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Biomedical subjects

E Greco

Publications and source records attributed to E Greco.

At least 19 recordsLinked to original sources
PubMed2003

Genetic control of serum IgE levels: a study of low molecular weight protein tyrosine phosphatase.

Protein tyrosine phosphatases (PTPases) have recently been recognized as important modulators of various signal transduction pathways in immune cells. Genetic polymorphisms have been described in genes codifying for members of this family of enzymes, and the genetics of PTPases is predicted to play an important role in the etiology of immune diseases and of their clinical variability. The low molecular weight protein tyrosine phosphatase (ACP1 or LMPTP) is one of the few PTPases with a known genetic polymorphism, and has been proposed to be associated with atopic dermatitis in a small sample from an Italian population. In this paper we describe the association of the ACP1 polymorphism with total IgE levels in two independent samples from English and Italian populations. In both the samples the mean value of serum IgE is lower among subjects carrying the BC genotype than in other ACP1 genotypes. The BC genotype is associated with the highest total ACP1 enzymatic activity. Our data suggest that one or both of the ACP1 isoforms exert an inhibitory role on some signal transduction pathway relevant for IgE hyperproduction.

DNA Primers↗
PubMed2003

Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI.

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.

Cell Nucleus↗
PubMed2002

Helicobacter pylori virulence genes and host IL-1RN and IL-1beta genes interplay in favouring the development of peptic ulcer and intestinal metaplasia.

Helicobacter pylori infection outcome might depend on genotypic polymorphisms of both the bacterium and the host. We ascertained: (1) the functionality of H. pylori oipA gene; (2) the polymorphism of the hostinterleukin (IL-1beta) gene (-31 C/T) and of the IL-1RN gene (intron 2 VNTR); (3) the association between the above genes and the histological and pathological outcome of H. pylori infection. One hundred and sixty-five H. pylori positive and 137 H. pylori negative subjects (23 gastric adenocarcinoma, 58 peptic ulcer, 221 gastritis) were studied. oipA was sequenced, IL-1beta was RFLP analysed. Antral and body mucosal biopsies were histologically evaluated. Functional oipA genes were correlated with cagA gene; both genes were significantly associated with gastritis activity, peptic ulcer and gastric adenocarcinoma. In these patients heterozygousIL-1RN 1/2 and IL-1beta C/T genotypes were more frequent than in gastritis patients. Intestinal metaplasia was associated with cagA, functional oipA and IL-1RN 2 allele. In conclusion, peptic ulcer and the preneoplastic intestinal metaplasia are associated with H. pylori virulence genes and with IL-1RN 2 host allele. An interplay between bacterial virulence factors and cytokines genotypes, is probably the main route causing H. pylori infection to lead to benign mild disease, benign severe disease or preneoplastic lesions.

Adenocarcinoma↗
PubMed2002

Low molecular weight PTP-IL-4RA interaction in atopy predisposition.

We recently described a protective effect of the low molecular weight protein tyrosine phosphatase (LMPTP) BC genotype, associated with the highest total enzymatic activity, against high serum IgE levels both in the English and the Italian populations. Here we test the hypothesis of a role of LMPTP in the negative modulation of IL-4 signal transduction checking for genetic interaction between interleukin-4 receptor alpha chain (IL-4RA) genetic polymorphisms and LMPTP polymorphism in the predisposition to high total IgE levels in the English population. We find a significant interaction between LMPTP polymorphism and the intracellular Gln/Arg polymorphism in position 551 of IL-4RA. Our data support the hypothesis of a direct or indirect biochemical interaction between LMPTP and IL-4RA resulting in different modulation of IL-4 signal transduction among joint genotypes.

Asthma↗
PubMed2002

IL-4 receptor alpha chain genetic polymorphism and total IgE levels in the English population: two-locus haplotypes are more informative than individual SNPs.

The IL-4RA locus encodes for the alpha chain of the IL-4 receptor, and is both a functional and positional candidate gene for atopy and allergic disease. Recently Ober et al. have shown that the study of haplotypes at multiple loci in the IL-4RA gene could be more informative than the separate study of single nucleotide polymorphisms (SNPs). One hundred and fifty subjects affected by atopic asthma and 150 healthy control subjects were studied in the English population (Oxford district). Subjects and controls were genotyped for the Ile50Val, Ser478Pro and Gln551Arg polymorphism of the IL-4 receptor alpha chain. The distribution of haplotypes 50-478 shows a highly significant association with IgE levels. In particular, the haplotype Val50/Pro478 is much less frequent in subjects with IgE levels > 100 U mL-1 than in those with IgE levels < 100 U mL-1. Furthermore, the distribution of haplotype 50-551 shows a weak association with IgE levels that is lacking for 478-551 haplotypes. A lower frequency of the Val50/Pro478 haplotype is also observed among asthmatic subjects as compared to healthy controls. With regard to individual SNPs (50 478 and 551), no significant association has been observed with IgE levels or with asthma, thus confirming the higher informative value of the haplotype analysis as compared to separate study on SNPs.

