxprogesterone. We have previously shown that the expression of this enzyme is not regulated by post-translational modification, but at the level of transcription. In this study we have established that the 20 alpha-HSD gene contains nine exons and have isolated a 2.5 kb promoter region. The transcription start site was identified and a TATA box was found. 5' deletions of this promoter significantly decreased basal promoter activity. Treatment with forskolin led to a dose dependent inhibition of the 2.5kg-20 alpha-HSD-luciferase construct. Computer analysis identified one CRE, two Nur77 response elements, two putative AP1 sites and one progesterone response element half-site. In summary, we have identified and partially characterized the promoter region of the rat ovarian 20 alpha-HSD and demonstrated that the regulatory elements for 20 alpha-HSD are present within a 2.5 kb 5' flanking region of the gene.

Interleukin-10 in preovulatory follicular fluid of patients undergoing in-vitro fertilization and embryo transfer.

PROBLEM: The present study aimed at investigating the presence of Interleukin-10 (IL-10) in preovulatory follicular fluid (FF) and its possible correlation with 17-beta-estradiol (E2) and progesterone (P) levels, and treatment outcome in patients undergoing in-vitro fertilization-embryo transfer (IVF-ET). METHODS: Twenty consecutive patients with tubal factor infertility who underwent oocyte retrieval for IVF-ET were assayed for pooled, preovulatory FF levels of IL-10, E2, and P. RESULTS: The mean FF levels of IL-10, E2, and P were 78.7 +/- 104.7 pg/ml, 2,787.0 +/- 726.1 pg/ml, and 1.5 +/- 0.8 ng/ml, respectively. No correlation was found between preovulatory FF concentration of IL-10, E2, oocyte number, oocyte fertilization rate, embryo quality, and pregnancy rate. The levels of IL-10 were found to be negatively correlated with P concentration, although not significantly (P = 0.057). CONCLUSION: Interleukin-10 exists in the preovulatory FF. Further investigations are needed to determine the role of IL-10 in the folliculogenesis.

Intrauterine signaling and embryonic implantation.

In mammals, the uterus is modified to be able to contain a pregnancy and nurture the developing embryo. In deciduate mammals, this is apparently due to formation of a special compartment, lined with decidual tissue, in which a semi-allogeneic (or even allogeneic, after embryo transplantation) pregnancy is accommodated. This review treats the mechanisms which have been evoked to explain the implantation of the egg and the decidualization of the implanting endometrium. At least three different neurochemicals have been considered to mediate induction of this response: histamine, prostaglandins and platelet-aggregating factor. Their importance is reviewed. The ability of the endometrium to transform into decidual tissue is contingent on the presence of the epithelium. The role of the epithelium is temporary, however, since it dies and is sloughed within a day of the induction. Studies of progesterone-dependent changes in the epithelial reaction to preimplantation pregnancy are considered.

Excess expression of uterine ribosomal protein genes P2 and S25 during the "implantation window" in the rat.

OBJECTIVE: The present study describes the isolation of "implantation-related" uterine cDNA clones. It further describes their temporal and spatial expression and their identification as the messengers for two ribosomal proteins: RP2 and RS25. METHODS: Libraries of cDNA, representing spayed rats treated with progesterone for 3 consecutive days and with estrogen for either 12 or 36 hours, were screened using homologous and heterologous probes. Two cDNA clones showing differential intensity of the signal were sequenced, the timing of their expression was analyzed by Northern analysis, and their spatial expression was visualized by in situ hybridization. RESULTS: The steady-state level of RP2 mRNA was temporally and spatially controlled by estrogen and progesterone. Whereas estrogen, alone or in combination with progesterone, stimulated this gene, the spatial distribution of the activation was different. Estradiol alone directed RP2 expression to the endometrial-myometrial border, whereas combined treatment increased epithelial staining and directed heavy expression to the stratum vasculare. In normal pregnancy, during the implantation-window period, RP2 was mainly expressed on the mesometrial side, with much less staining on the antimesometrial side. The steady-state levels of the RS25 messenger were elevated by estrogen alone or in combination with progesterone and were confined to the uterine epithelium. RS25 mRNA was evenly distributed throughout the uterine stroma during implantation. CONCLUSION: A developmental pattern of expression of RP2 is reported that corresponds temporally with the primary decidual response but is spatially expressed at the site of the secondary response.

Inhibition of decidual induction in rats by clomiphene and tamoxifen.

The objective of this study was to characterize the estrogen action that confers endometrial sensitization to nontraumatic deciduogenic stimuli by use of antiestrogens. Tamoxifen, ethamoxytriphetol, and clomiphene and its two component enantiomers inhibited decidual induction in pseudopregnant rats when administered 17 h before pyrathiazine. Unexpectedly, clomiphene (250 micrograms/rat) and tamoxifen (25 micrograms/rat) proved inhibitory at all times up to and including the time of induction. Clomiphene, administered in the hours preceding decidual induction, inhibited the increase of ornithine decarboxylase activity, which normally marks the end of the induction phase. Clomiphene had no inhibitory effect on the availability or receptor binding of progesterone. Clomiphene also inhibited implantation of blastocysts when administered at the time of their adherence to the uterus. The inhibition by antiestrogens of decidual induction could not be explained on the basis of the current understanding of mechanisms of estrogen action. The discrepancies were that no latent period between the time of antiestrogen administration and decidual induction was observed and no difference was observed in the inhibitory activities of the isomers of clomiphene.

Changes in gene expression following exposure of nulli-SCCl murine embryonal carcinoma cells to inducers of differentiation: characterization of a down-regulated mRNA.

cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells.

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.

Role of retinoids in differentiation and growth of embryonal carcinoma cells.

To study how retinoids promote differentiation and inhibit proliferation of embryonal carcinoma (EC) cells, we have followed their intracellular fate. Retinoic acid (RA) is effectively metabolized to more polar compounds by many EC lines. Unlike RA, retinol is slowly metabolized. Our inability to detect conversion of retinol to RA might indicate that the two retinoids elicit their effects on EC cells in different ways. Retinol added to cultures quickly appears in the nuclear fraction; the proportion associated with nuclei after detergent extraction is initially very low but increases with time. Retinol and RA might be translocated to nuclei by their respective binding proteins [cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP)]: isolated EC nuclei have specific, independent binding sites for both holoproteins but not their ligands. CRABP cannot be detected in the nucleoplasm of untreated EC cells, but activity is measurable after cells are exposed to RA. Interestingly, incubation with retinol promotes movement of both CRBP and CRABP into the nucleoplasmic fraction. Finally, we have demonstrated that brief exposure to RA dramatically reduces the cloning efficiency of EC cells. Since some cells are unaffected even by lengthy exposures to RA whereas the growth of their progeny is inhibited, we suggest that EC cells can become epigenetically refractory to RA.

Definition of period of induction of deciduoma in the rat using ornithine decarboxylase as a marker of growth onset.

The activity of uterine ornithine decarboxylase (ODC) was measured during the 24 hours after systemic induction of decidualization. Following a latent period of 2.5 hours, activity rose and reached a peak of 5.6 +/- 1.1 fold at 5 hours after induction. The activity then declined and a second, lower peak was seen at 21 hours. The increase in ODC activity was suppressed by treatment with cycloheximide (50 mg/kg) and dactinomycin, 500 microgram/rat, confirming that the enzyme response reflects protein synthesis. The increase and the base-line level of ODC activity were suppressed by ergocornine pretreatment (1 mg/rat) which disturbs the endocrine balance of pseudopregnancy. It was concluded that the growth and differentiation of decidual tissue began 2.5 hours after application of the induction stimulus.