PHOX2A and PHOX2B genes are highly co-expressed in human neuroblastoma.

The detection of PHOX2B mutations in a small proportion of patients affected with either familial or sporadic neuroblastoma (NB), has arisen interest on the possible pathogenic role of this gene in the disease determination. In this light, we have carried out a quantitative expression analysis of PHOX2B and its paralogue PHOX2A on a panel of NB cell lines and NB tumour samples to identify a possible differential expression between NB cells and their normal counterpart (adrenal medulla cells). Our results revealed that both PHOX2A and PHOX2B are over-expressed in tumour samples and NB cell lines. Particularly, the expression levels of the two genes in NB cell lines show a highly significant correlation, suggesting their possible synergistic role or a coordinated expression regulation. Furthermore, PHOX2 gene over-expression in NB tumours and cell lines suggests these genes may be widely involved in NB development through either a direct mechanism of up-regulation or a failure in maintaining proper transcript levels after embryonic development.

A diagnostic dilemma: endodontic lesion or odontogenic keratocyst? A case presentation.

AIM: To present a clinical case of odontogenic keratocyst (OKC) simulating a lateral periodontal cyst. SUMMARY: A 39-year-old female complaining of swelling and pain in the left mandibular premolar area was found to have a radiolucent lesion between teeth 34 and 35 (FDI). Both teeth had incomplete root fillings, and orthograde re-treatment of both premolars was performed. At 2-year follow-up, radiographic examination showed an increase in the radiolucent defect with respect to the previous examination. A surgical treatment of tooth 34 was then performed, with histological examination of the lesion. Histological features were consistent with an OKC, and the lesion was successfully treated by complete enucleation and application of Carnoy's solution. At 2-year follow-up, no clinical signs or symptoms were found and the radiolucent area had disappeared. KEY LEARNING POINTS: Odontogenic keratocysts may mimic endodontic lesions. Clinicians should carefully review their cases and consider surgical intervention with biopsy in cases that do not heal.

Apical plug technique using mineral trioxide aggregate: results from a case series.

AIM: To examine the clinical and radiographic appearance of teeth that suffered premature interruption of root development and were treated by an mineral trioxide aggregate (MTA) apical plug technique. SUMMARY: Eleven teeth with immature root apices in 11 patients were treated nonsurgically by the manual application of MTA in the apical portion of the root canal under microscopic vision. Follow-up evaluations were performed at 1-2 years after treatment. KEY LEARNING POINTS: Mineral trioxide aggregate appears to be a valid material to obtain periradicular healing in teeth with open apices and necrotic pulps. Ten out of 11 cases were associated with periradicular health at follow-up evaluation.

Half-dose depot triptorelin in pituitary suppression for multiple ovarian stimulation in assisted reproduction technology: a randomized study.

BACKGROUND: Pituitary suppression by depot GnRH agonist may be excessive for ovarian stimulation in assisted reproduction technology. This study compares the efficacy of standard and half-dose depot triptorelin in a long protocol. METHODS: A total of 180 patients were randomized into two groups using sealed envelopes. Pituitary desensitization was obtained in group 1 (90 patients) with half-dose (1.87 mg) triptorelin depot in the mid-luteal phase of their menstrual cycle, and in group 2 (90 patients) with full-dose (3.75 mg) triptorelin. RESULTS: There was no premature LH surge, with LH levels being lower in the full-dose group (1.04+/-0.05 versus 0.7+/-0.06 IU/l on the day of hCG). The number of FSH ampoules used was lower in group 1 (42+/-2 versus 59+/-3). The numbers of mature oocytes (10.1+/-0.54 versus 7.4+/-0.55), of fertilized oocytes (8.24+/-0.35 versus 6.34+/-0.37) and of embryos (7.8+/-0.36 versus 5.9+/-0.37) were significantly higher in group 1. No significant differences were found in pregnancy (38.8 versus 25.3%), implantation (22.6 versus 13.8%) or abortion (6.1 versus 5.0%) rates. Cumulative pregnancy (fresh plus frozen embryo transfers: 56.8 versus 35.4%) rate was significantly higher in group 1. CONCLUSION: A half-dose of depot triptorelin can be successfully used in ovarian stimulation for IVF and produce a higher number of good quality embryos with a good chance of implantation.

GnRH analogs: depot versus short formulations.

