HBsAg loss after peginterferon-nucleotide combination treatment in chronic hepatitis B patients: 5 years of follow-up.

Combining peginterferon-alfa-2a (pegIFN) with a nucleotide analogue can result in higher rates of HBsAg loss than either therapy given alone. Here, we investigated the durability of the response to combination therapy in chronic hepatitis B (CHB) patients after 5 years of follow-up. In the initial study, 92 CHB patients (44 HBeAg-positive, 48 HBeAg-negative) with HBV DNA >100 000 c/mL (~20 000 IU/mL) and active hepatitis were treated for 48 weeks with pegIFN 180 µg/week and 10 mg adefovir dipivoxil daily. For the long-term follow-up (LTFU) study, patients were followed up for 5 years after the end of treatment. At year 5, 70 (32 HBeAg-positive, 38 HBeAg-negative) patients remained in the study. At year 5, 19% (6/32) of HBeAg-positive patients and 16% (6/38) of HBeAg-negative patients lost HBsAg, and no HBsAg seroreversion was observed. The 5-year cumulative Kaplan-Meier estimate for HBsAg loss was 17.2% for HBeAg-positive patients and 19.3% for HBeAg-negative patients. Fourteen of sixteen patients who lost HBsAg at any time point during follow-up developed anti-HBs antibodies (>10 IU/L). At year 5, in total 63% (20/32) of HBeAg-positive and 71% (27/38) of HBeAg-negative patients were retreated with nucleos(t)ide analogues during follow-up. The cumulative Kaplan-Meier estimate for retreatment was 60% of patients at year 5. At year 5 of follow-up, 18% of CHB patients treated with pegIFN/nucleotide analogue combination therapy had durable HBsAg loss and 88% of these had developed anti-HBs antibodies.

Short communication: Circulating serotonin (5-HT) concentrations on day 1 of lactation as a potential predictor of transition-related disorders.

The monoamine serotonin (5-hydroxytryptamine; 5-HT) has been described as a homeostatic regulator of lactation. Recently, our laboratory determined that 5-HT is involved in the regulation of calcium and glucose homeostasis during the transition period in rodents. More specifically, we demonstrate that 5-HT is responsible for calcium mobilization from bone and upregulation of hepatic gluconeogenic enzymes and mammary gland glucose transporters. Our objective was to investigate the correlation between circulating 5-HT concentrations and circulating ionized calcium, parathyroid hormone-related protein (PTHrP), and glucose concentrations on d 1 postpartum. We also investigated the correlation between circulating 5-HT and milk fever and ketosis incidence and severity in multiparous Holstein cows at the onset of lactation. Blood samples were collected from 42 multiparous cows on d 1 of lactation and analyzed for 5-HT, calcium, glucose, and PTHrP. Milk fever (determined subjectively for each cow on d 1 postpartum) and ketosis incidence and severity (scale 1 to 4, determined objectively for each cow during the first 10 d postpartum) were recorded for all animals. Serum 5-HT was positively correlated with serum calcium and with plasma PTHrP (r>0.37). Serum 5-HT was negatively correlated with milk fever incidence and with ketosis severity (most severe ketosis incidence recorded during the first 10 d postpartum; r<-0.33). Serum calcium and plasma glucose concentrations were negatively correlated with milk fever and ketosis severity, respectively (r<-0.39). These data indicate that 5-HT potentially plays a role in the regulation of calcium and glucose homeostasis during the transition period in cattle, which we previously demonstrated in rodents. Increased circulating concentrations of 5-HT might decrease milk fever at the onset of lactation and ketosis severity during the first 10 d postpartum in dairy cows. Understanding this physiological axis could help describe the underlying mechanisms associated with these periparturient metabolic disorders in dairy cows.

Freeze-etch study of an unmodified lectin interacting with its receptors in model membranes.

