Biomedical subjects

E Cattaneo

Publications and source records attributed to E Cattaneo.

At least 55 records · Page 3Linked to original sources

PubMed1996

Activation of the JAK/STAT pathway leads to proliferation of ST14A central nervous system progenitor cells.

We were interested in whether central nervous system progenitor cells possess the signal transduction machinery necessary to mediate cytokine functions and whether this machinery can become activated upon stable expression of a particular cytokine receptor. For this purpose we utilized a previously obtained conditionally immortalized striatum-derived nestin-positive cell line (ST14A). We found that ST14A cells express Jak2, but not Jak1 or Tyk2. An identical pattern of expression was found in embryonic striatal tissue. To evaluate the susceptibility of these cytokine specific cytoplasmic transducers to activation, ST14A cells were stably transfected with the alpha and beta (AIC2A) chains of the murine interleukin-3 receptor. Four independent lines expressing both the alpha and beta receptor subunits were obtained. We found that cells from each of these lines were induced to proliferate upon exposure to interleukin-3. Dose response curve, antibody blocking experiments and binding studies revealed that the response was mediated by the reconstituted high affinity interleukin-3 receptor. Immunoprecipitation studies on these cells showed that Jak2 and Stat5 were being phosphorylated after stimulation of the reconstituted receptor. These results indicate that members of the JAK/STAT family of proteins are expressed in central nervous system progenitor cells and are susceptible to activation through stimulation of an exogenously expressed cytokine receptor, ultimately leading to cell proliferation.

Biomarkers↗

PubMed1996

Changes in beta amyloid precursor protein secretion associated with the proliferative status of CNS derived progenitor cells.

We characterized the secretion of amyloid precursor protein (APP) in rat primary neurons and conditionally immortalized central nervous system (CNS) derived progenitor cell lines, to evaluate their suitability as models for studies on APP metabolism. We observed that primary cells dissociated from different regions of the embryonic brain secreted progressively more APP during in vitro maturation. The increase in basal secretion resulted in an offset of the response to protein kinase C (PKC) activated secretion and was correlated with an increased PKC alpha expression. The same type of analysis was performed on CNS derived conditionally immortalized cells whose growth potential becomes restricted upon shifting of the culture conditions to a non-permissive temperature (39 degrees C), an event that coincides with their progression toward differentiation. With a pattern consistent with that observed in primary neurons, conditionally immortalized cells exposed to 39 degrees C showed an offset of the secretory response to activation by PdBu and also an increased expression of PKC alpha. These data suggest that APP secretion and its regulation in CNS derived cells is influenced by the proliferative status of the cells.

Amyloid beta-Protein Precursor↗

PubMed1996

Intracerebral tetracycline-dependent regulation of gene expression in grafts of neural precursors.

Tight control of the activity of a therapeutic gene introduced in vivo is a major issue in gene therapy research. Appropriate levels of expression may be crucial for gene correction. The tetracycline-sensitive regulatory system is highly effective for transcriptional regulation of foreign genes in mammalian cells. Here we report tight tetracycline-dependent regulation of a luciferase reporter gene transferred into the rat brain in the genetically modified neural precursor cell line ST14A as early as 2 days and until at least 6 days after transplantation. This is the first demonstration of the potential of this regulatory system for the modulation of the expression of therapeutic genes introduced into the central nervous system.

Animals↗

PubMed1996

Non-virally mediated gene transfer into human central nervous system precursor cells.

Lipofectamine-based transfection was used as a method of choice to deliver the bacterial beta-galactosidase gene into human central nervous system (CNS) precursor cells. We achieved a transfection efficiency of 7.4%. beta-Galactosidase expressing cells were shown to display both neuronal and glial phenotypes. We also delivered the temperature sensitive allele of SV40 Large-T antigen and obtained a high level of expression of the immortalizing oncoprotein in the cells. Colonies of Large-T antigen immunoreactive cells were indeed visible 10 days after transfection.