England↗
PubMed2002

Low dose prednisolone administration in routine ICSI patients does not improve pregnancy and implantation rates.

BACKGROUND: Glucocorticoids have been used in conjunction with zona dissection to improve pregnancy and implantation rates in IVF patients. The aim of this prospective randomized study was to evaluate the effect of low-dose prednisolone in addition to the standard protocol, on pregnancy and implantation rates in routine ICSI patients before and after embryo replacement. METHODS: A total of 313 patients in 360 consecutive cycles (patients <39 years old and with three or less than three ICSI attempts) performed at our centre were randomly assigned by computer-generated list to receive either prednisolone (10 mg/day in two divided doses), starting on the first day of ovarian stimulation and continuing for 4 weeks (group A), or no treatment (group B). RESULTS: The mean age, number of previously failed IVF attempts, basal FSH levels and the mean rank of trials were comparable between groups A and B. The mean (+/- SD) number of metaphase II oocytes retrieved (11.9 +/- 5.5 versus 12.0 +/- 5.1), 2-pronuclei fertilization rate (67.2 versus 65.8%), the pregnancy and the implantation rates were not different between the study and control groups (49.0 and 23.6% versus 50.0 and 23.3% respectively). CONCLUSION: Low-dose prednisolone treatment in addition to the standard protocol before and after embryo replacement does not appear to have a significant effect on pregnancy or implantation rates.

Adult↗
PubMed2001

Colorectal cancer metastatic phenotype stimulates production by fibroblasts of N-terminal peptide of type III collagen: clinical implications for prognosis.

In this study we assessed whether the serum levels of the N-terminal peptide of type III collagen (PIIIP), an index of type III collagen synthesis, are influenced by colorectal cancer stage, and whether "in vitro" fibroblast growth and PIIIP production could be altered by tumor tissues obtained from metastatic and nonmetastatic colorectal cancer. 208 colorectal cancer patients (115 colon and 93 rectum) were studied; 54 were stage I, 62 stage II, 37 stage III and 55 stage IV. PIIIP serum levels were significantly higher in stage IV as compared to all other patient groups. The 5-year survival of stage I, stage II, stage III and stage IV patients were 87%, 88%, 32% and 20%, respectively. In the subgroup of stage I and stage II patients considered together, PIIIP (> 0.5 U/ml), but not CEA (> 5 microg/l) serum levels, were predictive for survival. Fibroblast growth was significantly inhibited, while PIIIP production was significantly enhanced, when these cells were conditioned with colorectal cancer homogenates obtained from patients with distant metastases, than from those without distant metastases. In conclusion, colorectal tumors, when metastatic, stimulate fibroblasts' PIIIP synthesis and the serum levels of this peptide might predict patients' outcome after radical surgery.

Carcinoembryonic Antigen↗
PubMed2001

Laser-assisted intracytoplasmic sperm injection.

OBJECTIVE: To improve oocyte survival and fertilization rates after intracytoplasmic sperm injection (ICSI) in patients with inherent oocyte fragility. DESIGN: Pilot feasibility study and case report. SETTING: Private hospital. PATIENT(S): Infertile couple with repeated failures of ICSI caused by oocyte degeneration. INTERVENTION(S): Laser-assisted drilling of the zona pellucida followed by ICSI. MAIN OUTCOME MEASURE(S): Oocyte survival, fertilization, and pregnancy. RESULTS: In a couple with four previous ICSI failures because of poor oocyte survival (33.3%, 0%, 20%, and 18%), a fifth attempt using laser-assisted ICSI resulted in the survival of 8 oocytes out of 13 injected. Normal fertilization occurred in 5 oocytes, and a clinical pregnancy was established. CONCLUSION(S): Performing ICSI through a laser-drilled hole in the zona pellucida reduces the risk of oocyte damage related to deformation during the initial phase of the microinjection procedure. This modification of ICSI appears to be suitable for patients whose oocyte show inherent fragility and high degeneration rates after the standard ICSI procedure.

Adult↗
PubMed2001

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques.

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

Adult↗
PubMed2001

Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study.

BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50 IU/l FSH and 1 micromol/l testosterone. The frequency of spermatozoa showing DNA strand breakage and plasma membrane phosphatidylserine externalization was compared in before-culture and after-culture samples. The after-culture samples were used in assisted reproduction attempts. RESULTS: In a group of 11 azoospermic patients with at least two previous intracytoplasmic sperm injection (ICSI) failures, the incidence of DNA strand breakage was high in living testicular spermatozoa from before-culture samples, but significantly lower in after-culture samples (96 versus 30%, P < 0.001). The same applied to the incidence of phosphatidylserine externalization in the motile sperm subpopulation from the before-culture and after-culture samples (83 versus 6%, P < 0.001). Seven ongoing clinical pregnancies (six with fresh embryos and one with cryopreserved embryos) were established. CONCLUSIONS: Severe testicular sperm apoptosis may become a new indication for testicular tissue in-vitro culture before ICSI.

