Biomedical subjects

G A Johnson

Publications and source records attributed to G A Johnson.

At least 73 records · Page 4Linked to original sources

PubMed1999

Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes.

Interferon tau (IFNtau) is the antiluteolytic signal produced by the conceptus of ruminants. Intrauterine administration of recombinant ovine IFNtau suppresses expression of endometrial estrogen receptor (ER) and oxytocin receptor (OTR) in the luminal and superficial glandular epithelia to abrogate the production of luteolytic prostaglandin F(2alpha) (PGF(2alpha)) pulses. Subcutaneous (s.c.) injections of recombinant ovine (o) IFNtau appear to extend the interestrous interval by altering uterine PGF(2alpha) response to oxytocin. The present study tested the hypothesis that antiluteolytic effects of roIFNtau injected into the uterine lumen (paracrine) or s.c. (endocrine) are equivalent in suppressing expression of endometrial ER and OTR and inducing uterine expression of type I IFN-regulated Mx and ubiquitin cross-reactive proteins (UCRP). Sixteen cyclic ewes were fitted with uterine catheters on Day 5 (Day 0 = estrus), were assigned randomly to receive treatment with control proteins or roIFNtau (2 x 10(7) antiviral units/day) by either intrauterine or s.c. injections from Days 11 to 15, and were ovariohysterectomized on Day 16. Results indicated that expression of ER and OTR mRNAs in endometrial epithelium was suppressed by intrauterine but not by s.c. injections of roIFNtau. Intrauterine injections of roIFNtau increased expression of Mx and UCRP mRNA in the endometrium. Subcutaneous injections of roIFNtau increased endometrial Mx mRNA levels but not UCRP mRNA. Unexpectedly, intrauterine and s.c. injections of roIFNtau were equally effective in inducing expression of Mx and UCRP mRNA in the corpus luteum. Although s.c. injections of roIFNtau induced Mx mRNA in the endometrial epithelium, s.c. injections of roIFNtau did not abrogate activation of the uterine luteolytic mechanism by suppressing epithelial ER and OTR expression. Therefore, results of this study failed to support the assumption that endocrine roIFNtau mimics antiluteolytic effects of paracrine IFNtau to improve pregnancy rates in sheep.

Animals↗

PubMed1999

Ovine osteopontin: I. Cloning and expression of messenger ribonucleic acid in the uterus during the periimplantation period.

Trophoblast-derived interferon tau (IFNtau) acts on the endometrium to increase secretion of several proteins during the pregnancy recognition period in ruminants. One of these is a 70-kDa acidic protein that has not been identified. Our hypothesis was that the 70-kDa acidic protein is osteopontin (OPN). OPN is an acidic glycoprotein that fragments upon freezing and thawing or treatment with proteases including thrombin. OPN contains a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins to promote cell-cell attachment and cell spreading. Using antisera to recombinant human OPN, both 70-kDa and 45-kDa proteins were identified in uterine flushings from pregnant ewes by Western blotting. A clone containing the entire ovine OPN cDNA coding sequence was isolated by screening a Day 15 pregnant ovine endometrial cDNA library with a partial ovine OPN cDNA. In pregnant ewes, steady-state levels of OPN endometrial mRNA increased (P < 0. 01) after Day 17. In both cyclic and pregnant ewes, in situ hybridization analysis showed that OPN mRNA was localized on unidentified immune cells within the stratum compactum of the endometrium. In pregnant ewes, OPN mRNA was also expressed by the glandular epithelium. Results suggest that progesterone and/or IFNtau induce expression and secretion of OPN by uterine glands during the periimplantation period and that OPN may induce adhesion between luminal epithelium and trophectoderm to facilitate superficial implantation.

Amino Acid Sequence↗

PubMed1999

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.

Animals↗

PubMed1999

Development and characterization of immortalized ovine endometrial cell lines.

The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.6-0.8 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for more than 40 passages. Phase-contrast microscopy revealed that LE and GE cells exhibited a cobblestone morphology whereas immortalized ST cells were spindle shaped. The epithelial origin of LE and GE was confirmed by positive cytokeratin immunostaining, and ST cells were vimentin positive. All cell lines were negative for smooth muscle alpha-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFNtau) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFNtau. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium.