Cation Exchange Resins↗

PubMed1996

Complete nucleotide sequence of the Italian human T-cell lymphotropic virus type II isolate Gu and phylogenetic identification of a possible origin of South European epidemics.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II isolate (HTLV-II-Gu) from an Italian injecting drug user was obtained, representing the first entire sequence of a European HTLV-II isolate. The HTLV-II-Gu genome was more similar to the HTLV-IIb-NRA isolate (98.4%) and HTLV-IIb-G12 (98.2%) than to HTLV-IIa-Mo (95.2%). The classification of HTLV-II-Gu as subtype IIb was confirmed by restriction analysis. Just as for HTLV-IIa strain Mo, HTLV-IIb-Gu cultured lymphocytes produce two additional mRNAs generated through alternative splicing in the pX region. A phylogenetic analysis was performed by using the methods of neighbour-joining and parsimony with bootstrapping, and maximum likelihood. The different gene regions were analysed separately, comparing Gu with all other HTLV-II strains presently available. In the LTR, as well as in other genome regions, a clear separation between IIa and IIb was evident, and within the IIb subtype three clusters were present of which two were well supported; one contained exclusively Amerindian strains and the other included all Italian and Spanish strains together with two strains obtained from New York drug users. All data clearly showed that HTLV-IIa and IIb subtypes are closely related and are equidistant from HTLV-I, suggesting that both groups evolved simultaneously. The results suggest that HTLV-II-Gu and other IIb South European isolates were probably derived from North American IIb isolates. The data also indicate that sequence analysis is necessary to further classify IIa and IIb subtypes.

Base Sequence↗

PubMed1996

Fibroblasts of patients affected by Down's syndrome oversecrete amyloid precursor protein and are hyporesponsive to protein kinase C stimulation.

The present study investigates the ability of the pharmacologic activation of protein kinase C (PKC) to modulate amyloid precursor protein (APP) secretion in human skin fibroblasts from patients affected by Down's syndrome (DS). We assessed DS subjects at the Hospital Institute of Sospiro, Cremona, and at the Alzheimer's Disease Unit of the Sacred Heart Hospital in Brescia, and we subdivided them into nondemented (NDS) and demented (DDS) patients. All DS patients were trisomy 21 karyotype. DS fibroblasts had an increased content of APP immunoreactive material as revealed by immunocytochemistry analysis. The basal secretion of soluble APP was higher (+94.6%) in Down's cells with respect to controls. The observation on the fibroblasts prepared from DS is consistent with these patients' possessing an extra copy of the APP gene (mapped on chromosome 21) leading to increased APP expression. Phorbol-12,13-dibutyrate (PdBu, 9 to 150 nM) treatment promoted a dose-dependent increase of secreted APP in the conditioned medium of control fibroblasts. The peak response (+102.2%) was attained using 150 nM PdBu. In Down's fibroblasts, PdBu stimulated APP secretion already maximally at low concentrations (9 nM), but the peak response, due to the higher basal release, was lower on a percentage basis (+16.4%) than in control fibroblasts. The results indicate that in Down's fibroblasts the mechanisms controlling APP release are at least quantitatively altered. In addition, these results suggest caution when using information obtained from Down's patients to model Alzheimer's disease biochemical defects.

Adult↗

PubMed1995

Quantification of HTLV-II proviral copies by competitive polymerase chain reaction in peripheral blood mononuclear cells of Italian injecting drug users, central Africans, and Amerindians.

To better correlate the burden of human T cell leukemia virus type I (HTLV-I) and type II (HTLV-II) infection with diagnostic and prognostic markers, we developed a new competitive polymerase chain reaction (PCR) assay for the quantitative determination of proviral copy numbers in infected cells. A competitive plasmid was constructed that carried a 112-bp fragment from a highly conserved region of the HTLV tax gene and that was further modified by inserting a sequence of 24 bp. This competitive PCR assay system can be used for the quantification of HTLV-I and HTLV-II proviral DNA as demonstrated by using HTLV-I- and HTLV-II-infected cell lines and/or patient material. We determined the HTLV-II proviral load in peripheral blood mononuclear cells (PBMCs) of 11 Italian injecting drug users (IDUs) infected by this virus and in PBMCs of 10 seropositive Amerindian and Central African individuals from endemically infected ethnic groups. A great variation was observed in the number of HTLV-II proviral sequences in the PBMCs of Italian drug abusers, ranging from 5-10 to 16,239 copies/10(5) cells. There was no clear-cut correlation between proviral load, CD8 count, stage of HIV-1 infection, and therapy. A considerable variation in HTLV-II proviral load was also observed in PBMCs of Amerindians and Central Africans with no correlation between the amount of HTLV-II provirus and the geographic origin of the infected individuals.

