Biomedical subjects

G Migliaccio

Publications and source records attributed to G Migliaccio.

At least 19 recordsLinked to original sources

PubMed2026

The role of circulating tumor DNA (ctDNA) to detect minimal residual disease in locally advanced gastroesophageal carcinoma: the BUTTERFLY study.

BACKGROUND: Despite advances in perioperative and neoadjuvant strategies, patients with locally advanced gastroesophageal cancers remain at high risk of recurrence after curative intent treatment. No validated biomarkers are available to detect minimal residual disease (MRD) or to guide post-operative risk-adapted management. Circulating tumor DNA (ctDNA) has emerged as a noninvasive tool for disease monitoring; single-parameter or tumor-informed assays, however, may lack sensitivity in low-tumor burden settings. Multimodal, tumor-agnostic approaches may overcome these limitations. METHODS: The BUTTERFLY study is a prospective, multicenter observational study enrolling patients with stage II-III gastric, gastroesophageal junction, or esophageal cancer treated with perioperative chemotherapy or neoadjuvant chemoradiotherapy followed by surgery. It evaluates the diagnostic performance and prognostic value of an academic, tumor-agnostic, multimodal ctDNA assay for MRD detection and prognostic stratification. Serial plasma samples are collected from baseline through post-operative follow-up and at relapse. Cell-free DNA is analyzed using the Agnostic Liquid Biopsy Multimodal Advancement (ALMA) platform, integrating tumor fraction estimation, somatic copy number alterations, fragmentomic features, single-nucleotide variants, and whole-genome methylation profiling. Multimodal features are combined with clinical variables using machine learning-based models to enhance MRD detection and relapse risk stratification. The primary endpoint includes sensitivity and specificity of ALMA-defined ctDNA/MRD status at the 4-8 weeks after surgery landmark, whereas secondary endpoints assess diagnostic performance at other time points and associations between ctDNA status and dynamics with disease-free survival, overall survival, treatment response, and lead time to recurrence. FUTURE PERSPECTIVES: If validated, this tumor-agnostic, multimodal ctDNA approach may enable earlier molecular relapse detection and support personalized post-operative management strategies.

circulating tumor DNA (ctDNA)↗

PubMed2006

Development of humanized mice for the study of hepatitis C virus infection.

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

Animals↗

PubMed2003

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2.

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4+/-1.17), amplifying preferentially CD4(+) over CD8(+) T-cell subsets (FI=4.72+/-0.79 vs 2.73+/-1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4+/-2.27), but allowed preferential amplification of CD8(+) over CD4(+) T cells (FI=6.04+/-0.14 vs 1.67+/-0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor V(beta)-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67+/-1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

CD8-Positive T-Lymphocytes↗

PubMed2002

Circulating hematopoietic progenitor cells in a fetus with alpha thalassemia: comparison with the cells circulating in normal and non-thalassemic anemia fetuses and implications for in utero transplantations.

Our aim was to evaluate the number of progenitor cells circulating in an alpha-thalassemic fetus during its infusion in utero with paternal CD34(+) and adult red cells and to compare those values with those circulating in normal and non-thalassemic anemic fetuses of matched gestational age. The treatment of the alpha-thalassemic fetus has been described elsewhere. Fetal blood was obtained from normal and anemic fetuses by fetal blood sampling for diagnostic or therapeutic purposes according to a protocol approved by the human subject committee. The number of progenitor cells in fetal blood was estimated on the basis of the number of colonies they gave rise to in semisolid cultures. The alpha-thalassemic fetus, as did the other fetuses analyzed, contained high numbers (10(6)-10(7) depending on the age) of progenitor cells, values which were higher than the number (10(4)-10(5)) of paternal progenitor cells being transplanted. Progenitor cells with adult characteristics (adult kinetics of differentiation) were detected rapidly (10 min) after the CD34(+) cell infusion, but were not detectable 2-3 weeks after the transplant. These results indicate that adult progenitor cells do not have a numerical advantage when transplanted into alpha-thalassemic fetuses.

Antigens, CD34↗

PubMed2001

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Anemia↗

PubMed2001

Establishing an employee baseline purified protein derivative status in the division of immigration health Service Processing Centers.

