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G Bussolati

Publications and source records attributed to G Bussolati.

At least 127 records · Page 7Linked to original sources
PubMed1993

Nonspecific in situ hybridization reaction in neuroendocrine cells and tumors of the gastrointestinal tract using oligonucleotide probes.

Oligonucleotides used in in situ hybridization (ISH), regardless of their sequence specificity, bind to neuroendocrine (NE) cells in normal gastrointestinal mucosa and tumors. This nonspecific binding, presumably related to the presence in NE cells of hidden NH2 groups of obscure origin, can be prevented by acetic anhydride treatment of the sections. This is a routine step in several ISH protocols but not in all. This study emphasizes the need to establish safe protocols and controls to check the specificity of ISH procedures.

Base Sequence↗
PubMed1992

Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.

Alanine Transaminase↗
PubMed1992

Tumor types derived from epithelial and myoepithelial cell lines of R3230AC rat mammary carcinoma.

Epithelial and myoepithelial cells coexist in the rat R3230AC mammary tumor. To test the hypothesis that these two cell types constitute interactive but independent neoplastic populations, we obtained in vitro cell lines with epithelial or myoepithelial patterns and transplanted them in syngeneic animals. One stabilized line (EPI) and four cloned lines (A, C, D, E) with epithelial characteristics, confirmed by positive reactions for keratins in immunocytochemical and immunoblot tests, constantly gave rise in vivo to carcinomas, which, however, lacked structural and functional patterns typical of the original tumor. A fusiform shape and immunocytochemical characteristics of myoepithelial cells were observed in three clones (H, I, L), which in vivo gave rise to sarcomatous and mixed carcinosarcomatous neoplasms. These data are consistent with the above hypothesis and indicate that breast carcinomas derive from epithelial cells, while sarcomatous and carcinosarcomatous neoplasms can originate from myoepithelial cell proliferation. This study provides data suggesting myoepithelial cell involvement in the development of pathological entities occurring in the human breast and displaying mixed epithelial and stromal neoplastic components, i.e., cystosarcoma phylloides and sarcomatous metaplasia in carcinomas.

Animals↗
PubMed1992

Characterization of prolactin receptor in human brain and choroid plexus.

We have studied the binding of 125I-labeled human prolactin (PRL) to membranes from various regions of the human brain (hypothalamus, cerebral cortex, cerebellum and choroid plexus) derived from autopsy specimens. Among the various regions studied, the choroid plexus of both male and female subjects showed the highest specific binding and a clearly detectable specific binding was also observed in the hypothalamus of both sexes, whereas it was very low in other brain regions. No significant sex differences in PRL binding to various brain regions were observed except for the hypothalamus where a higher binding was seen in female subjects. The binding did not vary with the age of the subjects. Moreover, the cause of death and the time elapsed from death to autopsy in this study did not affect the binding significantly. The binding of 125I-labeled human PRL to hypothalamus and choroid plexus membranes from female specimens was inhibited in a dose-dependent manner by both unlabeled human and ovine PRL and by human growth hormone (GH), but not by other polypeptide hormones. Scatchard analysis of the binding revealed the presence of saturable binding sites with low capacity and high affinity for human PRL ligand. These results provide strong preliminary evidence for the presence of PRL binding sites in the human brain.

Animals↗
PubMed1992

Differential diagnostic patterns of lung neuroendocrine tumours. A clinico-pathological and immunohistochemical study of 122 cases.

A series of 3 tumourlets (TLs), 81 typical carcinoids (TCs), 14 atypical carcinoids (ACs) (well-differentiated neuroendocrine carcinomas, WDNCs) and 24 small cell-intermediate cell carcinomas (SCC-ICCs) of the lung were studied. Histopathological features were correlated with amine and peptide hormone immunoreactivity and with clinical data. All types of tumours expressed general neuroendocrine (NE) markers: Grimelius positivity and chromogranins were detected more frequently in well-differentiated (TLs, TCs) than in less well differentiated tumours [ACs (WDNCs) and SCC-ICCs] whereas neuron specific enolase (NSE) was prominent in the latter tumours. TLs and peripheral TCs were benign, often showing a paraganglioid pattern and frequently expressing gastrin-releasing peptide (GRP), which is present in the peripheral airways of normal lung. Central TCs were associated with lymph node metastases in 8.5% of the cases, frequently had a trabecular architecture, often associated with human milk fat globule 2 (HMFG2)-positive acinar and rosette-like structures, and were mainly immunostained for the alpha-subunit of human chorionic gonadotrophin (alpha-hCG) and serotonin. ACs (WDNCs) were associated with intrathoracic and/or extrathoracic metastases in 57.1% of the cases with a mortality rate of 35.7%. Their histological and cytological features were intermediate between those of TCs and SCC-ICCs. ACs (WDNCs) expressed serotonin and alpha-hCG less frequently than TCs. All SCC-ICCs were surgically treated and displayed a mortality rate of 91.6% with a mean survival of 10.2 months after operation. These tumours were characterized by high expression of HMFG2 and NSE, while the expression of both orthotopic (serotonin, GRP) and ectopic (ACTH) specific NE substances was very low. Since all TCs (either central or peripheral) had a favourable outcome, while about 36% of ACs (WDNCs) were fatal, the latter seem more appropriately designated "well-differentiated NE carcinomas". The differential diagnosis between different NE tumours of the lung is important and is mainly based on morphology. Both panendocrine and specific immunohistochemical markers are helpful in distinguishing the less aggressive, mostly benign varieties from the more malignant varieties.

