Absent right and persistent left superior vena cava: fetal and neonatal echocardiographic diagnosis.

Absence of the right superior vena cava (SVC) is a rare event occurring in patients without congenital cardiovascular abnormalities. Since absence of the right SVC is usually clinically silent, its diagnosis is mandatory prior to invasive medical or surgical procedures. We report two cases of echocardiographic diagnosis of absence of the right SVC with persistent left SVC and a large coronary sinus in structurally normal heart in a fetus of 20 weeks' gestation and in a newborn. The diagnosis was confirmed by transthoracic contrast echocardiography with intravenous injection of agitated saline into the right arm.

Detection of ornithine decarboxylase mRNA in human breast cancer MCF-7 cells by in situ RT-PCR.

We have studied the expression of ornithine decarboxylase (ODC) mRNA by in situ hybridization and in situ RT-PCR in human breast cancer MCF-7 cell line. In situ RT-PCR demonstrated the overexpression of ODC mRNA, localized over the cytoplasm, while a very low signal was detected by in situ hybridization. Our findings indicate that in situ RT-PCR could represent a useful tool to study different levels of ODC expression in normal and tumor tissues. Since ODC expression is regulated by c-Myc oncoprotein, this model could be useful to monitor in vivo the effects of new anti-neoplastic molecules, specific inhibitors of c-Myc.

Survey of Lyme disease in Abruzzo (Italy).

This study reports the diagnostic activity for the serological diagnosis of Lyme Disease (LD) in the patients of Abruzzo, a central region of Italy. During the period from August 1994 to July 1999, serological examinations for anti-Borrelia burgdorferi antibodies were performed on 1089 samples from 769 patients with symptomatology consistent with LD. Using an immunoenzymatic technique which was confirmed with Western Blot, 29 patients were diagnosed positive. Twenty-five of these patients contracted the disease in Abruzzo, two during a trip to the USA, one was from Molise and one from Marche. Overall the patients were young, 64% were women and residents of costal areas who frequently engaged in naturalistic activities. The most common symptoms were articular and one patient presented Bannwarth Syndrome. The various antibiotic therapies used gave good results in most cases. These are the first cases reported in literature for this region and for Molise. We believe that LD is underestimated, especially due to the favorable climatic and environmental conditions present in this region. Therefore, we suggest an intensification of clinical and epidemiological controls.

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.

Inhibition of cancer cell growth and c-Myc transcriptional activity by a c-Myc helix 1-type peptide fused to an internalization sequence.

c-Myc is a nuclear protein with important roles in cell transformation, cell proliferation, and gene transcription. It has been previously shown that a 14-amino acid (aa) modified peptide (H1-S6A,F8A) derived from the helix 1 (H1) carboxylic region of c-Myc can interfere in vitro with specific c-Myc DNA binding. Here, we have linked the above Myc-derived 14-aa peptide to a 16-aa sequence from the third helix of Antennapedia (Int). It has been repeatedly reported that this 16-aa Antennapedia peptide is able to cross mammalian cell membranes and to work as a vector for short peptides. Using fluorescent (dansylated or rhodaminated) peptides, we have shown that the fusion peptide with the Antennapedia fragment (Int-H1-S6A,F8A) but not the c-Myc derived fragment alone (H1-S6A,F8A) was capable of internalization inside MCF-7 human breast cancer cells. Int-H1-S6A,F8A and H1-S6A,F8A were the only two peptides capable of inhibiting coimmunoprecipitation of the c-Myc/Max heterodimer in vitro. We have treated (continuously for 10-11 days) MCF-7 cells with four different peptides: Int, H1-S6A,F8A, Int-H1-S6A,F8A, and Int-H1wt [a peptide differing from Int-H1-S6A,F8A by 2 aa (S6 and F8) in the H1 region]. In intact MCF-7 cells, Int-H1-S6A,F8A was the only active peptide capable of inducing the following biological effects: (a) inhibition of cloning efficiency on plates; (b) inhibition of cell growth and induction of apoptosis in subconfluent/confluent cells; and (c) inhibition of transcription of two c-Myc-regulated genes (ODC and p53). Int-H1-S6A,F8A was active in the 1-10 microM range. Int-H1-S6A,F8A may represent a lead molecule for peptidomimetic compounds that have a similar three-dimensional structure but are more resistant to peptidases and, therefore, suitable for in vivo treatment of experimentally induced tumors.