While the incidence of reportable tuberculosis in United States born persons declined, the number of cases among foreign-born persons increased by 6 percent in 1998. The Immigration and Naturalization Service (INS) processes about 95,000 undocumented aliens annually from countries with a high prevalence of tuberculosis. An effort was made to establish a baseline Purified Protein Derivative (PPD) status of employees of the Division of Immigration Health (DIH) and INS, Services Processing Centers (SPC). This was achieved through a special operation (project) of a one time, two-step, mass PPD testing of all SPC employees on all eleven sites in the United States and Puerto Rico. A reading of > 10mm was considered positive. The operation was optional and open to all SPC employees. Exclusion criteria for the study included a history of PPD skin testing within six months of the operation, past history of positive PPD test and past history of tuberculosis. Preliminary results from El Paso SPC, Texas, which is the largest SPC, showed that of the 148 employees which were tested (67 percent of all employees), 17 (11 percent) were PPD positive. Security officers constituted 100 percent of all positive cases. Eighty-one percent of the employees at El Paso are security officers, eighty-seven percent of whom participated in the study. Only 20 (23 percent) of administrative staff participated in the study. Results from El Paso are suggestive of differences in the pattern PPD positivity among SPC employees. The complete results of the study should provide sound evidence for formulating appropriate policies for establishing an effective employee tuberculosis prevention and surveillance program in the Service Processing Centers.

Emigration and Immigration↗

PubMed2001

Standardization of progenitor cell assay for cord blood banking.

Cord blood has proved itself, if correctly stored with rational criteria, an excellent source of stem cells for related and unrelated transplants. It has been recently proven that the factor which predicts the best the speed of engraftment in cord blood transplants in the dose of progenitor cells injected per kg of body weight of the recipient. This result has been obtained thanks to a careful standardization of the neonatal progenitor cell assay. This manuscript describes such a standardization realized as a joined effort by the Istituto Superiore di Sanità, Rome, and the pivotal cord blood bank founded as a feasibility study by the National Institutes of Health, Bethesda at the New York Blood Center.

Blood Banks↗

PubMed2000

Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells.

In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human beta-globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FACS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites.

Binding Sites↗

PubMed2000

A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1.

Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.

Amino Acid Sequence↗

PubMed2000

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.

We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568)

Animals↗

PubMed2000

Erythropoietin-dependent suppression of the expression of the beta subunits of the interleukin-3 receptor during erythroid differentiation.

To clarify how erythroid cells lose their response to interleukin-3 (IL-3), we analyzed the expression of the alpha (alpha(IL-3)) and beta (beta(IL-3)/beta(com)) subunits of its receptor in a panel of murine cell lines immortalized at different stages of hemopoietic differentiation. The panel was composed by the mast cell line 32D and by its granulo-monocytic (32D GM), granulocytic (32D G), and erythroid (32D Epo1.1 and Epo) subclones. The 32D Epo cells grow only in erythropoietin (EPO) while the Epo1.1 subclone grows in either EPO or IL-3. The phenotype of these cells is that of early (expression of globins and erythroid-specific carbonic anhydrase II) and late (also expression of the erythroid-specific band 4.1 mRNA) erythroblasts when they grow in IL-3 or EPO, respectively. All the cell lines expressed comparable levels of alpha(IL-3). In contrast, the expression of beta(IL-3)/beta(com) was restricted to cells growing in IL-3 and was barely detectable in 32D Epo and 32D Epo1.1 cells growing in EPO. When switched from EPO to IL-3, 32D Epo1.1 cells expressed 10 times more beta(IL-3)/beta(com) by rapidly activating (within 1 h) their transcription rate. When reexposed to EPO, 32D Epo1.1 cells first expressed (1-6 h) more beta(IL-3)/beta(com) (2 times) but suppressed such an expression at later time points (by 48 h). The beta(IL-3)/beta(com) mRNA half-life was also different when 32D Epo1.1 cells grew in EPO or IL-3 (2-3 h vs >5 h, respectively). These results indicate that EPO specifically induces transcriptional and posttranscriptional downmodulation of beta(IL-3)/beta(com) expression in late erythroid cells.

Animals↗

PubMed2000

Characterization of the T cell receptor repertoire of neonatal T cells by RT-PCR and single strand conformation polymorphism analysis.