Adult↗
PubMed2000

In-vitro maturation of immature human male germ cells.

Both meiotic and postmeiotic maturation events have been observed to occur in human male germ cells during in-vitro culture. The temperature of 30 degrees C, medium supplementation with follicle-stimulating hormone and testosterone and the maintenance of the original cell-cell associations within explanted segments of the testicular seminiferous tubules are common features of the most efficient culture systems. The in-vitro maturation processes are markedly accelerated as compared to the in-vivo situation, probably due to the abrogation of a checkpoint controlling the full assembly of molecules needed for spermiogenesis. Moreover, both meiotic and postmeiotic maturation processes can be reactivated in vitro in some cases with a complete block of the same processes in vivo. Healthy babies were born after micromanipulation-assisted fertilization with in-vitro matured elongated spermatids from men with complete in-vivo maturation arrest at the round spermatid stage and at the primary spermatocyte stage.

Cell Differentiation↗
PubMed2000

ME-PCR for the identification of mutated K-ras in serum and bile of pancreatic cancer patients: an unsatisfactory technique for clinical applications.

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.

Adult↗
PubMed2000

The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei.

OBJECTIVE: To compare the oocyte-activating ability of whole human round spermatids and their isolated nuclei. DESIGN: Prospective study using sibling oocytes from patients undergoing spermatid conception and intracytoplasmic sperm injection treatment cycles. SETTING: Private assisted reproduction laboratories and a university department. PATIENT(S): Couples with male infertility. INTERVENTION(S): Sibling oocytes were injected either with whole round spermatids or with their isolated nuclei, followed by artificial triggering of oocyte activation with calcium ionophore. Other sibling oocytes were injected either with isolated spermatid cytoplasm or with whole mature spermatozoa. MAIN OUTCOME MEASURE(S): Numbers of activated oocytes and cleaving embryos. RESULT(S): After oocyte activation was boosted with calcium ionophore, whole spermatids and isolated spermatid nuclei were equally effective in supporting oocyte activation and the formation of pronuclei, whereas no control oocyte was activated under the same conditions after injection of isolated spermatid cytoplasmic compartments. Cleavage rates were lower after the injection of isolated spermatid nuclei than after the injection of whole spermatids. CONCLUSION(S): The factor responsible for human oocyte activation after round spermatid injection is associated with spermatid nuclei. The requirement for the artificial trigger (calcium ionophore) suggests that this factor is identical to the male gamete activity previously characterized as calcium oscillator.

Calcium Signaling↗
PubMed2000

Immature germ cell conception-in vitro germ cell manipulation.

Experimental studies in laboratory animals have shown that successful conception can be achieved by fertilizing oocytes with immature male germ cells. This gave rise to the concept that immature germ cells recovered from the testes of azoospermic men with maturation arrest may be used for assisted reproduction. However, in contrast to using germ cells recovered from healthy animals, clinical application to the treatment of male sterility is burdened by inherent defects in germ cells attributable to underlying testicular pathology. The recent introduction of in vitro germ cell culture/manipulation techniques makes it possible, in some cases, to overcome the in vivo maturation arrest by allowing an additional meiotic and post-meiotic differentiation and the selective harvesting of cells devoid of apoptosis-related nuclear and cytoplasmic damage. These techniques enabled the first births of normal infants fathered by azoospermic men with maturation arrest at the primary spermatocyte stage and improved the efficacy of assisted reproduction in men with maturation arrest at the round spermatid stage.

Animals↗
PubMed2000

Criteria predicting the absence of spermatozoa in the Sertoli cell-only syndrome can be used to improve success rates of sperm retrieval.

In patients with non-obstructive azoospermia, testicular sperm extraction (TESE) is a method of choice to recover spermatozoa as a male therapeutic approach in intracytoplasmic sperm injection (ICSI) programmes. However, the efficacy of TESE in this indication is burdened by a frequent failure of sperm recovery, which renders useless both the invasive testicular intervention and ovarian stimulation of the patient's spouse. One of the most frequent pathological pictures characterizing complete absence of spermatozoa is germinal aplasia (Sertoli cell- only syndrome or SCOS). Two different histological patterns of SCOS have been already described during the past five decades. These two patterns can be characterized as the congenital (pure) and the secondary (mixed) forms. Both patterns, with different prognosis to retrieve spermatozoa by therapeutic testicular biopsy, are frequently confused when TESE is performed during ICSI programmes. Useful criteria to predict the absence of spermatozoa can be obtained by a definite recognition of the two typical histological patterns during the diagnostic testicular biopsy. The diagnosis of congenital or acquired SCOS can be refined by endocrine, chemical, immunohistochemical and molecular biology aids. Reduction of both sperm retrieval failure and unnecessary ovarian stimulation can be achieved by combination of these methods.

Forecasting↗
PubMed2000

Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes.

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.

Adenine↗