Mutation frequencies at codon 248 of the p53 tumour suppressor gene are not increased in colon cancer cell lines with the RER+ phenotype.

The replication-error positive (RER+) phenotype characterizes tumour cells with microsatellite instability. This 'mutator phenotype' is thought to induce spread mutations throughout the genome, thus increasing the risk of tumour development. Here we analyse spontaneously arising mutations at the tetranucleotide CCGG ( Msp I recognition site), at positions 14 067-14 070 of the p53 gene sequence, in three colon cancer cell lines, two with microsatellite instability and one without this characteristic. This restriction site covers hot-spot codon 248, which is often mutated in colon carcinomas. Using the Msp I RFLP-PCR assay we found that the mean mutation frequency at this site was not different among the cell lines considered. Taking the substitutions separately, none of the mutations involving codon 248 arose with significantly higher frequency in each of the RER+ cell lines (HCT116 and DLD1) compared with the RER-one (SW480). Only the CG transversion at nt 14 067 (codon 247) occurred with a slightly higher, but biologically insignificant, frequency in one of the RER+ cell lines (HCT116). Our in vitro data support the previously reported lack of correlation between microsatellite instability and p53 mutations in RER+ tumour specimens.

Mechanism of resistance to cisplatin in a human ovarian-carcinoma cell line selected for resistance to doxorubicin: possible role of p53.

A possible novel mechanism of cross-resistance to cisplatin (CDDP) in the doxorubicin-resistant ovarian-cancer cell line A2780-DX3, which displays atypical multidrug resistance, is presented. A2780-DX3 is found to be more resistant than the parental line A2780 in terms of CDDP-induced cytotoxicity and apoptosis. Resistance is not related to the amount of cross-links. Topoisomerase-II (topII) protein levels were similar in both cell lines, with lower cleavage activity in A2780-DX3 cells. The parental and the doxorubicin-resistant cells expressed the same level of c-erb2, which could be implicated in CDDP resistance. bcl2 was almost undetectable in both cell lines. At the same time, we found strong induction of p53, waf-1 and bax protein levels after CDDP treatment in the A2780, but not in the A2780-DX3, cell line. Treatment of both cell lines with mitomycin C (MMC), which acts with a mechanism different from CDDP, caused equal accumulation of p53 and induction of bax. We found that A2780-DX3 cells exhibit altered cellular localization of p53 protein in comparison with A2780. A significant proportion of p53 in A2780-DX3 cells was found in the cytoplasmic compartment, and CDDP treatment induced a functional p53 protein in the nucleus of A2780 much more strongly than in A2780-DX3, which coincides with an increase of transcriptional activity of p53 in treated A2780 cells. We propose that the cross-resistance to CDDP in the A2780-DX3 cell line may be due to inactivation of a CDDP-dependent p53-accumulation pathway.

A fast and simple in situ PCR method to detect human papilloma virus infection in archival paraffin-embedded tissues.

We describe a simple and fast method for the detection and localization of low copy numbers of HPV DNA in formalin-fixed and paraffin-embedded archival tissues. We have developed a protocol for direct bl situ-PCR in order to demonstrate its convenience in rapid and reproducible assessment of HPV infection in unknown biopsies. The morphological aspect of the tissues has been maintained, despite the multiple steps of fixation, permeabilization and thermal cycling, and positivity has been detected only in virus target cells.

Endogenous tumor necrosis factor enhances topoisomerase II inhibitors activity in human ovarian cancer cell lines.

In this study, we demonstrated that tumor necrosis factor (TNF), secreted endogenously by four human ovarian cancer cell lines (A2774, IGROV-1, OVCAR-8, SW626), is biologically active against L929 cells and its activity is specifically inhibited by anti-TNF antibodies. Its endogenous production is increased by treatment for 24 h with phorbol myristate acetate (PMA)/ Ionomycin (Iono). All cell lines express TNF high-affinity receptors and release only 60-kdalton soluble TNF receptor, both spontaneously and after stimulation with PMA/Iono. TNF endogenously secreted by human ovarian cancer cell lines is very efficient in potentiating the activity of DNA topoisomerase II inhibitors (doxorubicin, mitoxantrone, VP16). The activity of vinblastine and bleomycin is not potentiated and, more interestingly, cisplatin's activity is inhibited. In 24-h PMA/Iono-stimulated A2774 cells, mitoxantrone specifically generated more cleavable complexes than in unstimulated cells. This result could provide an important tool in the therapy of human ovarian cancer secreting TNF protein, previously considered as a negative prognostic factor.