Adult↗
PubMed1992

Primary oat cell (neuroendocrine) carcinoma of the breast. Report of four cases.

Four cases of oat cell (neuroendocrine) carcinoma of the breast are reported. Three patients died within 15 months of the diagnosis and the fourth patient is alive after 44 months. Histochemical, ultrastructural and mRNA markers of endocrine differentiation were present in three cases. These tumours show histological similarities to breast metastases of bronchial oat cell carcinoma, but a distinguishing feature is the presence of in situ ductal lesions. It appears that the breast is a further site which has to be added to the long list of extrapulmonary oat cell carcinomas.

Adult↗
PubMed1992

Antitumour activity of a sterically blocked ricin immunotoxin on a human colorectal adenocarcinoma grafted subcutaneously in nude mice.

We prepared a ricin-antibody conjugate, lacking the ability to bind the galactosidic residues of Sepharose 6B, a so-called blocked immunotoxin. The monoclonal antibody AR-3 was cross-linked to ricin through a thioether bond. Further studies showed that the immunoconjugate suppressed the tumour growth of HT-29 cells in intraperitoneally grafted nude mice, without showing any undesirable ricin toxicity. In this work, to demonstrate the therapeutic activity of the AR-3-ricin conjugate injected into mice bearing subcutaneous tumour, we first evalauted its pharmacokinetic behaviour and biodistribution. The behaviour of the immunoconjugate injected intravenously was almost intermediate between that of the antibody and ricin. Moreover, when the immunotoxin was intravenously administered to nude mice bearing subcutaneous tumour, no therapeutic effects appeared, in accordance with the relatively low permeability of the immunotoxin from the blood to the skin. In contrast, peritumoral treatment produced a strong reduction of the neoplastic nodules without substantial regrowth of the malignant cells. This result was also achieved when the immunotoxin treatment was performed on a well-established tumour. This finding was strictly related to the specifcity of the immunoconjugate, since the analogous treatment with an irrelevant immunotoxin showed therapeutic failure.

Adenocarcinoma↗
PubMed1992

Arguments against the prostatic origin of the R-3327 Dunning H tumor.

The Dunning tumor, originally described as a carcinoma of the rat dorsal prostate, has for long been used as an experimental model of prostatic cancer. We have recently presented a number of morphological findings that are incompatible with the prostatic origin of the H-subline of the Dunning tumor. In this paper, biochemical and immunohistochemical markers of rat prostate and mammary gland are studied in the R-3327 Dunning H tumor. Pieces of the H tumor were inoculated in male or lactating female rats. The electrophoretic protein pattern of Dunning tumor extracts was more similar to that of the mammary gland than the dorsolateral prostate. Proteins selectively appearing after metabolic labeling in Dunning tumors grown in lactating rats corresponded to labeled proteins in mammary glands from the same animals. Secretory proteins typical of the lateral prostate (SVS II) and dorsal prostate (transglutaminase) could not be detected immunohistochemically in the Dunning tumor. Western blot studies of tumor extracts and slot blot analysis of RNA preparations from the tumor confirmed the absence of SVS II and prostate specific transglutaminase from the Dunning tumor. On the other hand, the presence of mammary gland proteins such as milk fat globule membrane proteins, lactoperoxidase and lactalbumin were detected in the Dunning tumor by immunohistochemistry and Western blotting, but were absent from the dorsolateral prostate. Transferrin-mRNA, expressed in the male urogenital tract and also in the liver and other tissues, was detected in the mammary gland and Dunning tumor, but not in the dorsolateral prostate. The absence of mammary gland secretory beta-casein in the Dunning tumor was related to the elevated Ha-ras oncogene expression in the tumor, previously reported to suppress casein expression. The findings clearly demonstrate that the prostate cannot be the origin of the Dunning tumor, presently being used in prostatic cancer research. The designation prostatic adenocarcinoma for this tumor is therefore invalid. Furthermore, the data support our view that mammary gland might be the origin of the Dunning tumor, although the derivation from the bulbourethral or the parotid glands cannot strictly be excluded.

