Temporal, spatial and induced expression of chitinase in the spruce budworm, Choristoneura fumiferana.

Temporal, spatial and induced expression of Choristoneura fumiferana chitinase (CfChitinase) was studied using immunohistochemistry and Western blots. CfChitinase was detected in the integument, the midgut peritrophic membrane, the cuticular lining of the trachea, the spiracle, and salivary glands. The enzyme was expressed as larvae were preparing to molt from one instar to the next. The spatial and temporal expression patterns are consistent with its function in degrading chitin during the molting process. The 20-hydroxyecdysone agonist, tebufenozide (RH5992), induced the expression of the CfChitinase gene in the early stage of the sixth-instar larvae and the enzyme was detected in the epidermis and molting fluid 24 h post treatment.

Effect of RH-5992 on adult development in the spruce budworm, Choristoneura fumiferana.

The effect of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on adult development of the spruce budworm, Choristoneura fumiferana, was investigated by administering the compound intrahemocoelically to pupae on days 1-6 after pupal ecdysis. At concentrations of 200ng/pupa there was significant mortality but at doses of 50-100ng/pupa, the emerging adults displayed wing deformities which reduced their ability to mate and oviposit. Light microscopy of the pupal wings revealed that there was degeneration of the epithelial cells, reduction in the number of veins, precocious cuticle formation and inhibition of growth of normal wing scales. Injection of RH-5992 into pupae resulted in a dose dependent induction of mRNA for ecdysone-induced transcription factor, Choristoneura hormone receptor 3 (CHR3). These results suggest that the pupae respond to RH-5992 in a manner similar to larvae. However, the effects are not expressed overtly and are camouflaged by the pharmacological effects.

Mode of action of the ecdysone agonist tebufenozide (RH-5992), and an exclusion mechanism to explain resistance to it.

Spruce budworm larvae (Choristoneura fumiferana) upon ingesting tebufenozide (RH-5992) stop feeding and go into a precocious, incomplete molt, leading eventually to death. Like 20-hydroxyecdysone (20E), tebufenozide also acts at the receptor level and transactivates the expression of up-regulated genes but, because of its persistence, the down-regulated genes that are normally expressed in the absence of 20E are not expressed. While tebufenozide is lepidopteran-specific, an analog, RH-5849, is effective on dipterans. This is reflected in the respective effects of the two compounds on Cf-203 (C. fumiferana--203), a lepidopteran cell line and Dm-2 (Drosophila melanogaster--2), a dipteran cell line. Cf-203 cells accumulated [14C]tebufenozide and expressed CHR3 (Choristoneura hormone receptor 3), but Dm-2 cells excluded the material and did not express DHR3 (Drosophila hormone receptor 3). Using yeast ABC (ATP binding cassette) transporter mutants, we determined that PDR5 (pleiotropic drug resistance 5) was responsible for the exclusion. We discovered recently that older instars of the white-marked tussock moth (Orgyia leucostigma) are resistant to tebufenozide, perhaps as a result of such an exclusion system. We are currently cloning PDR5 (pleiotropic drug resistance 5), which is an essential step in studying the resistance mechanism.

Perifollicular Doppler flow and follicular fluid vascular endothelial growth factor concentrations in poor responders.

OBJECTIVE: To prospectively evaluate follicular fluid levels of vascular endothelial growth factor in women undergoing IVF cycles and to investigate the correlation of these levels with ovarian response to gonadotropins and with uterine or ovarian Doppler findings. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): 41 patients undergoing ART were divided into two groups according to response to ovarian stimulation protocols: poor responders (n = 18) and normoresponders (n = 23). INTERVENTION(S): Doppler analysis of perifollicular arteries and assay of follicular fluid vascular endothelial growth factor. MAIN OUTCOME MEASURE(S): During ovarian stimulation, patients underwent hormonal (E2), ultrasonographic (follicular number and diameter, endometrial thickness) and Doppler (uterine and perifollicular arteries) evaluation. Serum and follicular fluid concentrations of vascular endothelial growth factor were assayed in each female patient. RESULT(S): Compared with poor responders, more oocytes were collected and more embryos were transferred but follicular fluid levels of vascular endothelial growth factor levels were lower in normoresponders. Follicular fluid levels of vascular endothelial growth factor were inversely correlated with number of oocytes retrieved. Poor responders had significantly higher uterine and perifollicular Doppler flow resistances. The pregnancy rate per cycle was significantly higher in normoresponders (26%) than poor responders (6%). CONCLUSION(S): Elevated follicular fluid levels of vascular endothelial growth factor concentrations are associated with poor ovarian response and a very low pregnancy rate.

Allergic reaction to a highly purified urinary follicle stimulating hormone preparation in controlled ovarian hyperstimulation for in vitro fertilization.