Adolescent↗

PubMed1995

Selective in vitro blockade of neuroepithelial cells proliferation by methylazoxymethanol, a molecule capable of inducing long lasting functional impairments.

In order to characterize the antiproliferative effect of methylazoxymethanol neuroepithelial cells derived from the rat striata primordia at embryonic day 14 have been exposed to graded doses of this compound. It was found that methylazoxymethanol application to striatal neuroblasts elicits a blockade of cell proliferation at a dose which does not interfere with cell survival. By using synchronized cells and short term exposures to this compound, we found that the antiproliferative effect of methylazoxymethanol is strikingly correlated to the number of cells actively dividing in culture, thus indicating that the cells targeted by methylazoxymethanol must be in an active mitotic phase. To test for the selectivity of action of Methylazoxymethanol for dividing neuroblasts either cultures composed of mature proliferating astrocytes or muscle cells have been subjected to the same treatment. It has been observed that astrocytes proliferation was not affected by the dose of methylazoxymethanol shown to be effective on neuroepithelial cells. Finally we demonstrated that methylazoxymethanol is able only transiently to interfere with smooth muscle cell division, further supporting its selectivity of action within the developing CNS.

Animals↗

PubMed1995

Identification of IIa and IIb molecular subtypes of human T-cell lymphotropic virus type II among Italian injecting drug users.

The molecular characterization of two human T-cell lymphotropic virus type II (HTLV-II) isolates, Gu and Va, obtained from Italian injecting drug users (IDUs) has indicated that these isolates belong to the HTLV-IIb subtype. To establish whether Italian IDUs are also infected by the HTLV-IIa variant, sequencing of the gp21 env gene of proviral DNA from further patients was carried out. Two new isolates, Bo and Md, were found, which presented a divergence of 0.4-0.7% from the IIa prototype HTLV-II-Mo, thus indicating that they belong to the HTLV-IIa subtype. The results strongly support the existence of two distinct molecular subtypes of HTLV-II infecting Italian IDUs and demonstrate that, although the IIb subtype appears to be prevalent, there is the same variability for this virus in Europe as found in the United States. The Italian IIb isolates were also seen to encode an additional 25 amino acids at the C-terminal end of tax protein, as already shown for other IIb isolates. The identification of two HTLV-II molecular subtypes among Italian drug addicts will be useful in tracing the worldwide distribution of this virus and in further understanding its molecular structure and biology.

Amino Acid Sequence↗

PubMed1995

HTLV infection among Italian intravenous drug users and North African subjects detected by the polymerase chain reaction and serological methods.

Six hundred intravenous drug users (IVDUs) and two hundred North Africans were screened for human T-cell leukemia virus (HTLV) antibodies using several serological methods. Eighteen of the eighty-two HTLV-seropositive individuals were also tested by the polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), a non-isotopic method of immunoenzymatic detection of the amplified DNA. Of these eighteen subjects, eight IVDUs were found to be HTLV-II-positive by the PCR-DEIA, whereas all of the eighteen subjects were negative for HTLV-I. Western blot (WB) confirmed six of the eight HTLV-positive subjects, while the results of the remaining two were indeterminate. The results confirmed the PCR-DEIA as a rapid and an efficient method of discriminating between HTLV-I and HTLV-II infection, whereas serological tests, including the WB, have limitations in terms of specificity and sensitivity. Moreover, this study showed a higher frequency of HTLV seroreactivity in the Italian IVDU population than in previous studies and confirmed that HTLV-II is more frequent than HTLV-I in this population.

Africa, Northern↗

PubMed1995

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

BACKGROUND: The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. OBJECTIVES: In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). STUDY DESIGN: The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. RESULTS: In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. CONCLUSION: This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

Journal Article↗

PubMed1994

A short term analysis of the behaviour of conditionally immortalized neuronal progenitors and primary neuroepithelial cells implanted into the fetal rat brain.

