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J S Hunt

Publications and source records attributed to J S Hunt.

At least 19 recordsLinked to original sources
PubMed1995

Temporal and spatial expression of HLA-G messenger RNA in extraembryonic tissues of transgenic mice.

HLA-G, a nonclassical class I molecule, is expressed by trophoblasts, the only fetal cells in direct contact with maternal tissue. Results of previous experiments suggested that a 244-bp region located over 1 kb 5' from exon 1 is critical for extraembryonic expression of HLA-G in transgenic mice. We report here the production of HLA-G transgenic lines with a 6.0-kb HLA-G transgene that includes the 244-bp region. These lines exhibit copy number-dependent and developmentally appropriate transgene expression. One HLA-G transgenic line, G.3.2, exhibits extraembryonic HLA-G mRNA expression levels similar to those seen in human extraembryonic tissues. Studies of the cell-type-specific localization of HLA-G mRNA in placentas from the G.3.2 HLA-G transgenic mouse line show predominant localization of the HLA-G message in the spongiotrophoblast layer. This layer is in a similar anatomic location to the HLA-G-expressing human cytotrophoblast shell. The G.3.2 HLA-G transgenic mouse line should serve as an appropriate model for the study of HLA-G function at the maternal-fetal interface.

Animals↗
PubMed1995

Cellular localization and hormonal regulation of inducible nitric oxide synthase in cycling mouse uterus.

Nitric oxide (NO), a potent and versatile free radical, is synthesized in macrophages and mast cells as well as in other types of cells by the inducible form of nitric oxide synthase (iNOS). In this study, cells containing iNOS were identified in the uteri of cycling mice by using a rabbit antibody generated to an iNOS-specific peptide. Macrophages were identified in semiserial sections of the same tissues with the monoclonal antibody, F4/80, and mast cells were identified by toluidine blue staining. In tissue sections of uteri obtained from mice in the four stages of the estrous cycle (8 to 11 mice per stage), iNOS immunoreactivity was strongest in diestrus-I uteri and weakest in diestrus-II uteri. Myometrial mast cells and endometrial epithelial cells were prominent locations of iNOS, and specific protein was also present in myometrial smooth muscle and macrophage-like cells in the endometrial stroma. Because cyclic variations suggested regulation of iNOS expression by ovarian steroid hormones, studies were done using ovariectomized mice. Seven days after ovariectomy, immunoreactive iNOS was low but detectable in mast cells and luminal epithelial cells. In the uteri of ovariectomized, estradiol-17 beta (E2)-treated mice, mast cells were iNOS+ after 24 h whereas epithelial cells were negative; the reverse was observed in progesterone (P4)-treated mice. Both mast cells and epithelial cells were iNOS+ in the uteri of mice that had received a combination of E2 + P4. These results indicate that several types of uterine cells produce iNOS and that expression of this enzyme in specific cell lineages is governed by ovarian steroid hormones. The data are consistent with the postulate that NO derived from uterine leukocytes and other types of cells plays a role in uterine cyclicity and preparation for pregnancy.

Amino Acid Oxidoreductases↗
PubMed1995

Synthesis and granular localization of tumor necrosis factor-alpha in activated NK cells in the pregnant mouse uterus.

The synthesis and cellular localization of tumor necrosis factor-alpha (TNF-alpha) were studied in mouse GMG cells, which are activated NK cells in uterine decidual tissue during pregnancy. Synthesis of the protein was demonstrated in GMG cells on days 10 and 14 of pregnancy by in situ hybridization of TNF-alpha message. Immunostaining demonstrated that TNF-alpha protein was localized in the cytoplasmic granules of GMG cells at these times. The results suggest that the cytolytic activity of uterine NK cells may be due in part to TNF-alpha, and that this cytokine may be delivered to target cells intracellularly via transmembrane pores formed by perforin, which is also localized in uterine NK cell granules.

Animals↗
PubMed1995

Myometrial tumor necrosis factor alpha: cellular localization and regulation by estradiol and progesterone in the mouse.