Adenocarcinoma↗
PubMed1992

Immunohistochemical detection of estrogen and progesterone receptors in the normal urinary bladder and in pseudomembranous trigonitis.

According to recent studies showing the presence of estrogens receptors (ERs) in the human female lower urinary tract, we performed ER and progesterone receptor (PR) assays in fresh frozen and paraffin embedded biopsies taken from the urinary bladder. Fourteen females undergoing endoscopy during staging for gynecological cancer (endometrium, cervix, ovary) and 15 women complaining of recurrent abacterial cystitis (pseudomembranous trigonitis) were enrolled in the study as Group 1 and Group 2, respectively. After informed consent, they were submitted to cystoscopy, during which two biopsies were taken: one on the trigonum and the other on the bladder lateral wall. ERs were identified in both groups only in the trigonum (7/14 patients in Group 1 and 8/15 in Group 2), whereas the bladder lateral wall always stained negative. PRs were found at both sites in both groups (11/14 cases on the trigonum and 7/14 on the bladder lateral wall in Group 1; 11/15 and 2/15 respectively in Group 2). Morphological localization of PRs showed intense omogeneous staining in the nuclei of the stromal fibroblasts too. A clear correspondence between the presence of steroid receptors at the squamous metaplasia of the trigonum was observed. These data are discussed speculating about a possible endocrine pathogenesis of pseudomembranous trigonitis.

Adult↗
PubMed1992

Detection of hepatitis delta virus RNA by a nonradioactive in situ hybridization procedure.

A digoxigenin-tailed, synthetic oligodeoxynucleotide was used to detect genomic hepatitis delta virus (HDV) RNA in formalin-fixed, paraffin-embedded liver sections by a nonisotopic in situ hybridization (NISH) procedure. Twenty-three liver samples from chronically HDV-infected patients were studied. Eight liver specimens from humans and chimpanzees without markers of active HDV infection served as negative controls. In three samples, the NISH findings were compared with characteristic nuclear features and with the distribution of the HDV encoded antigen, HDAg, as detected by direct immunofluorescence. All samples from HDV-infected patients were positive for HDV RNA by NISH. The viral genome was exclusively observed within the hepatocytic nuclei. No enzymatic reaction was detected after hybridization with the negative controls. "Sanded" nuclei, a cytopathologic change associated with HDV infection, were HDV RNA-positive, but only a small percentage of infected cells showed that feature. Hepatocytes containing the HDV RNA were sometimes binucleated or exhibited giant nuclei. When HDAg and HDV RNA were sequentially detected within the same sections, the localization of the viral antigen almost completely overlapped with the expression of the HDV transcripts, and vice versa. In conclusion, detection of intrahepatic HDV RNA by NISH is a rapid, sensitive, and specific technique that is easily applicable to routine histopathology and allows correlation of HDV with the morphology of hepatocyte nuclei.

Antigens, Viral↗
PubMed1992

Expression of members of the chromogranin family in primary neuroblastomas.

Expression of the members of the chromogranin family [i.e., chromogranin A (CgA), chromogranin B, and secretogranin II (SgII)], the acidic proteins of the matrix of the chromaffin granules presently regarded as specific neuroendocrine markers, was investigated at gene and protein levels in a series of 14 cases of primary untreated neuroblastomas. Oligonucleotides and cRNA probes were employed for hybridization analysis of specific mRNAs (both by Northern blots and nonradioactive in situ hybridization); proteins were localized by immunocytochemistry. Expression of different amounts of each type of chromogranin was determined in all tumors. Cases found immunocytochemically negative were all positive by Northern blot and in situ hybridization. A better prognosis was associated with a higher relative expression of SgII; on the contrary, a worse outcome was observed in cases with a higher expression of CgA. These findings are consistent with the hypothesis that SgII is preferentially expressed in neuroblastomas undergoing neuronal differentiation. In cases of neuroblastomas, determination of expression levels of the different chromogranins in the tissues (and in the serum) can provide parameters of high diagnostic and prognostic value.

Adolescent↗
PubMed1992

Detection of cytomegalovirus by in situ hybridization using a digoxigenin-tailed oligonucleotide.