Animals↗

PubMed1999

Effects of recombinant ovine interferon tau, placental lactogen, and growth hormone on the ovine uterus.

Studies were conducted to determine effects of intrauterine administration of recombinant ovine interferon tau (IFNtau), placental lactogen (PL), and growth hormone (GH) on endometrial function. In the first study, administration of IFNtau to cyclic ewes for one period (Days 11-15) resulted in an interestrous interval (IEI) of approximately 30 days, whereas administration for two periods (Days 11-15 and Days 21-25) extended the IEI to greater than 50 days. Administration of IFNtau from Days 11 to 15 and of PL or GH from Days 21 to 25 failed to extend the IEI more than for IFNtau alone. In the second study, effects of IFNtau, PL, and GH on endometrial differentiation and function were determined in ovariectomized ewes receiving ovarian steroid replacement therapy. Endometrial expression of mRNAs for estrogen receptor (ER), progesterone receptor (PR), and oxytocin receptor (OTR) were not affected by PL or GH treatment; however, uterine milk protein mRNA levels and stratum spongiosum gland density were increased by both PL and GH treatments. Collectively, results indicated that 1) PL and GH do not regulate endometrial PR, ER, and OTR expression or affect corpus luteum life span; 2) down-regulation of epithelial PR expression is requisite for progesterone induction of secretory gene expression in uterine glandular epithelium; 3) effects of PL and GH on endometrial function require IFNtau; and 4) PL and GH regulate endometrial gland proliferation and perhaps differentiated function.

Animals↗

PubMed1999

Mechanism of action of interferon-tau in the uterus during early pregnancy.

Early pregnancy is maintained in ruminants through the actions of conceptus-derived interferon (IFN)-tau on the endometrium. IFN-tau alters uterine release of PGF2 alpha' which results in rescue of the corpus luteum and continued release of progesterone. The mechanism of action of IFN-tau includes inhibition of oestradiol receptors, consequent reduction in oxytocin receptors, activation of a cyclooxygenase inhibitor, and a shift in the PGs to favour PGE2 over PGF2 alpha' IFN-tau also induces several endometrial proteins that may be critical for survival of the developing embryo. One endometrial protein induced by pregnancy and IFN-tau has been identified as bovine granulocyte chemotactic protein-2 (bGCP-2). This chemotactic cytokine (chemokine) has been used as a marker to delineate IFN-tau from IFN-alpha responses in the endometrium. A second protein, called ubiquitin cross-reactive protein (UCRP), resembles a tandem ubiquitin repeat. UCRP becomes conjugated to cytosolic endometrial proteins in response to IFN-tau and pregnancy. Proteins conjugated to UCRP are either modulated or targeted for processing through the proteasome. The action of IFN-tau is mediated by induction of signal transducer and activator of transcription 1 (STAT-1), STAT-2 and interferon regulatory factor 1 (IRF-1) transcription factors. Induction of these transcription factors, the alpha chemokines and UCRP is the prelude to maternal recognition of pregnancy in ruminants.

Amino Acid Sequence↗

PubMed1998

In vivo magnetic resonance vascular imaging using laser-polarized 3He microbubbles.

Laser-polarized gases (3He and 129Xe) are currently being used in magnetic resonance imaging as strong signal sources that can be safely introduced into the lung. Recently, researchers have been investigating other tissues using 129Xe. These studies use xenon dissolved in a carrier such as lipid vesicles or blood. Since helium is much less soluble than xenon in these materials, 3He has been used exclusively for imaging air spaces. However, considering that the signal of 3He is more than 10 times greater than that of 129Xe for presently attainable polarization levels, this work has focused on generating a method to introduce 3He into the vascular system. We addressed the low solubility issue by producing suspensions of 3He microbubbles. Here, we provide the first vascular images obtained with laser-polarized 3He. The potential increase in signal and absence of background should allow this technique to produce high-resolution angiographic images. In addition, quantitative measurements of blood flow velocity and tissue perfusion will be feasible.

Angiography↗

PubMed1998

Magnetic resonance microscopy and histopathology: comparative approach of bromobenzene-induced hepatotoxicity in the rat.