Experiments are described in which liposomes bearing ganglioside or glycophorin as receptor were exposed to the native (unmodified) lectin, wheat germ agglutinin, and subsequently examined by freeze-etch electron microscopy. Visualized in this way in the absence of lectin, phosphatidylcholine bilayer membranes show no features attributable to the presence of small amounts of glycolipid. Similarly bilayers bearing glycophorin show no obvious etch face (outer surface) features attributable to that species, although they do have intramembranous particles associated with the hydrophobic interior. However, the otherwise smooth and featureless model membrane outer surface permitted ready visualization of bound lectin molecules, and thus the indirect localization of receptors. The lipid membrane itself in the vicinity of receptors was not visibly affected by lectin binding to glycolipid or glycoprotein. The only identifiable lectin-induced change was lateral redistribution of receptor glycoproteins. Ganglioside molecules showed some evidence of existing in small clusters; but their distribution was apparently unaffected by lectin binding. Clumps of lectin bound to glycophorin were found associated predominantly with fluid bilayer regions of phase separated membranes; and lectin distribution on the etch face correlated with intramembranous particle distribution in the fracture face. In contrast, ganglioside lateral distribution was not appreciably influenced by the host lipid matrix phase separation.

Glycolipid crypticity in membranes--not a simple shielding effect of macromolecules.

The potential of membrane-bound macromolecules for shielding glycolipids from involvement in specific binding events was considered in model membranes. Serum albumin and several Dextrans were covalently derivatized with oleic acid so that they adsorbed irreversibly to lipid bilayers. This provided a means of generating bilayer membranes with a considerable layer of attached material. Gangliosides dispersed in such membranes were subjected to attack by the enzyme, neuraminidase, in order to assess their "accessibility'. We were surprised to find that we could not demonstrate any significant reduction in ganglioside hydrolysis in phosphatidylcholine bilayers bearing extensive surface coats of protein or polysaccharide. We conclude that non-specific, physical shielding by macromolecules is an unlikely source of the often-observed "crypticity' of glycolipids at the cell surface. Consistent with this interpretation was a relative lack of headgroup motional restriction seen for spin-labelled ganglioside headgroups in the same bilayers and in cell membranes.

Evidence of a distribution difference between two gangliosides in bilayer membranes.

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM1 and GD1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM1 and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P beta' phase of this lipid might accentuate any behavioural differences between GM1 and GD1a. GM1 was found to exist preferentially in the 'trough' regions between P beta' ripples, while GD1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.

Headgroup behaviour of an uncharged complex glycolipid.

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide--which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.

Physical behaviour of glycolipids in bilayer membranes: distribution and accessibility.

A 3-dimensional scale drawing has been produced which purports to illustrate key features of glycolipid environment in the eukaryote plasma membrane. An attempt has been made to isolate two such features for separate study using bilayer model membranes. In particular, i) physical shielding of glycolipid headgroups by membrane-associated macromolecules has been investigated as a possible source of glycolipid crypticity; and ii) freeze-etch EM has been employed in an attempt to understand factors that control glycolipid receptor function and distribution. The first of these problems was approached by measuring sialic acid released (by the enzyme, neuraminidase) from gangliosides in lipid bilayers. The bilayers could be coated with a firmly attached layer of protein or high molecular weight polysaccharide in order to mimic the presence of macromolecules at a cell surface. In fact, surface coat material did not measurably reduce sialic acid release from gangliosides in fluid or rigid phosphatidylcholine membranes. The implication drawn is that non-specific shielding by surface glycoproteins is an unlikely source of glycolipid crypticity in cell membranes. The second problem was attacked by direct visualization of lectins and bilayer vesicles on the platinum-shadowed outer surface of large liposomes. To a first approximation bound lectins (and hence gangliosides) were equally distributed between coexisting fluid and rigid phospholipid domains. However the appearance was suggestive of a tendency for gangliosides to exist in small clusters. Lipid vesicles bearing lectin receptors were used to mark points of lectin-mediated membrane-membrane attachment on large liposomes. There was no obvious difference in the extent of such attachment to fluid and rigid domains.

Timing of final GnRH of the Ovsynch protocol affects ovulatory follicle size, subsequent luteal function, and fertility in dairy cows.