Conditionally immortalized (temperature-sensitive) striatal-derived neuronal progenitor cell lines and primary neuroepithelial cells were transplanted into the CNS of gestational day 15-16 rat fetuses using an 'in utero' surgical procedure. Each fetus received 2.5-3 x 10(4) donor cells previously labelled in vitro by incubation with 5-bromo-2'-deoxyuridine (BrdU). At 5 days following transplantation, 69% of the fetuses were still alive. Engrafted cells were detected by BrdU immunohistochemistry, and the appearance of the engrafted cells and the time course of Nestin and PCNA expression were measured at 6, 24, 64 h and 5 days after transplantation. The evolution of Large T-Antigen immunoreactivity in engrafted temperature-sensitive (ts) cells was also evaluated at the above time intervals. The results indicate that the majority of the implanted cells were aggregated into clusters 24 h after transplantation. These clusters were not visible at 6 h, when most of the cells were isolated. The clusters were located in both the ventricles and parenchyma. These findings were common to both ts cells and striatal primary neuroepithelial cells. At 64 h and 5 days, isolated cells associated with the germinal layer and scattered throughout the parenchyma were also found. In the clusters, Nestin expression decreased proportionally with time following transplantation. Furthermore, Large T-Antigen immunoreactivity disappeared from ts cells between 6 and 24 h after transplantation. Finally, measurements of the temporal evolution of PCNA expression within the clusters indicate a progressive reduction in the mitotic activity of the transplanted cells. The results demonstrate that striatal primary neuroepithelial cells and conditionally immortalized neuronal progenitors can survive, migrate and/or compartimentalize into clusters whilst changing their antigenic properties and ability to proliferate.

Animals↗

PubMed1993

Activated estrogen receptor mediates growth arrest and differentiation of a neuroblastoma cell line.

Several reports demonstrate estrogen receptor involvement in specific brain functions. In addition, estrogen receptors are expressed at early stages of brain development, suggesting that estrogens or related molecules may play an instructive role in the differentiation of specific brain areas. The lack of model systems in which these phenomena could be studied prompted us to develop a neuroblastoma cell line expressing the estrogen receptor. The cell line expresses the hormone receptor at levels compatible with a physiological activity. The activated estrogen receptor is capable of blocking proliferation of the cells without exerting toxic effects. Following growth arrest, the cells display a neuron-like morphology and express tau and synaptophysin, two proteins synthesized in differentiating neurons. The cell line generated will provide a valuable model system for molecular and biochemical studies of the activity of estrogens in neural-derived cells.

Cell Differentiation↗

PubMed1993

Molecular characterization of two isolates of human T cell leukaemia virus type II from Italian drug abusers and comparison of genome structure with other isolates.

The human T cell leukaemia virus type II (HTLV-II), whose pathogenicity is as yet unclear, was recently found to be associated with intravenous drug abuse in North America and Europe. HTLV-II was isolated from two Italian drug abusers belonging to the same cohort and coinfected with human immunodeficiency virus type 1. Two new isolates, HTLV-II Gu and Va, were established in a culture of BJAB cells, a continuous B cell line (Epstein-Barr virus-negative), and characterized by nucleotide sequence analysis of the long terminal repeat (LTR) and portions of the gag, env and X regions. These sequences were compared to those of the HTLV-II Mo isolate reported in the literature. No major variations were observed in important regulatory elements of LTR nor in the stem-bulge-loop configuration known to be essential for binding of rex protein. The results obtained from the sequence of the 1988 nucleotides examined indicated a 1.6% variability between the Gu and Va isolates and about 6% with respect to Mo. Notable differences were found in the structure of putative open reading frames of the X region when compared to those reported for the Mo isolate. Restriction analysis of proviral DNA of two isolates and comparison with the physical map of the Mo isolate confirmed the existence of genetic heterogeneity in the HTLV-II group and demonstrated that the new isolates Gu and Va belong to the HTLV-IIb subtype. The results of this study show that the new isolates have distinct features with respect to the Mo isolate though all important regulatory elements of the LTR appear to be well conserved.