The development of magnetic resonance (MR) microscopy has provided new approaches to histology and histopathology. Recent work has shown the promise of increased sensitivity in animal models of chemically induced hepatotoxicity. However, the field is so new that there is little experience to relate changes seen in MR micrographs to the more traditional optical images stained with hematoxylin and eosin. This work compares the sensitivity and reproducibility of MR microscopy with conventional histopathology in detecting bromobenzene-induced hepatotoxicity in the rat. A time-course study was undertaken to provide a range of histopathologies. Specimens were studied at 24, 48, 72, and 96 hours after exposure to 10% of the median lethal dose of bromobenzene. Using 4 animals per group (a total of 32 rats) added statistical significance to the study and defined a range of interanimal variability over 96 hours. This work shows that MR microscopy, besides being nondestructive and three-dimensional, is at least as sensitive as conventional hematoxylin-eosin staining in detecting bromobenzene-induced centrilobular lesions and recovery of the hepatocellular architecture in the rat. This study further suggests that, as we begin to understand the underlying mechanisms of contrast in MR histology, MR may, in fact, supply even higher specificity than more traditional studies: variations were observed in MR images of treated livers at a given time point that could be not be differentiated based on the grading of necrosis and inflammation on hematoxylin-eosin-stained sections.

Animals↗

PubMed1998

A fast spin echo technique with circular sampling.

This paper presents a fast spin echo (FSE) imaging method that employs circular sampling of k-space. The technique has been implemented on a 2 Tesla imaging system and validated on both phantoms and living animals. Experimental studies have shown that circular sampling can produce artifact-free FSE images without the need of phase correction. Although not fully explored, preliminary results also show that circular sampling may have advantages over the conventional rectilinear FSE in signal-to-noise ratio and imaging efficiency. A major disadvantage is the increased sensitivity to off-resonance effects. The authors expect that the FSE technique with circular sampling will find its applications in magnetic resonance microscopy, neuro-functional imaging, and real-time dynamic studies.

Animals↗

PubMed1998

MR microscopy of lung airways with hyperpolarized 3He.

A technique using hyperpolarized (HP) 3He to image the small airways of the lung by using moderate flip angles and a short scanning period during early inspiration is demonstrated. Flip angles (alpha) ranging from 10-90 degrees were used in guinea pig experiments with scanning during the entire inspiration period. A second series acquired data throughout a short window of the ventilatory cycle with alpha = 45 degrees. The success of the animal studies has motivated implementation of similar imaging techniques in the clinical arena. Human studies involved imaging over the total inspiration period with alpha approximately 10 degrees. The first series of guinea pig experiments demonstrated that larger flip angles (50-90 degrees) destroy the magnetization before it reaches the smaller airways. At moderate flip angles (20-40 degrees), airway branching down to the fourth generation was apparent. Fifth-order branchings were seen in the images of the second series. The trachea down to fourth generation pulmonary airway branching, along with some distal air spaces, was seen in the human lung images.

Animals↗

PubMed1998

Time-course imaging of rat embryos in utero with magnetic resonance microscopy.

Magnetic resonance (MR) microscopy was used to noninvasively investigate the development of live rat embryos in utero. The difficulty in making sequential observations of a developing mammalian embryo has frustrated developmental biologists for many years. Most current technologies analyze normal and abnormal development by observing end point phenotypes (in fixed specimens) rather than investigating the live embryo. MR microscopy was adapted to allow rat litters to be scanned three times each (at 1- to 3-day intervals) and has produced images of live developing embryos. It was demonstrated that repeated anesthesia and imaging protocols produced no gross malformations in the rat pups that were subsequently delivered and observed. Three-dimensional projection encoding with phase rewinders produced isotropic [256(3)] image data sets in about 30 minutes with excellent tissue contrast arising from steady-state effects in the amniotic fluid.

Animals↗

PubMed1998

Hyperpolarized 3He NMR lineshape measurements in the live guinea pig lung.

Spatially localized lineshapes of hyperpolarized (HP) 3He in guinea pig lungs have been measured in vivo. Three different axial slice locations, each containing different compositions of airway sizes and orientations, were studied. Gas peaks from major bronchi (2 ppm) and alveoli (-2 ppm) were distinguished. The gas phase spectra show structural features that are a result of frequency shifts caused by bulk magnetic susceptibility. For a given slice, the spectral lineshapes reflect the airway composition within the slice location, according to theory. The peak assignments given here also agree with previous studies done by Wagshul et al. with HP 129Xe. At each of the slice locations, data were acquired during two phases of the breathing cycle, resulting in a relative frequency shift of approximately 0.3 ppm in the superior slices. Spectra obtained over a number of breaths show the dynamics of the gas buildup in the lung and provide further evidence supporting the peak assignments.