A non-isotopic in situ hybridization procedure was used to detect cytomegalovirus (CMV) sequences within routinely fixed tissue. A digoxigenin-tailed oligonucleotide was hybridized to sections of specimens obtained at autopsy from 2 patients with CMV infection. Hybrids were revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. Serial sections were also assayed for the presence of CMV by in situ hybridization with a biotin-labelled cDNA probe and by immunohistochemistry and routinely stained for morphological evaluation. Results show that the two in situ hybridization procedures are equally sensitive but superior to the immunohistochemical detection of the viral antigen. Most cells positive for CMV DNA had the cytopathological features characteristic of CMV infection. A minor population of infected cells lacking morphological changes was also found. We recommend the routine application of the oligonucleotide-based assay because it is specific, easy and less expensive than other similar procedures.

Alkaline Phosphatase↗
PubMed1992

In situ hybridization in viral hepatitis.

In situ hybridization (ISH) is a sensitive and specific technique for detecting nucleic acids in intact cells. Visualization of the target sequences by autoradiography or immunohistochemistry allows their precise subcellular localization and quantitation. The application of ISH techniques has contributed to the understanding of the complex replicative cycle of hepatitis B virus. More recently, hepatitis delta and C virus replication has also been studied by this technique. ISH-based assays have finally been used to follow the replication of cytomegalovirus within the transplanted liver. Although ISH is a powerful tool for the molecular biologist, its clinical significance in the diagnosis and prognosis of human hepatitis virus infections has yet to be fully evaluated.

Hepacivirus↗
PubMed1992

Secretogranin II expression in Ewing's sarcomas and primitive neuroectodermal tumors.

Production of chromogranins, the acidic components of the chromaffin granules regarded as specific neuroendocrine markers, was analyzed by immunocytochemistry and hybridization (Northern blotting and in situ hybridization) in primary lesions and cell lines of Ewing's sarcomas, primitive neuroectodermal tumors (PNETs), and neuroblastomas. Antibodies and probes specific for chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) were used. Ewing's sarcomas and PNETs, unlike neuroblastomas, were negative for CgA and CgB. Two primary Ewing's sarcomas, one primary PNET (an Askin tumor), and one PNET cell line (TC32) were found to strongly express the SgII gene, as shown by the presence of specific mRNA. This result supports the hypothesis that some Ewing's sarcomas represent a most primitive form of neuroectodermal tumor; in addition, it indicates a diagnostic role of SgII in cases of Ewing's sarcomas and PNETs.

Adolescent↗
PubMed1992

Staining reaction for beta-galactosidase in immunocytochemistry and in situ hybridization.

beta-galactosidase, revealed by an indigo blue reaction product, represents a valid tracer in immunohistochemistry. Observations on the instability of the indigo precipitate led us to investigate this phenomenon. We conclude that avoiding of xylene, and mounting of the preparates in Histoclear (a xylene substitute) and Canada balsam (instead of synthetic resin mountants) yields a sharp and stable indigo precipitate. In addition, we propose a sensitive alternative staining procedure for beta-galactosidase, based on TNBT reduction and precipitation. These reactions can be used both in immunohistochemistry and in in situ hybridization.

Galactosidases↗
PubMed1992

Cytological analysis of benign breast disease.

The different histological lesions of benign breast disease (BBD) are the result of interactions and regional prevalence of epithelial, myoepithelial, apocrine, and "null" (undifferentiated) cell types. We conducted an immunocytochemical analysis on 14 cases of BBD. Specific markers were employed to identify the different cell types; proliferation in these cells was revealed either by bromo-deoxyuridine uptake or PCNA ("cyclin") localization. The results indicate that apocrine cells do not undergo proliferation, representing therefore a terminally differentiated cell. The question related to the preneoplastic potential of BBD and on which cell type might possibly represent the precursor of in situ cancerous lesions remains unanswered. However, our data tend to exclude that such a putative proliferating precursor might be represented by apocrine or myoepithelial cells or even by the epithelial cells featuring epitheliosis.

Adult↗
PubMed1991

Expression of the gastrin-releasing peptide gene in carcinomas of the breast.

Expression of gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian bombesin, has been investigated at gene and protein level in a series of 28 primary breast carcinomas, in 6 mammary cancer cell lines and in one transplantable rat mammary carcinoma. Moderate to strong expression of GRP mRNA was detected in 5 breast carcinomas by Northern blot analysis with a pre-pro-GRP probe; 4 other cases were weakly reactive. Two of these cases also gave a specific immunocytochemical reaction for GRP, controlled with absorption experiments. Correlation with NE differentiation [as shown by chromogranins (Cg) and/or NSE and/or Grimelius positivity] was low, since only one case of breast carcinoma co-expressed GRP and Cg mRNAs. Breast cancer cell lines and a rat carcinoma gave negative results. GRP production in breast cancer did not appear to bear prognostic implications, but longer follow-up periods are needed to confirm these data. As shown in small-cell lung cancer, GRP might be involved in autocrine growth control mechanisms of a group of breast carcinomas.

Animals↗