The expression of tumor necrosis factor alpha (TNF) protein in mouse myometrial cells on each day of the estrous cycle and after ovariectomy, with and without replacement of estradiol (E2), progesterone (P4), or E2 + P4, was investigated. TNF protein was assessed by immunocytochemistry. In addition, the numbers of mast cells and the numbers of TNF-containing mast cells were determined under each condition. The total number of mast cells fluctuated during the estrous cycle; and at each stage, essentially 100% of the mast cells exhibited TNF immunoreactivity. In contrast, after ovariectomy and after ovariectomy with E2 replacement, only approximately 50% of the mast cells contained immunoreactive TNF. P4 treatment resulted in further decline in mast cell TNF whereby after 24 h of P4, only 3% of the mast cells were TNF-positive and by 72 h of treatment, no TNF-positive mast cells could be detected E2 and P4 in combination increased the total number of mast cells, and in a pattern reminiscent of normal cycling myometrium, over 90% of the mast cells exhibited TNF immunoreactivity after 24 h of treatment. TNF protein was detectable in muscle cells throughout the estrous cycle, with weak immunostaining observed on proestrus and more intense immunostaining on estrus, diestrus-I, and diestrus-II. After ovariectomy, only light immunostaining was observed in the muscle cells. Immunoreactive TNF was detected in muscle cells after each type of steroid treatment. E2 and E2 + P4 treatments resulted in a biphasic pattern of immunoreactive TNF expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A cautionary note.

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Abortion, Habitual↗
PubMed1995

Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death.

The low-affinity receptor for leukemia inhibitory factor (LIFR) interacts with gp130 to induce an intracellular signal cascade. The LIFR-gp130 heterodimer is implicated in the function of diverse systems. Normal placentation is disrupted in LIFR mutant animals, which leads to poor intrauterine nutrition but allows fetuses to continue to term. Fetal bone volume is reduced greater than three-fold and the number of osteoclasts is increased six-fold, resulting in severe osteopenia of perinatal bone. Astrocyte numbers are reduced in the spinal cord and brain stem. Late gestation fetal livers contain relatively high stores of glycogen, indicating a metabolic disorder. Hematologic and primordial germ cell compartments appear normal. Pleiotropic defects in the mutant animals preclude survival beyond the day of birth.

Animals↗
PubMed1995

Tumor necrosis factor-alpha in the female reproductive tract.

Tumor necrosis factor-alpha (TNF), originally identified as an inflammation-associated cytokine, is synthesized throughout the female reproductive tract as well as in placentas and embryos. Development, female sex steroid hormones, and lipopolysaccharide influence expression of this gene. The functions of TNF may be determined in part by differential expression of the two species of TNF receptors, both of which seem to be regulated by female sex steroid hormones. Evidence has accumulated that supports a role for this potent, pleiotropic cytokine in autocrine and paracrine processes central to reproduction, including gamete and follicle development, steroidogenesis, uterine cyclicity, placental differentiation, development of the embryo, and parturition.

Animals↗
PubMed1994

Identification and cellular localization of unique interferon mRNA from human placenta.

Although constitutive expression of trophoblast or pregnancy-associated interferon (IFN) has long been recognized, their cDNA sequences have been determined for only ruminant ungulates. Here we show a human trophoblast IFN (htIFN) cDNA whose nucleotide sequence is very similar (85% identity) to that of ovine and bovine trophoblast IFNs, IFN tau s. Like ruminant IFN tau s, htIFN cDNAs contain an open reading frame of 195 codons including a signal sequence of 23 amino acids, resulting in a mature polypeptide of 172 amino acids. The deduced amino acid sequence of htIFN shares 73, 62, and 56% identities with ovine IFN tau, human IFN alpha II, and human IFN alpha I, respectively. However, the expression of htIFN is not limited to a specific period of pregnancy because transcripts of htIFN genes are detected in human lymphocytes, cells obtained by amniocentesis (amniocytes), first trimester, and term placentas. Human trophoblast IFN mRNA is localized mainly in extravillous trophoblasts cells of placental villi, particularly in the migrating cytotrophoblasts cells, which eventually replace maternal endothelial cells in spiral arteries of the decidua. Both sense and antisense mRNAs for human IFN alpha II are localized in the outer layer of villous structures. Coexistence of these mRNAs at the placental villi throughout pregnancy suggests that, in addition to a role in placental cell growth and differentiation, IFNs may play a role protecting the fetus in viral environments.

Amino Acid Sequence↗
PubMed1994

Gestation-related expression of the interferon-gamma receptor gene in mouse uterine and embryonic hematopoietic cells.