We report a case of general hypersensitivity-like allergic reactions to intramuscular injections of highly purified urinary follicle stimulating hormone (uFSH-HP) successfully managed by using intramuscular recombinant FSH (rFSH). The patient underwent a first cycle of in vitro fertilization (IVF) and controlled ovarian hyperstimulation (COH) was achieved with a combination of gonadotropin releasing hormone against (GnRH-a) and uFSH-HP. Because, after oocyte recovery, no fertilization occurred, the couple subsequently entered an intracytoplasmic sperm injection (ICSI) program. During the COH, the woman developed general hypersensitivity-like allergic reactions with itching, redness and swelling. Although there was regular follicular growth, the allergic symptoms worsened and, on day 8 of COH, the stimulation cycle was suspended. A few months later, the patient entered a new ICSI cycle. COH was achieved by using a combination of GnRH-a and rFSH. The cycle was completed and the patient did not report any allergic reaction. To avoid allergic reaction to the protein components of the urine-derived FSH preparations, the use of rFSH is suggested in those patients who present local and/or general hypersensitivity-like allergic reactions.

Human embryo development and pregnancies in an homologous granulosa cell coculture system.

PURPOSE: Our purpose was to determine the effects of the coculture of embryos on human granulosa cells (GCs) in patients in the first cycle of IVF-ET treatment and in patients with repeated implantation failures and to investigate the presence of specific proteins in a 48-hr GC conditioned medium and the GC ultrastructural characteristics. METHODS: Eighteen patients with tubal or idiopathic infertility were enrolled in this study: 7 patients (Trial 1) were in the first cycle of IVF-ET treatment and 11 patients (Trial 2) had repeated implantation failures (one to five). Embryos from each patient were cocultured randomly either on homologous granulosa cells or on a conventional culture medium. RESULTS: At the end of the coculture period (day 5 or 6), 50% of the embryos (Trial 1) reached the blastocyst stage, with respect to 35% in Trial 2. The pregnancy rate per retrieval was 14.2 and 9%, respectively, in Trial 1 and in Trial 2. Many conditioned media showed proteins of 24-29 kDa. and some of them showed additional proteins of 90 kDa. The ultrastructural analysis of GCs showed healthy, metabolically active, protein-synthesizing, and mostly steroidogenic cells. CONCLUSIONS: GC cultures improve embryo development but not pregnancy rates both in Trial 1 and in Trial 2.

Successful induction of ovulation using highly purified follicle-stimulating hormone in a woman with Kallmann's syndrome.

OBJECTIVE: To describe a woman with Kallmann's syndrome who was treated successfully with highly purified FSH to achieve ovulation induction and pregnancy. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 32-year-old woman with Kallmann's syndrome who had been treated with oral contraceptives to prime secondary sex characteristics and genital organs since the age of 16 years. INTERVENTION(S): Highly purified FSH was administered intramuscularly for a total dose of 3,825 IU. MAIN OUTCOME MEASURE(S): Follicle number and diameter. RESULT(S): Three follicles with a diameter of > 1.7 cm and an endometrial thickness of 8 mm were observed. A clinical pregnancy, which subsequently was spontaneously aborted, was obtained. CONCLUSION(S): In primed patients with Kallmann's syndrome, highly purified FSH may be a useful alternative to pulsatile GnRH or menopausal gonadotropins to achieve ovulation induction and pregnancy.

Technical aspects of oocyte cryopreservation.

Since the successful development in the mouse, the oocyte cryopreservation has been applied with varying success to a number of different species including the human. The recently reported successes in terms of pregnancies obtained by human oocyte cryopreservation are encouraging. Several studies typically reported different rates of survival (20-80%), fertilization (30-60%) and cleavage (32-100%). This variability of results throws some doubts on the usefulness of oocyte cryopreservation in IVF treatment cycles. It remains to be determined whether the relatively different success rates reported in literature, mainly in terms of survival rate, are due to methodological differences. We tried to investigate the effect of some factors on the oocyte survival rate after thawing: the presence or absence of cumulus oophorus and the exposure time of the oocytes to cryoprotectant. We suggest that a combination of several factors including both morphological and biophisical ones can affect the oocyte survival rate.

Polycystic ovary syndrome: it is always bilateral?

