Acoustic radiation force impulse (ARFI) elastography in acute liver failure: necrosis mimics cirrhosis.

Acoustic radiation force Impulse (ARFI) technology correlates shear-wave velocity with fibrosis. It can differentiate between advanced fibrosis and normal tissue in chronic liver disease. However, specificity is impaired by cholestasis, inflammation or oedema in acute hepatitis. In patients with acute liver failure (ALF) necessitating liver transplantation ARFI has not been evaluated yet. We investigated 3 patients with ALF and compared their ARFI results to those of healthy controls (n = 33) and cases with liver cirrhosis (n = 21). In the 3 ALF patients shear-wave velocities were 3.0, 2.5, and 2.7 m/s, respectively. These results were significantly increased compared to those of healthy controls (median: 1.13 m/s; p < 0.001) and similar to those of cirrhotic individuals (median: 2.93 m/s). Two individuals underwent liver transplantation. Explants showed massive necrosis, but no signs of chronic liver disease. Patient 3 recovered spontaneously and showed decreasing ARFI results during follow-up. In conclusion, hepatic necrosis can mimic liver cirrhosis at ARFI evaluation in ALF patients and this impairs the specificity of ARFI.

Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells.

Previous studies have suggested a role for glucocorticoids in the differentiation of the acinar pancreas. We have now used the rat tumor cell line AR42J, derived from the acinar pancreas, to directly study this effect of glucocorticoids in vitro. The steroid hormones dexamethasone, corticosterone, aldosterone, and progesterone, but not estrogen, increased both the amylase content and the number of secretory granules of these cells. The potencies of the steroids were directly related to their effectiveness as glucocorticoids; dexamethasone was the most potent hormone and gave maximal effects at 100 nM. Morphometric analyses revealed that dexamethasone increased the volume density of granules 5.5-fold from 0.20 +/- 0.08 to 1.10 +/- 0.20% (n = 4) of the cytoplasmic volume. Dexamethasone treatment also increased the volume density of rough endoplasmic reticulum 2.4-fold from 1.20 +/- 0.09 to 2.86 +/- 0.30% (n = 5) of the cytoplasmic volume. After 48 h of dexamethasone treatment the cellular content of amylase increase eightfold from 2.8 +/- 0.4 to 22.6 +/- 3.8 U/mg protein (n = 6). This effect of dexamethasone was discernible after 12 h of incubation and approached maximal stimulation after 72 h of incubation. The increases in cellular amylase content were due to increased amylase synthesis as shown by specific immunoprecipitation of [35S]methionine-labeled proteins. Moreover, in vitro translation of cellular mRNA indicated that dexamethasone treatment increased amylase mRNA. Dexamethasone treatment also led to increased secretion of amylase in response to the secretagogue cholecystokinin. These data indicate, therefore, that glucocorticoids induce a more highly differentiated phenotype in AR42J pancreatic cells, and they suggest that glucocorticoids act via the enhanced transcription of specific mRNAs for acinar cell proteins.

Insulin and other stimulants have nonparallel translational effects on protein synthesis.

Isolated pancreatic acini from streptozocin-induced diabetic rats were used to study the role of insulin on the synthesis of specific cellular proteins. When acini were incubated with 0-100 nM insulin for 2 h and then pulsed with [35S]methionine, a dose-dependent increase in [35S]methionine incorporation into total cellular proteins was observed. When acinar cell lysates were subjected to gel electrophoresis, 12 major newly synthesized protein bands were resolved. Insulin (100 nM) increased the incorporation of [35S]methionine into all bands but with significantly different rates, varying from 84 to 216% of control. Next, specific antibodies to amylase, trypsin, ribonuclease, myosin, and lactate dehydrogenase (LDH) were used to evaluate the biosynthesis of known proteins. Insulin stimulated labeled amino acid incorporation into amylase by 148% over control. Insulin stimulated the synthesis of trypsinogen to a similar degree, but ribonuclease synthesis showed a significantly smaller increase of 53% over control. Insulin stimulated myosin and LDH synthesis by 169 and 184%, respectively. A differential pattern of protein synthesis was also observed when acini were treated with two other stimulators of protein synthesis, cholecystokinin and hemin. Both of these stimulators had a reduced effect on ribonuclease synthesis compared with amylase and trypsinogen synthesis but failed to increase myosin synthesis. When the RNAs extracted from control acini and acini treated with 100 nM insulin were translated in vitro, the proteins synthesized were quantitatively similar. This study therefore indicates that insulin has translational effects on acinar protein synthesis, and these effects are nonparallel for various specific acinar cell proteins.