Gonadotropin releasing hormone agonists (GnRH-a) are widely used in controlled ovarian hyperstimulation (COH) for assisted reproduction (ART). Two different formulations are now available: short formulations and depot formulation. Some authors have suggested that depot GnRH-a induce a too high pituitary suppression and have put forward protocols using reduced GnRH-a doses. A reduced dose of daily triptorelin is enough for pituitary suppression during ovarian stimulation but provides no significant improvement in IVF cycle outcome when compared with depot formulation in normally responding women. However, it seems to improve ovarian response and overall results in poor responding patients. Low doses of short GnRH-a allow shorter treatment, requiring lower amounts of gonadotropins. This possibility should be considered in view of its economic advantage.

Melting temperature analysis as quantitative method for detection of point mutations.

Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K-ras gene in DNA of subjects with colorectal cancer. The K-ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K-ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.

Use of primed in situ labeling (PRINS) for the detection of telomeric deletions associated with mental retardation.

Rearrangements involving the telomeric regions of human chromosomes are often associated with mental retardation. These rearrangements, however, are difficult to detect using conventional cytogenetic techniques. We propose the use of primed in situ (PRINS) labeling as an alternative to fluorescence in situ hybridization because it is very fast, reproducible, and simple to perform. Sixty-five children with unexplained mental retardation were studied using PRINS technology; two of them were shown to have a telomeric deletion.

Effect of duration of estradiol replacement on the outcome of oocyte donation.

PURPOSE: To investigate if duration of estrogenic endometrial stimulation can affect recipient pregnancy rate in an ovum donation program. METHODS: Each recipient received micronized 17 beta-estradiol orally in a steadily increasing dosage from 2 to 6 mg daily over a period of time varying from 5 to 76 days until oocyte were available for donation. Recipients (520 patients for a total of 835 transfer cycles) were retrospectively divided into five groups depending on the duration of E2 administration. RESULTS: No significant difference was seen in pregnancy and implantation rates between groups. There was a higher number of miscarriages in Group A (41%), p < 0.05 vs. Group B (15%), and vs. Group E (1%). Age, number of pregnancies and miscarriages, or implantation rate in donors (327 women aged < 35 years) were similar in all the five groups. CONCLUSIONS: Endometrial receptivity is tolerant to a wide duration of E2 treatment (until 2 months), while waiting for oocytes available for donation, but best results are achieved with a treatment range of 11 to about 40 days.

Effect of reduced dose of triptorelin at the start of ovarian stimulation on the outcome of IVF: a randomized study.

BACKGROUND: Partial pituitary desensitization using gonadotrophin-releasing hormone (GnRH) agonists may be sufficient in women undergoing controlled ovarian hyperstimulation for assisted reproduction. However, the minimal effective agonist dose remains to be determined. The aim of the study was to investigate the effect of a reduced daily dose of triptorelin, administered at the start of ovarian stimulation, on the results of IVF and intracytoplasmic sperm injection. METHODS: A total of 132 patients was randomized in two groups. Pituitary desensitization was obtained in group 1 (66 patients) with a single 3.75 mg injection (i.m.) of triptorelin. In group 2, 66 patients received 100 microg triptorelin daily, which was then reduced to 50 microg at the start of follicle-stimulating hormone (FSH) stimulation. RESULTS: No significant differences were found in terms of pregnancy rate per transfer (38% in group 1 versus 34.9% in group 2), implantation rate (20.2 versus 18%) and abortion rate (8.3 versus 9.1%). The number of FSH ampoules used, as well as the number of days stimulation required, was significantly reduced in group 2 (41 +/- 26 versus 46.6 +/- 25.3, P < 0.03 and 11 +/- 1.3 versus 11.8 +/- 1.5, P < 0.002 respectively). No significant differences were seen in oestradiol concentrations and in follicle number, in the quantity of oocytes collected and fertilized, or in the number of embryos obtained or transferred. CONCLUSION: A reduced dose of triptorelin is enough for pituitary suppression during ovarian stimulation but provides no significant improvement in IVF cycle outcome when compared with depot formulation. The possibility of a shorter treatment protocol requiring lower amounts of gonadotrophins should be considered in view of its economic advantage.

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues.

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.

Cellular localization of human herpesvirus 8 in nonneoplastic lymphadenopathies and chronic interstitial pneumonitis by in situ polymerase chain reaction studies.