Comparison between novel steroid-like and conventional nonsteroidal antioestrogens in inhibiting oestradiol- and IGF-I-induced proliferation of human breast cancer-derived cells.

1. This study has two specific aims: (a) to compare the antioestrogenic activity of two steroidal analogues of 17 beta-oestradiol, the 7 alpha-alkylamide, ICI 164,384 and the 7 alpha-alkylsulphinylamide, ICI 182,780, with that of the triphenylethylene-derived compound 4OH-tamoxifen on a pool of human breast cancer cell lines (HBCCL) with a range of hormonal responsiveness and acquired anti-oestrogen resistance and (b) to investigate the ability of such antioestrogens to modulate the potent breast carcinoma growth-stimulatory activity of the 'IGF-I system'. 2. For the chemosensitivity investigations we used a long-term colorimetric and the short-term thymidine incorporation assay; we analysed IGF-I in conditioned media by a radioimmunoassay, IGF-I mRNA in the cells by RT-PCR and molecular species of IGF-I-binding proteins, secreted in conditioned media, by Western ligand blot. IGF-I receptors were assayed on cell monolayers by binding studies and by Scatchard analysis, we calculated KD, Bmax and sites/cell. 3. Our results indicate that ICI 182,780 and ICI 164,384 are 1.5-5.5 fold more potent than 4OH-tamoxifen in inhibiting the basal proliferation of oestrogen-receptor positive (ER+) breast cancer cell lines. Moreover we demonstrate the capacity of ICI 182,780 and ICI 164,384 to reduce, in a time-dependent fashion, oestrogen- and/or IGF-I-stimulated growth of ER+cell lines, possibly by negatively interfering with an IGF-I-like material secretion and IGF-I-receptor number. 4. Our data provide the first evidence that, on ER+human breast carcinoma cell lines, steroidal antioestrogens inhibit cell growth and modulate the IGF-I mitogenic system. The mechanism of this latter effect has yet to be identified.

Oncosuppressor genes: mechanisms of inactivation for the retinoblastoma gene and p53.

Mutations that affect oncosuppressor genes contribute to transformed phenotype. The recessive characteristics of these genes require mutations on both alleles. For this reason alterations of the oncosuppressor genes can be transmitted with the germ line. In this review we focus on mechanisms of inactivation of the retinoblastoma gene and p53. The products of these genes are nuclear phosphoproteins involved in controlling cellular proliferation. Inactivated Rb genes have been found in several tumor types. The phosphorylation of the Rb gene product, p105Rb, is regulated by cyclin-dependent kinases in accordance with the cell cycle. Hypophosphorylated wild-type p105Rb is tightly bound to the nuclear matrix and seems to be critical in inhibition of cellular proliferation. Hyperphosphorylation is a physiological mechanism of inactivation of p105Rb. The p53 gene product is a transcriptional factor that blocks the progression of cell division. Mutations in the p53 gene are frequently found in many human cancers and are localized in the highly conserved region of the gene. The protein product of the mutated gene looses its function as a negative regulator of cellular proliferation. The wild-type protein can also be inactivated by forming stable complex with a mutated p53 protein or with the SV large T antigen.

Detailed ultrasound as a screening method for craniospinal abnormalities.

High resolution diagnostic ultrasound was assessed as a screening method for craniospinal anomalies during the second trimester of pregnancy in a population at low risk for neural tube defects (83,403 mothers). The effectiveness of the test was about 60% and the failure rate mainly due to late attendance. In a subgroup (9325) where the screening purposes were satisfactorily fulfilled, the detection rate (87%) was substantially greater. The significance of the results and the cost/benefit ratio, especially compared with serum alpha-feto protein screening services, are then discussed.

Multiple malignant tumors of the uterus.

A case of a 59 year old patient with a diagnosis of uterine double carcinoma, namely of the cervix and of the corpus is reported. Pathological criteria of diagnosis and pathogenetic hypotheses are then discussed.