Macrophages and natural killer (NK)-like cells are the major hematopoietic cell populations in the cycling and pregnant mouse uterus and are also found in the embryo. In order to evaluate potential receptivity of these cells to interferon-gamma (IFN-gamma), tissues taken from cycling and pregnant mice were tested for IFN-gamma receptor (IFN-gamma R) mRNA and protein. Macrophages were identified immunohistochemically by using the specific monoclonal antibody F4/80. NK cells were identified by their large size, distinctive intracellular granules, and binding of a monoclonal antibody to the common leukocyte antigen. In cycling uteri, the abundance of IFN-gamma R mRNA relative to an invariant message (glyceraldehyde-3-phosphate dehydrogenase) increased during progression of the hormonally regulated estrous cycle. IFN-gamma R mRNA in situ hybridization signals were slightly higher in macrophage-like than in other types of endometrial stromal cells. In pregnant uteri, the highest proportions of IFN-gamma R mRNA were observed at gestation day (g.d.) 16. Specific message and protein were present in uterine macrophages by g.d. 7 and in NK cells by g.d. 9. IFN-gamma R expression in both lineages remained stable through the balance of pregnancy. In embryos, IFN-gamma R mRNA increased between g.d. 14 and 16. Specific transcripts were present in many cells at g.d. 14, but none were detected in embryonic liver macrophages until g.d. 16. The results of this study suggest relationships between IFN-gamma R expression and ovarian hormones as well as cell maturation and support the postulate that IFN-gamma receptor-ligand interactions may improve the ability of uterine and embryonic hematopoietic cells to perform specific tasks during gestation.

Animals↗
PubMed1994

Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry.

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.

Adenocarcinoma↗
PubMed1994

Differential responses of phenotypically distinct rat trophoblast cell lines to MHC class I antigen-inducing cytokines.

Phenotypically distinct rat trophoblast cell lines, the Rcho-1 and R8RP.3 cells, were compared for their responses to cytokines known to induce major histocompatibility (MHC) class I antigens, tumour necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma). Cell enzyme immunosorbent assays and flow cytometry experiments showed that only IFN-gamma could induce RT1 class I antigens on the Rcho-1 cells. Non-adherent cells were slightly less responsive than adherent, giant cell-like Rcho-1 cells. By contrast, RT1 class I antigens on the R8RP.3 cells were induced by both TGF-beta 1 and IFN-gamma. The cytokines also had different effects on mitochondrial enzyme activity in the two lines. TNF and TGF-beta 1 mRNAs were demonstrated in both lines by using Northern blot hybridization. Rcho-1 but not R8RP.3 cells contained two TNF messages (approximately 2.2, 1.9 kb). Steady state levels of transcripts from the TNF gene, and, to a lesser extent, the TGF-beta 1 gene, were increased in cultures of Rcho-1 cells that contained high proportions of giant cells. Thus, phenotypically distinct rat trophoblast cell lines do not respond identically to TNF, TGF-beta 1 or IFN-gamma, transcription of cytokine genes does not prevent the cells from responding to paracrine cytokine signals, and the cells contain novel TNF transcripts that might be important in cell maturation or differentiation.

Animals↗
PubMed1994

Immunologically relevant cells in the uterus.

Macrophages and a special subset of lymphocyte natural killer (NK) cells populate the uteri of cycling humans, mice, and rats. After implantation, major changes take place that have important functional implications. The macrophages and NK cells increase in number, are redistributed into specific uterine compartments, and exhibit markers consistent with cell activation. Activation enhances macrophage and NK cell production of a wide range of pleiotropic, multifunctional polypeptide growth factors, reactive oxygen intermediates, and bioactive lipids. Thus, activated uterine hematopoietic cells are equipped to perform certain immunological and nonimmunological functions within their microenvironments that could have major influences on the course of pregnancy.

Animals↗
PubMed1994

Mouse endometrial tumor necrosis factor-alpha messenger ribonucleic acid and protein: localization and regulation by estradiol and progesterone.