Synchronization of ovulation (Ovsynch) is an effective method for controlling time of first and subsequent AI in lactating dairy cows. However, validation of the original Ovsynch program did not include testing the optimal time to deliver the final treatment of GnRH. In Experiment 1, the effect of administering the final dose of GnRH on the same day as prostaglandin F2alpha (PGF2alpha) administration was tested. Lactating dairy cows (n = 218) were randomly assigned to receive either Ovsynch (OV; cows were given 100 microg GnRH, then 7 days later cows were administered 25mg PGF2alpha followed by a subsequent treatment of 100 microg GnRH 2 days after the PGF2alpha or the modified version of Ovsynch (MOV; cows were given 100 microg GnRH, then 7 days later cows were administered 25mg PGF2alpha followed immediately with 100 microg GnRH). In both treatment groups, AI took place 16 h after the final administration of GnRH. In Experiment 2, cows (n = 457) were randomly divided into four treatment groups that were administered GnRH 0, 12, 24 and 36 h following PGF(2alpha). The 36 h treatment group served as control. Pregnancy diagnoses were performed by palpation per rectum 36 days post-AI in Experiment 1 and by ultrasonography on Day 28 in Experiment 2. In Experiment 1, pregnancy rate/AI (PR/AI) was greater (P<0.025) in OV versus MOV. In a subset (n = 85), percentage of cows with both synchronized ovulations and regressed CL following administration of PGF2alpha were similar (P>0.1) between OV and MOV, respectively. All cows that became pregnant in the MOV subset group showed regression of the CL in response to the PGF2alpha. Diameter of the ovulatory follicle at the time of final GnRH administration was greater (P<0.05) in OV versus MOV. In Experiment 2, the synchronization rate was once again similar among treatments (P>0.28). There was a linear effect of treatment on follicle size (P<0.05) and PR/AI (P<0.0001) as time increased between administration of PGF2alpha and GnRH, with the greatest PR/AI at 36 h. There was a trend for a greater percentage of cows with short luteal phases in the 0 h group (P<0.10). In summary, delivering the final treatment of GnRH of the Ovsynch program at the same time as PGF2alpha, or in the 24h following PGF2alpha, resulted in lower fertility compared to controls.

Use of critical interactive thinking exercises in teaching reproductive physiology to undergraduate students.

In higher education, increasing emphasis is being placed on the use of new technologies in the classroom. However, the emphasis needs to be placed on methods that truly enhance understanding and knowledge retention. Class discussions help students understand and retain information previously presented in lecture format. Furthermore, if students are challenged to critically evaluate, communicate, and defend their ideas, knowledge retention and understanding will increase even more. Critical interactive thinking exercises (CITE) were employed at two different universities to enhance student knowledge retention and promote the development of critical thinking. Applicability of CITE to undergraduate learning was assessed over a 3-yr period in the undergraduate reproductive physiology courses at Michigan State University and the University of Missouri-Columbia. For each exercise, students were challenged to prepare a one-page, double-spaced composition addressing an incompletely understood phenomenon or problem-solving situation related to the reproductive system. In preparing their compositions, students were encouraged to use information previously presented in lecture plus outside information to develop their ideas. Students were required to formulate and defend a hypothesis or approach to the problem presented. At the subsequent class period, students were divided into groups of three to four, in which they interactively discussed their ideas. Each group member was challenged to defend his or her hypothesis and explanation and to persuade other group members to adopt their ideas. Each group then arrived at a consensual opinion that was presented during a discussion by the entire class. The class then debated the merits of each group's hypothesis or explanation and the supporting arguments presented. At first, the students were apprehensive about the CITE, particularly about communicating and defending ideas with their classmates. However, course evaluations showed that 131 out of 137 students considered the CITE a positive experience that enhanced learning. Additionally, 131 out of 137 students reported that the CITE enhanced their critical thinking skills. We feel that the use of CITE in teaching reproductive theory to undergraduate students fosters critical thinking skills, communication skills, and knowledge retention. The general concept can be readily applied to courses in other subject areas in the animal sciences.

Inhibition of intrafollicular PGE2 synthesis and ovulation following ultrasound-mediated intrafollicular injection of the selective cyclooxygenase-2 inhibitor NS-398 in cattle.

