Biomedical subjects

M A Caligiuri

Publications and source records attributed to M A Caligiuri.

At least 37 records · Page 2Linked to original sources

PubMed2001

High affinity interleukin-3 receptor expression on blasts from patients with acute myelogenous leukemia correlates with cytotoxicity of a diphtheria toxin/IL-3 fusion protein.

Diphtheria fusion proteins are a novel class of agents for the treatment of chemotherapy resistant AML. We prepared DT(388)IL3 composed of human interleukin-3 (IL3) fused to the catalytic and translocation domain of diphtheria toxin (DT(388)) and assessed its activity on patient AML blasts. The number and affinity of IL3 receptors in circulating blasts was measured using a radiolabeled IL3 agonist (SC-65461). Ninety-two percent of patients' blasts had both high and low affinity IL3 receptors. DT(388)IL3 cytotoxicity (>1 log cell kill) was seen in nine of 25 samples (36%). There was a significant correlation between DT(388)IL3 log cell kill and blast high affinity IL3 receptor density (P=0.0044). These results show that specific high affinity IL3 binding is one factor important in the sensitivity of patients' leukemic blasts to DT(388)IL3.

Acute Disease↗

PubMed2001

The biology of human natural killer-cell subsets.

Human natural killer (NK) cells comprise approximately 15% of all circulating lymphocytes. Owing to their early production of cytokines and chemokines, and ability to lyse target cells without prior sensitization, NK cells are crucial components of the innate immune system. Human NK cells can be divided into two subsets based on their cell-surface density of CD56--CD56(bright) and CD56(dim)--each with distinct phenotypic properties. Now, there is ample evidence to suggest that these NK-cell subsets have unique functional attributes and, therefore, distinct roles in the human immune response. The CD56(dim) NK-cell subset is more naturally cytotoxic and expresses higher levels of Ig-like NK receptors and FCgamma receptor III (CD16) than the CD56(bright) NK-cell subset. By contrast, the CD56(bright) subset has the capacity to produce abundant cytokines following activation of monocytes, but has low natural cytotoxicity and is CD16(dim) or CD16(-). In addition, we will discuss other cell-surface receptors expressed differentially by human NK-cell subsets and the distinct functional properties of these subsets.

Animals↗

PubMed2001

Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia.

Amplification of the CBFbeta/MYH11 fusion transcript by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) has been used to detect minimal residual disease (MRD) and assess the risk for disease relapse in inv(16)(p13q22) acute myeloid leukemia (AML). This strategy has, however, produced conflicting results and because of an uncertain predictive value, its use in the clinical setting cannot be recommended. The objective of the current study was to evaluate if quantification by Real Time RT-PCR could be useful to determine levels of CBFbeta/MYH11 fusion transcripts predictive of clinical outcome in inv(16)(p13q22) AML at diagnosis or during remission. Bone marrow (BM) samples from 16 patients with inv(16) AML enrolled on a German multicenter trial (AML HD93) were analyzed for levels of CBFbeta/MYH11 fusion transcripts by Real Time RT-PCR at diagnosis (n= 14), during remission (n= 10) and at relapse (n=6). The CBFbeta/MYH11 transcript copy number in each sample was normalized to copies of an internal control housekeeping transcript (ie 18S). The copy number measured at diagnosis or relapse were 3 to 4 log higher that those measured during remission, following completion of induction treatment. A high CBFbeta/MYH11 transcript copy number at diagnosis had a significant correlation with a high percentage of BM blasts (Spearman's coefficient = -0.66; P= 0.03), and a borderline correlation with a short complete remission (CR) duration (Spearman's coefficient = -0.51; P= 0.07). No difference in levels of CBFbeta/MYH11 fusion transcripts measured during intensification therapy was found between patients destined to relapse and those who continued in CCR (P= 0.75). Following completion of the entire chemotherapy program, patients that during CR showed a CBFbeta/MYH11 fusion transcript copy number >10 had a significantly shorter CR duration (P= 0.002) and higher risk for disease relapse (P= 0.05) than patients with a CBFbeta/MYH11 fusion transcript copy number <10. The results of the current study, therefore, suggest that it is possible to determine in remission samples a threshold of CBFbeta/MYH11 transcript copy number above which relapse occurs and below which continuous CR is likely.