OBJECTIVES: To study the cellular localization of human herpesvirus 8 (HHV-8) in rare cases of HHV-8 infection from Italy that are associated neither with human immunodeficiency virus (HIV) infection nor Kaposi's sarcoma (KS). METHODS: The presence and distribution of HHV-8-infected cells was investigated by direct in situ polymerase chain reaction (PCR) in the lymph node tissues from 2 patients with reactive lymphadenopathies with florid follicular hyperplasia and increased vascularity and in the lung tissue from 1 patient with chronic interstitial pneumonitis. RESULTS: HHV-8 was localized in lymphoid and monocyte-macrophage cells scattered in the interfollicular regions of both lymph nodes but not in endothelial cells. In the lung tissue, HHV-8 was found in the inflammatory cells infiltrating the interalveolar interstitium, in endothelial cells of the pulmonary vasculature, and in rare pneumocytes. CONCLUSIONS: HHV-8 can infect nonneoplastic lymph nodes of immunocompetent subjects, and the distribution of infected cells outside of the germinal centers resembles that of Epstein-Barr virus (EBV)-infected cells in the lymph nodes in the course of infectious mononucleosis. Endothelial cells and pneumocytes may be a target of HHV-8 infection out of the KS setting, at least in the presence of a chronic inflammatory process.

Births after transcervical gamete intrafallopian transfer with a falloposcopic delivery system.

OBJECTIVE: To evaluate the safety and efficiency of a new delivery system to perform transcervical GIFT. DESIGN: Evaluation of pregnancy rate (PR), miscarriage rate, ectopic pregnancy rate, and delivery rate. SETTING: Institute of Obstetrics and Gynecology, Reproductive Endocrinology Unit, Infertility and IVF Center. PATIENT(S): Twenty-five patients with patent tubes documented by laparoscopy plus falloposcopy. INTERVENTION(S): Superovulation was induced with GnRH analogue and FSH. Under laparoscopic control, transcervical cannulation of the tube was done using a linear everting catheter incorporating direct falloposcopic vision of the tubal lumen. Two lengths of everting catheter (3 and 6 cm) were used providing either isthmic-ampullary or midampullary placement of the inoculum. A comparison was done in terms of ease of access and transfer, falloposcopic observations, and PRs between the groups. MAIN OUTCOME MEASURE(S): Efficacy was established by evaluating the PR, miscarriage rate, ectopic pregnancy rate, and delivery rate. RESULT(S): The PR was 28% (with no differences between the lengths of everting catheters). No ectopic pregnancies occurred. The abortion rate was 28.6% and the delivery rate was 20%. Neither tubal perforation nor other complications occurred during the procedure. CONCLUSION(S): Falloposcopic GIFT is safe and efficient and may be a less invasive alternative than laparoscopic transfer.

Quantitation of feline immunodeficiency proviruses in doubly infected cats using competitive PCR and a fluorescence-based RFLP.

A nested polymerase chain reaction assay, which amplifies a region of the gag gene, was developed for the direct detection of feline immunodeficiency virus (FIV) DNA sequences in the blood of infected cats. This method detects as few as ten copies of a plasmid containing the whole genome of the FIV-Pet isolate on agarose gel. To distinguish two FIV isolates in double infected cats, we devised an RFLP analysis on PCR amplified products exploiting sequence differences in the gag gene of the two strains. To quantitate the two strains, a fluorescent inner-sense primer was used in the second amplification step. Amplicons were subsequently digested, heat-denatured and loaded on a polyacrylamide gel in an automated DNA sequencer. The proportion of the two isolates was determined using the laser-excited fluorescence of labelled strain specific fragments. These data were used to extrapolate the numbers of proviral genomes from the total viral load as estimated by using a competitive PCR assay. These sensitive and specific assays complement virological detection of FIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

Partial nucleotide sequencing of six subtype 2c hepatitis C viruses detected in Italy.

The great majority of 121 hepatitis C virus (HCV) isolates obtained from 117 Italian patients with community-acquired infection could readily be typed by genotype-specific PCR. Subtype 1b was dominant (74 isolates); subtypes 2b, 2a, and 1a followed, with 19, 14, and 8 isolates, respectively. The six isolates that remained untyped by this method were classified as subtype 2c on the basis of sequence analysis of PCR amplicors obtained from the core and NS5 genes. These findings indicate that HCV subtype 2c has a relatively high prevalence in Italy. Sequencing the core region from positions 160 to 259 is sufficient to distinguish subtype 2c from other known HCV genotypes.

Comparison between depot leuprorelin and daily buserelin in IVF.

OBJECTIVES: To compare the effects of depot and daily forms of GnRH analogs in IVF programs. METHODS: One hundred seventeen patients undergoing IVF, with no severe male factor, were randomized between two treatment groups. Pituitary desensitization was obtained in group 1 (60 patients) with a single IM injection of leuprorelin (3.75 mg), and in group 2 (57 patients) with buserelin (0.3 mg SC twice daily). In a subgroup of 10 patients (5 for the depot form and 5 for the daily form) several GnRH tests were performed to investigate pituitary desensitization. RESULTS: No differences were found in the time to reach desensitization. Resumption of pituitary activity occurred in 7 days with the daily form and in about 2 months with the depot form. No significant differences were found in the stimulation pattern, oocyte quality, percentage of fertilization. The pregnancy rate per transfer was slightly, but not significantly, better in the depot group (29.4% vs 25.9%). Implantation rate (11.9% vs 12.3%) and the percentage of miscarriages (26.6% vs 28.5%) were similar. CONCLUSION: Depot and daily forms of GnRH analogs are equally effective in superovulation induction for IVF. Considering improved patient compliance and preference, depot forms are advantageous.