Synthesis and secretion of rat pancreatic proteins by Xenopus laevis oocytes.

An in vivo translation system, the Xenopus laevis oocyte, was employed to study the synthesis and secretion of pancreatic proteins. RNA was purified from normal and diabetic rat pancreas and normal rat liver by use of guanidine isothiocyanate lysis and cesium chloride gradient centrifugation. The presence of functional mRNA was documented by translation in a reticulocyte lysate that yielded precursors of all major secretory proteins, i.e., slightly higher Mr than proteins synthesized in situ by pancreatic acini. Mature X. laevis oocytes were then microinjected with either total RNA or purified mRNA. When oocytes were subsequently incubated with 35S-methionine, pancreatic secretory proteins or hepatic albumin could be immunoprecipitated from oocyte lysate with specific polyclonal antibodies against amylase, trypsin, ribonuclease, and albumin. Amylase was shown to be enzymatically active. Moreover, oocytes released pancreatic secretory proteins into the medium when injected with pancreatic RNA in a time-dependent manner. Only the mature form of amylase was secreted and secretion was not regulated by secretagogues. When a comparison was made after injection of RNA from diabetic pancreas known to contain altered amounts of individual mRNAs, there was a decrease in amylase and an increase in trypsinogen synthesis in oocytes that was comparable to the results of cell free translation. The oocyte expression system, therefore, should be useful not only for studies of protein synthesis but also for processing and secretion.

The quality of human embryo growth is improved when embryos are cultured in groups rather than separately.

OBJECTIVE: To determine the effect of culturing human embryos in groups on cleavage rates, morphology grades, and embryo scores when compared with embryos cultured singly. DESIGN: Prospective. SETTING: The IVF-ET program of the Pennsylvania State University, Department of Obstetrics and Gynecology, Hershey Medical Center, Hershey, Pennsylvania. PATIENTS: Fifty-five infertile women who each had at least five zygotes underwent IVF-ET. INTERVENTIONS: Zygotes from each patient were allocated to be cultured singly and in groups. MAIN OUTCOME MEASURES: Cleavage rate, morphology grade, and embryo score. RESULTS: Grouping embryos significantly enhanced cleavage rates and embryo scores but not morphology grade as compared with embryos grown singly. Additionally, the size of the groups correlated positively with cell number and embryo score but not the morphology grade. CONCLUSION: Culturing human embryos in groups enhances the quality of their growth by increasing the cleavage rates and embryo scores. Because pregnancy rates are improved by transferring embryos with higher embryo scores, coculturing human embryos may be a way of enhancing pregnancy rates.

A randomized comparison of the methods of sperm preparation for intrauterine insemination.

OBJECTIVE: To compare the effectiveness of three methods of sperm preparation for IUI during superovulation of infertile women. DESIGN: Randomized assignment of one of three sperm preparation methods. SETTING: University infertility practice. PATIENT(S): Infertile couples undergoing superovulation and IUI. INTERVENTION(S): The method of preparation of sperm for IUI during superovulation was assigned randomly to double centrifugation, multiple-tube swim-up, or Percoll density gradient. MAIN OUTCOME MEASURE(S): Total number and percent recovery of motile sperm, percent of recovered sperm with normal morphology, and cycle fecundity. RESULT(S): No method of sperm preparation provided better cycle fecundity than the others despite differences in sperm recovery. CONCLUSION(S): Double centrifugation, multiple-tube swim-up, and Percoll density gradient sperm preparation for IUI yield similar cycle fecundity rates.

ART in women 40 and over. Is the cost worth it?

OBJECTIVE: To determine the efficacy and cost of assisted reproductive techniques in older women (40 years or older) in comparison to younger women (less than 30 years old). STUDY DESIGN: Retrospective review of records from one university-based infertility practice. RESULTS: Women 40 years or older were significantly less likely to achieve pregnancy with human menopausal gonadotropin (hMG)/intrauterine insemination as compared to women under age 30. The older women were also significantly less likely to achieve pregnancy with in vitro fertilization (IVF). The use of donor oocytes resulted in the highest pregnancy rates in older women. Costs per cycle were similar, however, for both groups. CONCLUSION: Older women will consume an equal amount of medical resources per cycle in infertility treatment as compared to younger women (aged < 30 years). However, older women utilizing assisted reproductive techniques are four to five times less likely to achieve pregnancy than the younger group. This poor prognosis for success in older women adds significantly to the mean cost per pregnancy as compared to younger women. Donor oocytes may be the most cost-effective option for achieving pregnancy in older women.