Adolescent↗

PubMed2001

GM-CSF and IL-2 induce specific cellular immunity and provide protection against Epstein-Barr virus lymphoproliferative disorder.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a potentially life-threatening complication in immune-deficient patients. We have used the severe combined immune deficient (SCID) mouse engrafted with human leukocytes (hu-PBL-SCID) to evaluate the use of human cytokines in the prevention of EBV-LPD in vivo. Daily low-dose IL-2 therapy can prevent EBV-LPD in the hu-PBL-SCID mouse, but protection is lost if murine natural killer (NK) cells are depleted. Here we demonstrate that combined therapy with human GM-CSF and low-dose IL-2 is capable of preventing EBV-LPD in the hu-PBL-SCID mouse in the absence of murine NK cells. Lymphocyte depletion experiments showed that human NK cells, CD8(+) T cells, and monocytes were each required for the protective effects of GM-CSF and IL-2 combination therapy. This treatment resulted in a marked expansion of human CD3(+)CD8(+) lymphocytes in vivo. Using HLA tetramers complexed with EBV immunodominant peptides, a subset of these lymphocytes was found to be EBV-specific. These data establish that combined GM-CSF and low-dose IL-2 therapy can prevent the immune deficiencies that lead to fatal EBV-LPD in the hu-PBL-SCID mouse depleted of murine NK cells, and they point to a critical role for several human cellular subsets in mediating this protective effect.

Animals↗

PubMed2001

In situ determination of T-cell receptor beta expression patterns.

A definitive diagnosis of T-cell lymphoma may be contingent on the rearrangement profile of the T-cell receptor. This is most accurately done by molecular analysis of the beta-chain of the T-cell receptor (TCR beta) by Southern blotting hybridization that requires unfixed tissue. We describe a reverse transcriptase in situ PCR (RT in situ PCR) method that permits the target-specific direct incorporation of the reporter nucleotide into the different transcripts that comprise the TCR beta, using paraffin-embedded, formalin-fixed tissue. Each of the 25 possible V beta segment rearrangements was documented in three lymph nodes with nonspecific lymphadenitis, with clonal expansion evident in a case of metastatic melanoma. Monoclonal expression was documented in seven tissues diagnostic of a T-cell lymphoma. We analyzed five additional tissues for which a definitive diagnosis of T-cell vs B-cell lymphoma could not be rendered on the basis of histological, immunohistological, and flow cytometric analysis. RT in situ PCR for TCR beta expression with CD3 co-labeling demonstrated which of these lesions was a B-cell-rich T-cell lymphoma. We conclude that the RT in situ PCR methodology will allow the routine determination of monoclonal vs multiclonal expression patterns of the TCR beta using archival paraffin-embedded tissues.(J Histochem Cytochem 49:139-145, 2001)

Diagnosis, Differential↗

PubMed2001

Ontogeny and expansion of human natural killer cells: clinical implications.

Our knowledge of NK cells and their critical role in the innate immune system has increased enormously since their discovery several decades ago. However, it is only within the last 10 years that rational cytokine therapies, such as those utilizing low doses of IL-2, have been successful in expanding NK cells in patients with cancer and/or immunodeficiency. Such experiences in vivo have highlighted the importance of basing immunotherapeutic strategies on the known cellular and molecular properties of the targeted cell population. Recent advances in our understanding of the physiologic factors and events that orchestrate NK cell ontogeny, including IL-15 and receptor tyrosine kinase ligands to c-kit and flt3, provide novel therapeutic possibilities for cytokine therapy. This review summarizes our current understanding of human NK cell ontogeny, and links this knowledge to ongoing and future clinical strategies for the endogenous expansion of NK cells in patients with cancer and/or immunodeficiency.

Animals↗

PubMed2000

Characterization of Epstein-Barr virus-infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response.