Genes, Viral↗

PubMed1991

Identifying and manipulating neuronal stem cells.

Fetal brain tissue has been shown to have clear behavioral effects when transplanted into adult lesioned brains. These results have focused attention on the cell types of the embryonic brain. Transplantation experiments using primary cells are beginning to define the plasticity of these cells and the times when they become committed to specific neuronal fates. Growth factors have been defined that regulate the proliferation of these cells in culture. Cell lines have been established that express stem cell properties and that are capable of differentiation when implanted into the developing brain. In this article we review this work on mammalian neuroepithelial stem cells and discuss how these studies might contribute to the therapeutic use of brain transplants.

Animals↗

PubMed1991

Sensitivity and specificity of anti-HIV ELISA employing recombinant (p24, p66, gp120) and synthetic (gp41) viral antigenic peptides.

We have developed two immunoassay systems, one designated HIV (p24, p66, gp41) ELISA that uses as antigens the immunodominant epitopes mixed from each of three major groups of HIV-1 proteins: the core (p24), the pol (p66) and the env (gp41) gene. The other immunoassay system consists of four separate ELISAs for detection of single antibodies to HIV gag gene (p24), HIV pol gene (p66) and HIV env gene (gp41 and gp120). In the present study 200 specimens from patients with AIDS and 200 specimens from patients with ARC were repeatedly positive by HIV (p24, p66, gp41) ELISA. 1425 specimens from HIV drug addicts positive at W.B. were positive at HIV (p24, gp41, p66) ELISA. In addition, 60 samples that were indeterminate by W.B., were repeatedly positive at HIV (p24, p66, gp41) ELISA. The sensitivity and specificity of HIV (p24, p66, gp41) is estimated to be 100%. In this study 1507 specimens from HIV drug addicts, positive at W.B., were all positive (more than one test positive) at HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA used in combination. 135 samples from HIV positive drug addicts, positive at standard ELISA but indeterminate at W.B., were positive by HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA using the same criteria as in W.B. interpretation. The specificity (defined in terms of percentage of non-reacting persons in a low risk population) of HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA, HIV gp120 ELISA is 100%. In this work we demonstrated that: a) HIV (p24, p66, gp41) ELISA could be used as an adjunct or reliable alternative to standard ELISA for detection or confirmation of HIV antibodies in human sera; b) the specificity and sensitivity of antibodies to p24, p66, gp41, gp120 by ELISA used alone and/or in combination, is equal to or greater than W.B.

AIDS-Related Complex↗

PubMed1991

Seroepidemiology of HIV infection: an early survey in a peripheral area of Uganda.

At the end of 1985, when the AIDS epidemic was in its early stages in Uganda, a survey was carried out in a peripheral area of the country. Sera were collected from groups of people, and examined for the presence of HIV infection. The results show a very limited number of positive cases, present only among sexually active subjects. High specificity and sensitivity in the laboratory tests was shown by the Western blot technique.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Detection of antibodies to p24 and gp41 epitopes of HIV by ELISA using the recombinant core protein (p24) and envelope synthetic protein (gp41).

We have developed a system consisting of two separate ELISA, one designed to detect antibodies to HIV gag gene (p24) and the other to detect antibodies to HIV env gene (gp41). The antigen used in these ELISA was produced as recombinant DNA-derived proteins expressed in E. coli for HIV gag gene (p24) and synthetic peptide for the HIV env gene (gp41). These HIV (env-gag) ELISA, that provide independent determinations of the antibody response to the core and envelope proteins, are highly specific and sensitive. In this work we have demonstrated that determinations of antibodies such as those to p24 and gp41 by HIV (env-gag) ELISA are among the criteria for a confirmation procedure, and sensitivity one (gp41) and/or both these determination should be equal or greater than the sensitivity of W.B. In addition, the procedure should be objective and standardized and the antigen source used should be different from that adopted in the "classical" W.B. and screening test. In view of these considerations, this HIV (env-gag) ELISA could be used as a reliable alternative to W.B. for confirmation of antibody detection.

AIDS Serodiagnosis↗