Animals↗

PubMed1998

Signal dynamics in magnetic resonance imaging of the lung with hyperpolarized noble gases.

The nonequilibrium bulk magnetic moment of hyperpolarized (HP) noble gases generated by optical pumping has unique characteristics. Based on the Bloch equations, a model was developed describing the signal dynamics of HP gases used in magnetic resonance imaging (MRI) of the lung with special consideration to the breathing cycle. Experimental verification included extensive investigations with HP 3He and 129Xe during both inspiration and held breath in live guinea pigs. Radial acquisition was used to investigate the view variations with a temporal resolution of 5 ms. Agreement between theoretical predictions and in vivo results was excellent. Additionally, information about effects from noble gas diffusion and spin-lattice relaxation was obtained. In vivo results for T1 were 28.8 +/- 1.8 s for 3He and 31.3 +/- 1.8 s for 129Xe. Comparison with in vitro data indicated that relaxation in the pulmonary gas space is dominated by dipolar coupling with molecular oxygen. The results provide a quantitative basis for optimizing pulse sequence design in HP gas MRI of the lung.

Animals↗

PubMed1998

Laparoscopic paraaortic lymphadenectomy using laparosonic coagulating shears.

With marked innovations in endosurgical instrumentation, operative laparoscopy to include lymphadenectomy has become feasible and has a valuable role in the management of gynecologic malignancy. We used laparosonic coagulating shears (LCS) for laparoscopic paraaortic lymphadenectomy in two women with cervical carcinoma. Operating times for the laparoscopic portion were 55 and 65 minutes and blood loss was 20 and 30 ml, respectively. No surgical complications were encountered. Lymphatic tissues were evaluated histologically and no thermal artifacts were identified. The major advantage of the ultrasonically activated scalpel of the LCS is the ability to cut and coagulate tissues simultaneously without electrical current. The LCS may afford the surgeon a greater margin of safety than unipolar electrocoagulation scissors by eliminating potential thermal and electrical injury to vital structures. Ultrasonic-activated technology deserves extended clinical investigation in laparoscopic lymphadenectomy to substantiate our preliminary findings, as well as to explore its potential in gynecologic oncology.

Adult↗

PubMed1998

Pregnancy and interferon-tau induce conjugation of bovine ubiquitin cross-reactive protein to cytosolic uterine proteins.

Conceptus-derived interferon-tau (IFN-tau) induces bovine endometrial ubiquitin cross-reactive protein (UCRP) mRNA and protein on Days 15-21 of pregnancy. Bovine UCRP retains the Leu-Arg-Gly-Gly C-terminal sequence of ubiquitin that ligates to and directs degradation of cytosolic proteins. The objectives of the present experiments were to determine whether UCRP became conjugated to endometrial cytosolic proteins during early pregnancy and in response to recombinant bovine (rbo) IFN-tau. Ubiquitin (8 kDa), UCRP (17 kDa), and conjugates thereof (> or = 30 kDa) were quantitated using Western blotting and densitometry. Endometrial ubiquitin and its conjugates did not differ between Day 18 pregnant and nonpregnant cows, or between control and rboIFN-tau-treated (25 nM) explant cultures (Day 14; nonpregnant). Bovine UCRP was induced in endometrium from pregnant as compared with nonpregnant cows. Conjugation of endometrial proteins to UCRP was induced in pregnant as compared to nonpregnant cows. Recombinant boIFN-tau induced UCRP and its conjugates in cultured endometrial explants from nonpregnant cows. It is concluded that UCRP, in response to rboIFN-tau, becomes conjugated to endometrial cytosolic proteins during early pregnancy. The regulation of uterine proteins by UCRP may be integral to the maintenance of early pregnancy in ruminants.

Amino Acid Sequence↗

PubMed1998

Complex induction of bovine uterine proteins by interferon-tau.