OBJECTIVE: To evaluate whether patients with unilateral polycystic ovary showed different ovarian and uterine blood flow from those with bilateral polycystic ovaries, and to investigate whether there was a correlation between the ultrasonographic aspect and different hormonal parameters. DESIGN: An observational study. SUBJECTS: Sixteen patients with unilateral polycystic ovary and twenty patients with bilateral polycystic ovaries underwent clinical, biochemical, gray-scale and color Doppler ultrasonographic evaluation. METHODS: The following parameters were evaluated: hormonal (luteinizing hormone (LH), follicle stimulating hormone (FSH), LH/FSH concentration ratio, estradiol, prolactin, androstenedione, testosterone), clinical (body mass index, Ferriman-Gallwey score), ultrasonographic (ovarian volume, number and distribution of subcapsular follicles, stromal score) and Doppler (uterine artery and intraparenchymal vessel pulsatility index, ovarian stromal vascularization), in oligomenorrheic patients in the early follicular phase (cycle days 3-5) or in amenorrheic patients at random. RESULTS: Significantly higher androstenedione plasma levels and LH/FSH concentration ratios were observed in bilateral polycystic ovaries. In unilateral polycystic ovaries, gray-scale and color Doppler ultrasonography showed different features in the affected and the unaffected ovary, similar to the appearance of a polycystic and normal ovary, respectively. CONCLUSION: Polycystic ovary syndrome does not predetermine a single ultrasonographic and Doppler pattern.

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.

Uterine and cerebral vascularization in postmenopausal women treated with hormone-replacement therapy.

The effects of hormone-replacement therapy on the Doppler flow parameters of the ophthalmic artery in postmenopausal women were studied and compared with those registered at the level of the internal carotid and uterine arteries. Fifty-seven postmenopausal patients were submitted to continuous estradiol transdermal supplementation and 12-day courses of medroxyprogesterone acetate. During the estrogen phase of hormone-replacement therapy all patients underwent (at 1, 3 and 6 months after the beginning of hormone-replacement therapy) transvaginal ultrasonographic evaluation of the pelvic organs and of endometrial thickness. On the same day, they underwent color Doppler analysis of the blood flow impedance of the uterine, internal carotid and ophthalmic arteries. Estradiol plasma concentrations were assayed on the day that ultrasonographic and Doppler examinations took place. The pulsatility index of all the arteries improved, from baseline values, during the therapy and attained stable values compared to those after the first month of treatment. Furthermore, at the level of the internal carotid and ophthalmic arteries, a significant increase of the peak systolic blood flow velocity (Vmax) was observed over the 6 months of therapy. Doppler studies of the ophthalmic artery are capable of affording specific and precise pathophysiologic information to assess peripheral intracranial blood flow variations. Furthermore, such studies may be useful in monitoring hormone-replacement therapy effects on cerebral perfusion.

Hormonal replacement therapy and urinary problems as evaluated by ultrasound and color Doppler.

OBJECTIVE: To determine how hormone replacement therapy modifies bladder vascularization and urinary symptoms. STUDY DESIGN: Twenty-eight postmenopausal women with urinary symptoms (day-time frequency > 8; nocturia > 1; urgency and/or dysuria) were analyzed before and after 1, 3 and 6 months of hormone replacement therapy. The patients underwent transvaginal ultrasound evaluation of the pelvic organs and endometrial and bladder wall thickness. Transvaginal color Doppler analysis of blood flow impedance of the uterine and intramural bladder wall arteries was performed in all cases. RESULTS: Hormone replacement therapy significantly increased bladder wall and endometrial thickness. This result was associated with significant improvements in uterine and bladder wall vascularization. Urinary symptoms also improved during therapy. CONCLUSION: The study of bladder wall thickness and vascularization provides additional information regarding the beneficial effect of hormone replacement therapy on lower urinary tract symptoms in postmenopausal women.

Studies of the nucleopolyhedrovirus infection process in insects by using the green fluorescence protein as a reporter.

A recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) expressing the green fluorescence protein (GFP) under the control of the AcMNPV polyhedrin promoter was constructed to study the spatial and temporal regulation of baculovirus infection in a permissive host. Larvae that ingested AcMNPV-GFP showed localized expression of GFP in the midgut epithelial cells, as well as hemocytes, at 24 h postinfection. The presence of fluorescence in these tissues indicated not only that the virus was replicating but also that the very late viral proteins were being synthesized. Secondary infection occurred within the tracheal cells throughout the body cavity, confirming earlier reports, and these foci of infection allowed entry of the virus into other tissues, such as the epidermis and the fat body.

Oocyte cryopreservation.

Cryopreservation of human oocytes has been employed with little success in clinical practice, even though it may solve the legal and ethical problems linked to embryo freezing. Various attempts to cryopreserve human oocytes have mostly been unsuccessful, leading to low oocyte survival rates after thawing, and the search for an optimal protocol for oocyte cryopreservation remains elusive. A preliminary study was undertaken to evaluate some of the factors influencing the survival rate of human oocytes and the efficiency of intracytoplasmic sperm injection (ICSI) as an insemination procedure. A total of 38 women with tubal infertility were enrolled in the study. The cryopreservation procedure consisted of a slow freeze-rapid thawing technique using 1,2 propanediol and sucrose as cryoprotectants. The overall oocyte survival rate was approximately 60%. A better survival rate was obtained when the oocytes were cryopreserved in the presence of partially removed cumulus oophorus rather than in the presence of totally enzymatically removed cumulus oophorus. The cryoprotectant concentration and the equilibration time also appear to influence the oocyte survival rate. ICSI may be an efficient method of achieving a satisfactory outcome in terms of fertilization in cryopreserved human oocytes. Embryonic morphological quality does not seem to be compromised by cryopreservation. In conclusion, these data show that cryopreservation may ensure that the integrity of the human oocyte is adequate for normal fertilization and embryo development.