We have analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) the individual non-germ line configurations of the T cell receptor (TCR) Vbeta chains expressed by T cells from eight individual cord blood specimens. cDNA from each cord blood was amplified using a common primer coupled with a primer specific for each of 22 variable elements of the Vbeta chain family and the amplified fragments were separated under high resolution conditions. With cDNA from adult blood (as a control), all of the TCR chains were amplified as a smear consistent with the extensive polyclonality of adult T cells. In contrast, a heterogeneous pattern of amplification was observed with cDNAs from cord blood: only 26.7+/-21.9% of the 22 Vbeta chains analyzed were amplified as a smear. The majority of them were amplified as a discrete number of bands (up to 10) (in 68.2 +/-18.7% of samples) and some of them as a single fragment (4.0+/-7.8%). Only one of the eight samples analyzed expressed the majority (72.7%) of its Vbeta chains as a smear, consistent with an adult-like TCR repertoire. In conclusion, cord blood expressed, on average, a less complex TCR repertoire than adult blood.

Adult↗

PubMed2000

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries.

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

Antibodies↗

PubMed2000

Effects of cell banking manipulations on ex vivo amplification of umbilical cord blood.

The small volume of placental/umbilical cord blood (PUCB) collectable restricts the use of these stem cells to pediatric transplantation. To extend the use of PUCB to adult recipients, many laboratories are investigating the feasibility of ex vivo PUCB expansion. The present study analyses the effects that PUCB banking cell manipulations (cell sedimentation, cryopreservation and thawing, mononuclear and CD34+ cell isolation) have on the number, viability and ex vivo expansion potential of PUCB cells. The results presented indicate the necessity of an open discussion on whether procedures used for handling the cells in PUCB banks can be extrapolated or not as such to the clinical use of ex vivo expanded PUCB.

Adult↗

PubMed1999

Lineage-restricted expression of protein kinase C isoforms in hematopoiesis.

The pattern of expression of several protein kinase C (PKC) isoforms (alpha, betaI, delta, epsilon, eta, and zeta) during the course of hematopoietic development was investigated using primary human CD34(+) hematopoietic cells and stable cell lines subcloned from the growth factor-dependent 32D murine hematopoietic cell line. Each 32D cell clone shows the phenotype and growth factor dependence characteristics of the corresponding hematopoietic lineage. Clear-cut differences were noticed between erythroid and nonerythroid lineages. (1) The functional inhibition of PKC-epsilon in primary human CD34(+) hematopoietic cells resulted in a twofold increase in the number of erythroid colonies. (2) Erythroid 32D Epo1 cells showed a lower level of bulk PKC catalytic activity, lacked the expression of epsilon and eta PKC isoforms, and showed a weak or absent upregulation of the remaining isoforms, except betaI, upon readdition of Epo to growth factor-starved cells. (3) 32D, 32D GM1, and 32D G1 cell lines with mast cell, granulo-macrophagic, and granulocytic phenotype, respectively, expressed all the PKC isoforms investigated, but showed distinct responses to growth factor readdition. (4) 32D Epo 1.1, a clone selected for interleukin-3 (IL-3) responsiveness from 32D Epo1, expressed the epsilon isoform only when cultured with IL-3. On the other hand, when cultured in Epo, 32D Epo1.1 cells lacked the expression of both epsilon and eta PKC isoforms, similarly to 32D Epo1. (5) All 32D cell lines expressed the mRNA for PKC-epsilon, indicating that the downmodulation of the epsilon isoform occurred at a posttranscriptional level. In conclusion, the PKC isoform expression during hematopoiesis appears to be lineage-specific and, at least partially, related to the growth factor response.

Animals↗

PubMed1999

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice.

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.

Animals↗

PubMed1999

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses.

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.

Animals↗

PubMed1999

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and leukemia inhibitory factor (LIF) protect the repopulating ability of purified murine hematopoietic stem cells in serum-deprived cultures stimulated with SCF and IL-3.

The engraftment potential of murine stem cells (HSC) is greatly reduced when these cells are expanded in vitro with stem cell factor and interleukin-3. We have evaluated if the addition of MIP-1 alpha or LIF to these cultures would protect the ability of murine wild type HSC to engraft the stem cell defective W/Wv recipient. In this transplantation model red and white blood cell reconstitution is assessed by hemoglobin electrophoresis and c-kit PCR genotyping, respectively. The results obtained indicate that both MIP-1 alpha and LIF protect, at least transiently, the HSC repopulating ability in vivo in spite of the modest expansion in the number of nucleated and progenitor cells observed.

Analysis of Variance↗