Ultrasound-mediated intrafollicular injection and aspiration procedures were used to investigate the ability of the selective cyclooxygenase-2 inhibitor, NS-398, to inhibit intrafollicular PGE2 synthesis and suppress ovulation in dairy cattle. Follicular growth and timing of the preovulatory gonadotropin surge were synchronized in 55 Holstein cows and the position of the ovulatory follicle was determined by daily ultrasound scanning. Preovulatory follicular fluid was aspirated from the largest follicle in four animals at 0, 6, 12, 18, and 24 h after GnRH injection (n = 20). The remaining 35 animals were subjected to ultrasound-mediated intrafollicular injection of NS-398 (10 microM final concentration; n = 19) or diluent (n = 16; controls). At 24 h after GnRH injection, follicular fluid was harvested from a subset of NS-398- (n = 9) and diluent-treated animals (n = 6). The remaining NS-398- and diluent-treated animals were subjected to ultrasonography every 6 h for 36 h after intrafollicular injection, and then daily through d 7 of the subsequent luteal phase to monitor ovulation and corpus luteum development. Follicular fluid PGE2 concentrations were increased following GnRH injection and reached a maximum at 24 h (P < 0.05). Follicular fluid PGE2 concentrations were decreased in NS-398- vs. diluent-treated follicles (7.2 vs. 52.2 ng/mL respectively; P < 0.05), but progesterone concentrations did not differ. Intrafollicular injection of NS-398 also inhibited follicle rupture (P < 0.001). All 10 control animals ovulated within 30 h of GnRH injection. Nine out of the ten NS-398-injected animals failed to ovulate. The NS-398-injected follicles developed morphological and endocrine characteristics resembling luteinized, unruptured follicles. Thus, intrafollicular PGE2 synthesis and follicle rupture, but not luteinization, were inhibited in cattle following ultrasound-mediated intrafollicular injection of NS-398. Ultrasound-mediated intrafollicular injection of NS-398 is a useful tool for mechanistic studies of intrafollicular regulation of the ovulatory process in cattle.

Fertility of lactating dairy cows treated with Ovsynch after presynchronization injections of PGF2 alpha and GnRH.

Synchronization of ovulation (Ovsynch) using GnRH and PGF2 alpha allows control of follicle growth, corpus luteum regression, and ovulation, but resulting pregnancy rates vary. This study examined whether presynchronization to allow initiation of Ovsynch during diestrus would improve pregnancy rates at timed artificial insemination (AI). Lactating dairy cows (n = 427), 69 to 75 d postpartum, were randomly assigned to two groups by parity. Control cows received Ovsynch (GnRH, d 0; PGF2 alpha, d 7; GnRH, d 9; timed AI 16 h after second GnRH). Treated cows received presynchronization injections of PGF2 alpha and GnRH, 10 and 7 d, respectively, before starting Ovsynch. Pregnancy diagnoses were performed 36 d after AI. Progesterone (P4) concentrations from a subset of cows (n = 84) were determined in serum samples collected on d 0, 3, and 7 of Ovsynch. Presynchronization increased the percentages of cows with > or = 1 ng/ml serum P4 compared with control cows at first injection of GnRH (d 0; 93 vs. 56%) and on d 3 (90.7 vs. 51.2%) during Ovsynch. On day of PGF2 alpha, d 7 during Ovsynch, percentages of cows with > or = 1 ng/ml serum P4 were similar (95.3%, treated vs. 82.9%, control) but more treated cows had > or = 2 ng/ml serum P4 (95.3 vs. 63.4%). However, pregnancy to timed AI was similar between treated (41.5%) and control cows (38.3%). Cows with above-average milk production had greater pregnancy rate (45.8 vs. 33.8%) compared with lower producing cows. Although presynchrony increased the proportion of cows with luteal function at onset of Ovsynch, pregnancy rate to timed AI was not improved. Cows with above-average milk production had greater fertility at timed AI than herdmates with lower milk production.

Circumferential alignment of muscle cells in the tunica media of the human brain artery.

Our purpose was to measure how structurally coordinated is the network of muscle cells in the brain artery. Vessels from 7 autopsies were fixed with glutaraldehyde and formalin at physiologic pressure. We embedded each artery alongside a block of liver, formed into a rectangular prism, prepared it for light microscopy and stained the sections with haematoxylin and eosin (HE). The angle of cutting the arterial segments was determined with the aid of the block of liver tissue as a Cartesian reference. We measured the directional alignment of vascular smooth muscle using the centrally located nucleus as a vector of orientation. The end coordinates of the profiles of the nuclei (appearing dark with the HE stain) were recorded on a digitizer tablet, and analysis was done as suggested by a previous modelling study. The method provides an average alignment from the collective measurements on the hundreds of nuclei in each histological section. Data from 10 arteries (approximately 22,000 nuclei) from 17 sections showed that brain arteries have highly oriented medial muscle cells aligned circumferentially (average magnitude of 1.3 +/- [SD] 1.5 degrees from true cross section), with a helical variation (along the artery) of +/- 7.9 degrees and a variation in the spiral direction of +/- 5.4 degrees, i.e. a three-dimensional variation from nucleus to nucleus of +/- 10 degrees.