A neutralizing antibody-inducing peptide of the V3 domain of feline immunodeficiency virus envelope glycoprotein does not induce protective immunity.

Specific-pathogen-free cats, immunized with a 22-amino-acid synthetic peptide designated V3.3 and derived from the third variable region of the envelope glycoprotein of the Petaluma isolate of feline immunodeficiency virus (FIV), developed high antibody titers to the V3.3 peptide and to purified virus, as assayed by enzyme-linked immunoassays, as well as neutralizing antibodies, as assayed by the inhibition of syncytium formation in Crandell feline kidney cells. V3.3-immunized animals and control cats were challenged with FIV and then monitored for 12 months; V3.3 immunization failed to prevent FIV infection, as shown by virus isolation, anti-whole virus and anti-p24 immunoglobulin G antibody responses, and positive PCRs for gag and env gene fragments. Sequence analysis of the V3 region showed no evidence for the emergence of escape mutants that might have contributed to the lack of protection. The sera of the V3.3-hyperimmunized cats and two anti-V3.3 monoclonal antibodies neutralized FIV infectivity for Crandell feline kidney cells at high antibody dilutions but paradoxically failed to completely neutralize FIV infectivity at low dilutions. Moreover, following FIV challenge, V3.3-immunized animals developed a faster and higher antiviral antibody response than control cats. This was probably due to enhanced virus replication, as also suggested by quantitative PCR data.

Comparison between depot and standard release triptoreline in in vitro fertilization: pituitary sensitivity, luteal function, pregnancy outcome, and perinatal results.

OBJECTIVE: To compare the effects of depot and standard release form of triptoreline in superovulation induction in IVF programs. DESIGN: One hundred ninety-six patients undergoing IVF were randomized between two treatment groups. Pituitary desensitization was obtained in group 1 (102 patients) with a single IM injection of 3.75 mg D-Trp-6-luteinizing hormone-releasing hormone (LH-RH) and, in group 2 (94 patients), with daily SC administration of 0.1 mg D-Trp-6-LH-RH. In a subgroup of 11 patients, a series of GnRH tests was performed to investigate pituitary desensitization and, in another subgroup of 12 patients, a study of luteal phase steroid profile was performed. In an additional 23 patients, a series of GnRH tests were performed to investigate pituitary desensitization during the late follicular and midluteal phases. RESULTS: No differences were found in the time necessary to reach desensitization (11.3 +/- 1.03 versus 11.3 +/- 1.45 days; mean +/- SEM), whereas resumption of pituitary activity takes place in 7 days after the discontinuation of the daily form and in about 2 months after discontinuation of the depot form. No differences were found in the duration of stimulation, number of FSH ampules, E2 levels, and number of follicles (11.7 +/- 0.68, versus 12.2 +/- 0.68) on hCG administration day and the total oocytes collected (9.1 +/- 0.6 versus 9.2 +/- 0.64). Oocyte quality, percentage of fertilization and cleavage, pregnancy rate per transfer (28.7% versus 25.6%), and miscarriages (about 30%) were similar in the two protocols. No difference was found in hormonal levels during the luteal phase. In both groups there was a high incidence of multiple pregnancy. CONCLUSION: Comparable results can be achieved with both long-acting and standard-release forms of GnRH analogs in patients undergoing assisted reproduction in terms of follicular stimulation and abortion rates despite differences in the duration of pituitary suppression.

Competitive polymerase chain reaction for quantitating feline immunodeficiency virus load in infected cat tissues.

To quantitate FIV provirus copy numbers present in tissue of infected cats, we have applied a competitive polymerase chain reaction (cPCR) recently described for HIV. The method consists in coamplifying a fixed amount of the DNA to be examined with graded copy numbers of a DNA competitor incorporating a short deletion and bearing the same primer recognition sequences. These conditions ensure almost identical thermodynamic and amplification efficiency for both template species but permit a prompt recognition of the two amplification products by gel electrophoresis. Since the amounts of the two amplicons are dependent on relative initial template concentrations, the number of FIV genomes in the sample can be calculated by densitometric analysis of the electrophoretic bands. After validation, the method has been applied to study the provirus loads in the tissues of cats infected with the Pisa-M2 isolate of FIV.