Exon 3 of the alpha folate receptor gene contains a 5' splice site which confers enhanced ovarian carcinoma specific expression.

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1-chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5' and 3' deleted variants of the P1-CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5' splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma.

Functional effect of point mutations in the alpha-folate receptor gene of CABA I ovarian carcinoma cells.

The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.

Interferon-gamma potentiates interleukin (IL)-6 and tumor necrosis factor-alpha but not IL-1beta induced by endotoxin in the brain.

Because interferon-gamma (IFN gamma) is present in the central nervous system during neurologic diseases associated with inflammation, its effect on endotoxin-induced cytokines was studied. Cerebrospinal fluid (CSF) and serum levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF alpha), their messenger RNA expression in brain areas (hypothalamus, hippocampus, and striatum) and in spleen were evaluated 2 and 8 h after endotoxin [lipopolysaccharide (LPS), 25 microg/rat i.c.v.], IFN gamma (2.5 microg/rat i.c.v.) or after their coadministration in rats. CSF and serum IL-1beta levels were increased by LPS alone and IFN gamma coadministration did not furtherly increase them. IFN gamma potentiated LPS effect on IL-6 and TNF alpha levels in both CSF and serum. LPS and IFN-gamma coadministration did not alter IL-1beta messenger RNA expression induced by LPS in brain areas and in spleen, but it potentiated that of IL-6 and TNF alpha. The present in vivo data show that i.c.v. coadministration of LPS and IFN gamma results in a potentiation of cytokine production (IL-6 and TNF alpha) which may trigger a cascade of events relevant to neurodegenerative processes. This action is independent of IL-1beta because the production of this cytokine is not altered by IFN gamma treatment.

Beta-amyloid fragment potentiates IL-6 and TNF-alpha secretion by LPS in astrocytes but not in microglia.

The effect of a peptide homologous to the biologically active fragment of beta amyloid 25-35 (beta 25-35) was studied on interleukin 6 (IL-6) and tumour necrosis factor (TNF-alpha) secretion induced by lipopolysaccharide (LPS) in primary rat astrocytes and microglia. Twenty-four hour exposure to LPS (50 ng/ml) induced IL-6 and TNF-alpha both in astrocytes and in microglial cells, while the effect of beta 25-35 (50 microM) per se was negligible in both cell types. In microglial cells, the application of beta peptide did not alter the production of either cytokine induced by LPS. However, beta 25-35 strongly amplified the production of both IL-6 and TNF-alpha in astrocytes. These findings confirm the complex interaction between cytokines and amyloidogenesis in Alzheimer's disease and indicate that astrocytes rather than microglia respond to the beta amyloid fragment, suggesting that these cells may be actively involved in cytokine-mediated events in AD.

[Importance of the analysis of the carcinoembryonic antigen in clinical oncology]. / Importanza del dosaggio dell'antigene carcino-embrionario in oncologia clinica.

330 patients (126 with gastric neoplasms, 204 with large bowel carcinoma) were submitted to serial assays to evaluate the possible relations between C.E.A. levels, pathologic stage and histologictype of the neoplasm and to define the usefulness of the C.E.A. test in monitoring the followup of the patients with gastrointestinal neoplasms. From our experience it ensues that C.E.A. test positivity (C.E.A. greater than or equal to 5 ng/ml, according to the method employed) is higher in colon neoplasms in comparison with gastric neoplasms, in the adenocarcinomas compared with the anaplastic forms. Besides, the study of the relationship with the pathologic stage points out the scanty usefulness of the C.E.A. test in the early diagnosis of gastroenteric neoplasms (Dukes A-B-C1 = 29.2%; CH stage = 88.1%). The use of C.E.A. test during the follow up seemed us of fundamental importance. We observed that: a) after radical surgery, 72% of the patients showed a normalization of C.E.A. values; b) there is a significant relationship between clinical course and C.E.A. as it can predict, sometimes several months earlier, the occurrence of relapses and metastases; c) there is also a close relationship (P less than 0.001) between the modifications of the antigen under chemotherapy and the clinical response. At present, C.E.A. seems to play, above all, fundamental role in choosing a correct treatment after radical surgery or in modifying the chemotherapeutic treatment in non surgical cases or in non radically resected patients.

Clinical usefulness of CEA assay.

CEA is a normal constituent of fetal cells, which can reappear in entodermally-derived neoplasms, following a de-repression process. In order to evaluate the correlation between CEA serum levels, pathological stage and histological type of tumour, and to define the usefulness of CEA in monitoring the patients with gastroenteric neoplasms, 177 patients (62 with gastric heteroplasia and 115 with large bowel carcinoma) were tested for CEA; 83 patients had pre-surgical CEA tests. It was observed that CEA positivity (serum levels greater than or equal to 5 ng/ml, employing the technique here outlined), is higher in colonic than in gastric neoplasms, and in adenocarcinomas in comparison with undifferentiated forms, moreover depending on the pathologic stage. 108 patients were tested for CEA several times and a correlation between CEA variations and clinical evolution was observed. This investigation bears out the importance to monitor the patients with gastrointestinal neoplasms with CEA test, before and after surgery, to evaluate the usefulness of surgery and, when necessary, chemotherapy and, eventually, to adopt more suitable chemotherapeutic modalities.

Correlation between clinical course and CEA levels during chemotherapy.

In order to evaluate the correlation between clinical progress and CEA levels in gastroenteric tumours, particularly during chemotherapeutic treatment, CEA assay was performed on 330 patients (126 with gastric neoplasms, 204 with large bowel carcinoma). 175 out of these had a pre-operative assay. Moreover CEA test positivity (CEA larger than or equal to 5 ng/ml according to the technique employed by us) is higher in colon neoplasms compared with gastric neoplasms and in adenocarcinomas in comparison with undifferentiated forms; besides it depends on the pathologic stage. In colon tumours CEA test showed a higher positivity for left than for right forms (66.6% versus 38%). 240 patients were followed up with repeated CEA assays: the following observations were made: a - After radical surgery 72% of the patients shows normalized CEA values. b - In 198 patients who underwent radical surgery, not requiring chemotherapy, there was a close correlation between CEA levels and clinical evolution in 98% of the cases. c - 60 out of 68 patients (88%) submitted to chemotherapy for advanced neoplasms show a close correlation between CEA response to the chemotherapy and clinical response (p less than 0.001). These investigations stress, above all, the importance of CEA test to monitor the treatments performed (surgical and chemotherapeutic) and to adopt, eventually, more effective chemotherapeutic modalities.

Significance of the presence of the various subtypes for the preparation of HBAb.

The superficial Australia antigen is essentially bound to the small particles and possesses different antigenic specificities 'a', 'd', 'g', 'w', and 'r'. The population which we tested was characterized by the major frequency (more than 90%) of the 'ag' subtype with respect to the 'ad' subtype. The doses have been achieved using the solid phase RIA, modified according to Ling, in which the specificity of the antisera anti 'a', 'd' and 'g' was obtained by means of the absorption and dissociation of the antigen-antibody complexes. The investigations which we carried out to establish the avidity of the various anti-HBs anti-sera used for research into the Au antigen, have established that in general the avidity is maximal for the anti-ad and minimal or absent for the anti-ag. From this arises the necessity for the preparation of an anti-HBs anti-serum 'pool' consisting of healthy and hepatitic donors, with an optimal relationship between the subtypes 'ad' and 'ag'. The subsequent research carried out using the true subtypes in order to establish a relationship between the subtype and the presence of the anticore antibody on RIA revealed the lack of any correlation.