Lateral distribution of gangliosides in bilayer membranes: lipid and ionic effects.

Native Ricinus communis agglutinin (RCA 60) has been used as a visual marker to localize the ganglioside GM1 under a variety of conditions in model membranes. By employing the technique of freeze-etch electron microscopy, it was possible to resolve membrane structural details down to some 3 nm. The most striking feature was frequent existence of the marker lectin in clusters--presumably reflecting the underlying presence of clustered receptor. This feature persisted at glycolipid concentrations from 0.5 to 7 mol %. It was visible in membranes of single pure phospholipids above and below their phase transition temperatures, in membranes of mixed phospholipids, and in membranes containing cholesterol. The presence of cations, including Ca2+, was not seen to alter the pattern of lectin binding at a resolution of 3 nm. In pure dipalmitoyl phosphatidylcholine, a lining-up of glycolipid in the "troughs" between ripples in rigid lipid was apparent, in agreement with a similar phenomenon reported by Tillack et al for a neutral glycosphingolipid in pure dimyristoyl phosphatidylcholine [1982: Biochim Biophys Acta 691:261-273]; but this feature was not evident in host matrices composed of several different lipids.

Coalignment of the muscle cell and nucleus, cell geometry and Vv in the tunica media of monkey cerebral arteries, by electron microscopy.

We undertook to ascertain how well aligned is the rod-shaped nucleus within the spindle-shaped cell of vascular muscle in order that we might use the darkly staining nucleus in histological sections to indicate precisely the directional alignment of the cell. We fixed cerebral arteries from five monkeys under physiological pressure and embedded portions of the tissue so that mid-plane longitudinal sections of the arteries were obtained; the circumferentially arranged muscle cells were cut in cross-section. From the electron micrographs we obtained the cross-sectional profile of the cell and its nucleus, determining that the centre of the nucleus was on average 9.5 +/- 5.8% (SD) away from the centre of the cell (expressed as a ratio of the cellular diameter). We calculated the alignment between the cell and nucleus to be from 0 to 3 degrees, and obtained a volume fraction of 59% for muscle tissue in the tunica media of these arteries.

Gonadotrophin surge-induced upregulation of mRNA for plasminogen activator inhibitors 1 and 2 within bovine periovulatory follicular and luteal tissue.

The serine proteinases, tissue-type (tPA) and urokinase (uPA) plasminogen activator, are implicated in the ovulatory processes via their ability to convert plasminogen to its active form, plasmin. One mechanism for regulation of plasmin-directed ovarian extracellular matrix remodelling during follicle rupture and corpus luteum formation is through inhibition of plasminogen activation by the plasminogen activator inhibitors (PAI-1 and PAI-2). The effect of the preovulatory gonadotrophin surge on the temporal and spatial regulation of expression of PAI-1 and PAI-2 mRNA and PAI activity in preovulatory bovine follicles and new corpora lutea collected at 0, 6, 12, 18, 24 and 48 h after a GnRH-induced gonadotrophin surge was examined. Both PAI-1 and PAI-2 mRNAs were upregulated markedly after the gonadotrophin surge, with the highest expression observed in follicles collected at about the time of ovulation (24 h) and in corpora lutea (48 h). PAI-1 mRNA was localized primarily to the thecal layer of preovulatory follicles. In contrast, PAI-2 mRNA was localized specifically to the granulosa cell layer. Significant PAI activity was detected in follicle extracts, but temporal or spatial differences in PAI activity were not detected in response to the gonadotrophin surge. These results indicate that PAI-1 and PAI-2 mRNAs are upregulated in preovulatory bovine follicles after the gonadotrophin surge in a cell-specific way. Regulation of PAI-1 and PAI-2 may help to control plasminogen activator activity associated with ovulation and early corpus luteum formation.