Concomitant primary infection of the midgut epithelial cells and the hemocytes of Trichoplusia ni by Autographa californica nucleopolyhedrovirus.

We have constructed a modified Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to express the green fluorescent protein (GFP) under the polyhedrin promoter and used it to study the infection process of AcMNPV in Trichoplusia ni larvae. T. ni larvae that ingested the virus showed localized expression of GFP in the midgut epithelial cells and the hemocytes at 12 h post infection (hpi). The presence of GFP-related fluorescence in the midgut columnar cells indicated that the virus was not only replicating, but also synthesizing the late viral proteins. Studies using the transmission electron microscope showed that the virus infected the midgut columnar cells. At the same time a proportion of the parental virus travelled through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, entered the hemocoel and infected the hemocytes. This resulted in the simultaneous infection of the midgut epithelial cells and the hemocytes. Subsequently, the budded virus (BV) released from the infected hemocytes into the hemolymph caused secondary infection within the tracheal epithelial cells. The virus then rapidly spread through the tracheal system allowing the infection of a variety of other tissues such as the epidermis and the fat body.

Synthesis of the same two proteins prior to larval diapause and pupation in the spruce budworm, Choristoneura fumiferana.

Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those that terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36-48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.

CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut cell line.

Morphological and molecular changes produced by Autographa californica nuclear polyhedrosis virus (AcMNPV) infection in a permissive cell line, IPLB-SF-21AE (SF-21), of Spodoptera frugiperda and a nonpermissive cell line, FPMI-CF-203 (CF-203), of Choristoneura fumiferana are described. CF-203 cells inoculated with AcMNPV showed a DNA ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. Typical virus replication and occlusion body (OB) production were seen in SF-21 cells inoculated with AcMNPV and no apoptosis-like symptoms were observed. mRNA for the apoptosis suppressor gene p35 was detected 9 hr later in AcMNPV-inoculated CF-203 cells than in SF-21 cells. Only a trace amount of mRNA for the AcMNPV-inhibitor of apoptosis homologue (Ac-iap) gene and no mRNAs for the late genes, AcMNPV-polyhedrin (Ac-polh) and AcMNPV-p10 (Ac-p10), were detected in AcMNPV-inoculated CF-203 cells. Inoculation of CF-203 cells with CfMNPV at least 12 hr prior to inoculation with AcMNPV prevented apoptosis-like cell death, and mRNAs for Ac-iap, Ac-polh, and Ac-p10 genes were expressed resulting in successful virus replication and OB production.

Photosensitized release of von Willebrand factor from cultured human endothelial cells.

Cultured endothelial cells from the human umbilical vein were incubated with low concentrations (1 microgram/ml) of the photosensitizer Photofrin II. Following a sublethal light exposure, a light dose-dependent release of von Willebrand factor (vWf) into the culture medium was observed. Analysis of the multimeric composition of the released protein indicated that it originated from the intracellular pool of large vWf multimers stored in the Weibel-Palade bodies. This release was detected as early as 1 h postirradiation. Release was inhibited at low temperature and was dependent upon the presence of extracellular calcium. Photosensitization resulted in an influx of calcium whose time course paralleled vWf release from the cells. Since vWf mediates platelet adhesion to the vascular subendothelium, it is possible that its photochemically stimulated release in vivo could contribute to platelet thrombus formation observed in tissue following photodynamic therapy.

Demonstration of HBsAg as the antigen component in circulating immune complexes detected by peg-solid phase test.

The development of an enzyme-linked immunosorbent assay to identify HBsAg as the antigen component within circulating immune complexes using immobilized polyethylene glycol (PEG) is described. The method utilizes, on one hand, the ability of PEG to bind stably to plastic supports and, on the other, to precipitate circulating macromolecules. This method is easily performed, very cheap, quick and, above all, it helps define the biological nature of the immune complexes. HBsAg can be revealed as the antigen component of HBsAg/anti-HBs soluble immune complexes at concentrations of at least 20 ng/ml and either in antigen or antibody excess. Our results indicate that HBsAg circulates in a complexed form in 47% of HBsAg chronic carriers and in 10.7% of patients with liver disease who are positive for serum antibody to hepatitis B surface antigen (anti-HBs) and to core antigen (anti-HBc). None of the other groups of patients in the study had circulating HBsAg in the complexed form.