Tumor necrosis factor-alpha (TNF) messenger RNA (mRNA) and protein have been identified in the uteri of cycling rats, mice, and women. In this study, a mouse model was used to investigate the cell-specific expression and regulation of the TNF gene in the endometrium. Uteri from cycling and ovariectomized hormone-reconstituted mice were tested by in situ and Northern blot hybridizations as well as by immunohistochemistry. Cyclic variations were observed in TNF mRNA. Although weak to undetectable during proestrus, estrus, and diestrus-I, TNF mRNA was present in both epithelial and stromal cells on diestrus-II. TNF protein was observed in the endometrium on each day of the estrous cycle, primarily in the epithelial cells. Stromal TNF immunoreactivity was observed only during diestrus-I. Seven days after ovariectomy, TNF mRNA and protein were undetectable in the endometrium. Specific message and protein were restored in both epithelial and stromal cells after administration of 17 beta-estradiol (E2), progesterone (P4), and E2 plus P4. E2 treatment resulted in a biphasic pattern of TNF mRNA expression. mRNA was present in epithelial cells 1 and 6 h posttreatment, was not detectable after 24 h, and then was present in both the epithelium and stroma after 72 h. In contrast, TNF mRNA was detectable at all time points after P4 administration. TNF mRNA was localized to both the epithelium and stroma until 72 h of P4 treatment, when stromal TNF mRNA was no longer detectable. TNF mRNA was also present at all time points examined after the combination E2 and P4 treatment. TNF mRNA was invariably localized to the epithelium; however, stromal mRNA fluctuated over the course of treatment. TNF mRNA was in the stroma 1 and 24 h post-E2 plus P4 treatment, but was not present at the 6 and 72 h points. In general, the pattern of TNF protein matched that of the TNF mRNA, with a few exceptions. Twenty-four hours after E2 treatment, TNF mRNA was not detectable; however, TNF protein was present in the epithelium. The opposite was observed 24 h after E2 plus P4 administration when the epithelium and stroma contained TNF mRNA; however, no protein was observed. In addition, with the exception of 72 h of E2 treatment, stromal TNF protein was not abundant, although TNF mRNA was often detected. In summary, the results of this study indicate that both epithelial and stromal cells contain TNF mRNA and protein and that E2 and P4 exert individual influences on the cell-specific expression of TNF transcripts and protein.

Animals↗
PubMed1994

Expression of HLA class II-associated peptide transporter and proteasome genes in human placentas and trophoblast cell lines.

Expression of HLA class I antigens is closely controlled in the placental trophoblast cells, which interface directly with maternal cells during pregnancy. In this study, the possibility that peptide transporter (TAP-1, TAP-2) or proteasome (LMP7) genes might be involved in regulating antigen expression in these or other cells that comprise placentas was investigated. Analysis by Northern blot hybridization showed that transcripts from all three genes were present in samples of first trimester and term placental RNA. TAP-1 and TAP-2 messages were consistently more abundant in early than in late gestation placentas, whereas the reverse was observed for LMP7 mRNA. Futher experiments were done on two trophoblast cell lines. One line, Jar, is negative for HLA class I, and the second, JEG-3, expresses HLA-G as well as other HLA class I genes. Both Jar and JEG-3 cells contained TAP-1, TAP-2 and LMP7 mRNA. With the exception of LMP7 in JEG-3 cells, message from all three genes was increased by treating the trophoblast cells with interferon-gamma. While no evidence was collected to support the postulate that the HLA class I negative status of some trophoblast cell subpopulations could be related to absent or dysfunctional TAP-1, TAP-2 or LMP7 mRNA, the data are consistent with the postulate that placental cell expression of HLA class I antigens could be influenced by the availability of peptide transporters and proteasome components.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Cellular distribution of HLA-G mRNA in transgenic mouse placentas.

The human MHC class I gene, HLA-G, is unique among members of the class I gene family in that it is nonpolymorphic, and expression is primarily restricted to extraembryonic tissues. To examine regulatory elements that direct tissue- and cell lineage-specific expression of this gene, transgenic mice expressing HLA-G have been established. In this study, in situ hybridization was used to evaluate the cellular distribution of HLA-G mRNA in transgenic placentas. Extraembryonic tissues were obtained at gestation day 12.5 from embryos that had been microinjected with either 6.0 or 5.7 kb of HLA-G genomic DNA and had been transferred into pseudopregnant HLA-G transgenic mice or Swiss mice. The 6.0 kb transgene contained an additional 250 bp at the extreme 5'-end of the upstream region. Genotype of the recipient had no discernable effect on the cellular distribution of HLA-G mRNA. HLA-G mRNA was present in both trophoblast and mesenchymal cells in transgenic placentas carrying 6.0 kb of genomic HLA-G, a pattern strikingly similar to that of HLA-G message distribution in early gestation human placentas. By contrast, in placentas from embryos carrying 5.7 kb HLA-G DNA, specific mRNA was found primarily in mesenchymal cells at the base of the placenta. Thus, the 6.0 genomic fragment contains elements capable of directing HLA-G expression in placentas, and is particularly influential in the trophoblastic cell lineage.

Animals↗