Interferon-tau (IFN-tau) is released by the conceptus and induces two uterine proteins during early pregnancy: ubiquitin cross-reactive protein (UCRP) and granulocyte chemotactic protein-2 (GCP-2). The present experiments were designed to determine whether detection (Western blot) of cytosolic UCRP and release of GCP-2 could be used to examine IFN-tau signal transduction in cultured endometrial explants and primary epithelial cells. Recombinant (r) type 1 IFNs (rboIFN-tau and rboIFN-alpha; 5, 25, 100 nM) induced UCRP, but only rboIFN-tau induced GCP-2 in explant culture. Recombinant boIFN-tau and conceptus secretory proteins containing native IFN-tau induced UCRP and GCP-2 in cultured primary epithelial cells. All concentrations of rboIFN-alpha (25, 50, 100 nM) induced UCRP, but only the highest concentration induced GCP-2 in cultured primary epithelial cells. Interestingly, phorbol ester (100, 500, 1000 ng/ml) induced GCP-2, but it had no effect on UCRP. Because type 1 IFNs induce UCRP, IFN-tau probably interacts with the janus kinase (Jak)-associated IFN-alpha receptor to phosphorylate signal transducers and activators of transcription (STAT) and/or interferon regulatory factor-1 (IRF-1). However, IFN-tau-specific induction of GCP-2 may involve a variant type 1 receptor subunit or activators of transcription that are associated with protein kinase C and the Jak/STAT/IRF-1 pathway.

Animals↗

PubMed1998

A phase II trial of isotretinoin and alpha interferon in patients with recurrent squamous cell carcinoma of the cervix: a Gynecologic Oncology Group study.

From January 1993 through January 1996, 37 patients with unresectable squamous carcinoma of the cervix were entered on study and scheduled to receive oral isotretinoin 1 mg/kg per day with subcutaneous alpha interferon 6,000,000 units/day. A course was defined as 4 continuous weeks of therapy. The mean number of four-course cycles delivered was 1.8. One patient was ineligible because of wrong cell type and two were never treated. Thus, 34 patients were evaluable for toxicity. Eight patients were inevaluable for response. Five did not receive a complete 4-week course and three did not have additional tumor measurements; thus 26 were evaluable for response. Prior radiotherapy had been given to 25 patients and prior chemotherapy to 23 patients. There was no grade 4 neutropenia. The incidence of Gynecologic Oncology Group (GOG) grade 3 granulocytopenia and thrombocytopenia was 8.8% and 5.8%, respectively. Six patients (17.6%) developed grade 3 or worse nausea and vomiting. Four (11.7%) patients developed grade 3 neurologic symptoms. There were no complete responses and one partial response. The overall response rate was 3.8% (95% confidence interval, 0.1-19.6%). In this pretreated population, isotretinoin and alpha interferon in the dose and schedule employed exhibit minimal activity.

Adult↗

PubMed1998

Measuring the progression of foreign-body reaction to silicone implants using in vivo MR microscopy.

We used in vivo magnetic resonance (MR) microscopy to follow the growth of fibrous capsule as a foreign body reaction to silicone implants in rats. Anesthetized rats were imaged 1, 7, 14, and 28 days after silicone-coated MR imaging coils were sutured to their neck muscles. On the twenty-eighth day, rats were sacrificed and coils and adjacent tissues were removed en bloc and fixed in formalin, reimaged with MR, and sectioned for conventional histology. Three-dimensional (3-D) spin-echo [3DFT] acquisition gave in-plane resolution of 32 x 32 microns in vivo and 16 x 16 microns ex vivo. All MR images showed a diffuse band of elevated signal intensity between the silicone of the coil and adjacent tissue. The border of the hyperintense band was thin and not well defined at seven days post-implantation. From 7-28 days, the band showed relatively homogeneous signal intensity and its thickness increased 44% on the rectus muscle side and 78% on the subcutaneous side. The capsule thickness determined either by MR in vivo and ex vivo microscopy or conventional histology was not significantly different, and there was a significant correlation between thickness measurements among those methods. MR in vivo microscopy provides sufficient resolution and spatial information to serially evaluate the growth of the foreign body fibrous capsule over time, thus achieving greater accuracy and consistency in measurements.

Animals↗