Use of an endoscopic suturing device (the "ESD") to treat patients with gastroesophageal reflux disease, after unsuccessful EndoCinch endoluminal gastroplication: another failure.

BACKGROUND: Endoluminal gastroplication, using the EndoCinch procedure, has emerged as a potential endoscopic antireflux therapy. Although initial results have been promising, the long-term durability of the treatment is uncertain due to suture loss. A new endoscopic suturing device, the "ESD," has been developed that promises excellent visibility and endoscopic control. The aim of this study was to evaluate prospectively the feasibility and efficacy of the ESD method after EndoCinch failure. METHODS: The study involved 20 patients with gastroesophageal reflux disease (GERD), who had been initially treated with an EndoCinch procedure, but had relapsed after a median of 7.5 months, with lost or dysfunctional sutures and with reflux symptoms that required proton pump inhibitor (PPI) treatment. Using the ESD, at least three plications were created at the gastroesophageal junction. Patients underwent endoscopy, 24-hour pH monitoring and esophageal manometry before treatment and 6 months afterwards. In addition, reflux symptoms as well as quality-of-life scores were assessed (using the SF-6 and GERD-HRQL scales). RESULTS: The ESD procedure (median procedure time 45 min) was performed successfully in all patients without major complications. After 6 months only one patient (5 %) still had all sutures in situ, while no remaining sutures could be detected in 3/20 (15 %). No significant changes in reflux esophagitis; 24-hour pH monitoring results (median pH < 4/24 h9.9 % vs. 12.3 %; P = 0.60); manometry findings (median lower esophageal sphincter pressure 7.2 mm Hg vs. 9.9 mm Hg; P = 0.22); PPI use; or reflux esophagitis could be detected after 6 months. While reflux symptoms improved (heartburn severity score 30 vs. 48, P < 0,05), no changes in quality-of-life scores were detected. CONCLUSIONS: Endoluminal gastroplication using the ESD is an easy and safe, but unfortunately ineffective procedure for endoscopic GERD treatment. Endoluminal gastroplication techniques clearly need refinements before these therapies can evolve as a treatment option for GERD patients.

Long term failure of endoscopic gastroplication (EndoCinch).

INTRODUCTION: Endoluminal gastroplication (EndoCinch; Bard) has been introduced as an endoscopic treatment option in gastro-oesophageal reflux disease (GORD) patients with promising short term results. However, little is known about the long term efficacy of endoscopic suturing. The aim of this study was to evaluate prospectively the long term outcome after EndoCinch. PATIENTS AND METHODS: A total of 70 patients treated with EndoCinch at a single referral centre were studied prospectively. All patients were interviewed using a standardised questionnaire regarding their symptoms and medication prior to and 18 months after EndoCinch. In addition, follow up included endoscopy, 24 hour pH monitoring, and oesophageal manometry. RESULTS: The procedure was well tolerated without major short or long term complications. Eighteen months after EndoCinch, 56/70 patients (80%) were considered treatment failures as their heartburn symptoms did not improve or proton pump inhibitor medication exceeded 50% of the initial dose. Endoscopy showed all sutures in situ in 12/70 (17%) patients while no remaining sutures could be detected in 18/70 (26%). In 54 and 50 patients examined, respectively, no significant changes in 24 hour pH monitoring (median pH <4/24 hours, 9.1% v 8.5%; p = 0.82) or lower oesophageal sphincter (LOS) pressure (7.7 v 10.3 mm Hg; p = 0.051) were observed while median LOS length slightly increased (3.0 to 3.2 cm; p<0.05). CONCLUSION: Endoscopic gastroplication (EndoCinch) is a safe and minimally invasive endoscopic treatment for GORD with reasonable short term results. In contrast, long term outcome is disappointing, probably due to suture loss in the majority of patients. Therefore, technical improvements to ensure suture durability are mandatory before endoscopic suturing can evolve as a therapeutic option for GORD treatment.

Patterns of prohormone processing. Order revealed by a new procholecystokinin-derived peptide.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.