Post-transplantation lymphoproliferative disease (PTLD) is associated with Epstein-Barr virus (EBV). Quantitative and qualitative differences in EBV in peripheral blood mononuclear cells (PBMCs) of PTLD patients and healthy controls were characterized. A quantitative competitive polymerase chain reaction (QC-PCR) technique confirmed previous reports that EBV load in PBMCs is increased in patients with PTLD in comparison with healthy seropositive controls (18 539 vs 335 per 10(6) PBMCs, P =.0002). The average frequency of EBV-infected cells was also increased (271 vs 9 per 10(6) PBMCs, P =.008). The distribution in numbers of viral genome copies per cell was assessed by means of QC-PCR at dilutions of PBMCs. There was no difference between PTLD patients and healthy controls. Similarly, no differences in the patterns of viral gene expression were detected between patients and controls. Finally, the impact of therapy on viral load was analyzed. Patients with a past history of PTLD who were disease-free (after chemotherapy or withdrawal of immunosuppression) at the time of testing showed viral loads that overlapped with those of healthy seropositive controls. Patients treated with rituximab showed an almost immediate and dramatic decline in viral loads. This decline occurred even in patients whose PTLD progressed during therapy. These results suggest that the increased EBV load in PBMCs of PTLD patients can be accounted for by an increase in the number of infected B cells in the blood. However, in terms of viral copy number per cell and pattern of viral gene expression, these B cells are similar to those found in healthy controls. Disappearance of viral load with rituximab therapy confirms the localization of viral genomes in PBMCs to B cells. However, the lack of relationship between the change in viral load and clinical response highlights the difference between EBV-infected PBMCs and neoplastic cells in PTLD.

Adult↗

PubMed2000

Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferon-gamma production and STAT-mediated signal transduction.

The administration of therapeutic doses of recombinant cytokines to patients with malignant disease can be complicated by systemic toxicities, which in their most severe form may present as a systemic inflammatory response. The combination of interleukin (IL)-18 and IL-12 has synergistic antitumor activity in vivo yet has been associated with significant toxicity. The effects of IL-18 plus IL-12 were examined in a murine model, and it was found that the daily, simultaneous administration of IL-18 and IL-12 resulted in systemic inflammation and 100% mortality within 4 to 8 days depending on the strain employed. Mice treated with IL-18 plus IL-12 exhibited unique pathologic findings as well as elevated serum levels of proinflammatory cytokines and acute-phase reactants. The actions of tumor necrosis factor-alpha did not contribute to the observed toxicity, nor did T or B cells. However, toxicity and death from treatment with IL-18 plus IL-12 could be completely abrogated by elimination of natural killer (NK) cells or macrophages. Subsequent studies in genetically altered mice revealed that NK-cell interferon-gamma mediated the fatal toxicity via the signal transducer and activator of transcription pathway of signal transduction. These data may provide insights into methods of ameliorating cytokine-induced shock in humans. (Blood. 2000;96:1465-1473)

Animals↗

PubMed2000

Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: evidence for its fusion with MLL in acute myeloid leukemia.

We have identified a gene at 11q23, telomeric to MLL, that encodes a guanine nucleotide exchange factor (GEF). This gene is transcribed into a 9.5-kb mRNA containing a 4.6-kb ORF. By Northern analysis, it was found to be expressed in all human tissues examined including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon, and minimally in thymus. Analysis of the predicted protein sequence indicates that it has strong homology to several members of the family of Rho GEFs that includes such oncogenes as Dbl, Vav, Tiam, and Bcr. A patient with primary acute myeloid leukemia (AML) and a karyotype of 51,XY,+8,+19,+3mar was found to have the 5' end of MLL at exon 6 fused in-frame with the 3' end of almost the entire ORF of this gene, which we named LARG for leukemia-associated Rho GEF. Transcriptional orientation of both genes at 11q23 is from centromere to telomere, consistent with other data that suggest the MLL-LARG fusion resulted from an interstitial deletion rather than a balanced translocation. LARG does not appear to have any homology with other MLL partner genes reported thus far. Thus, LARG represents an additional member of the GEF family and a novel MLL fusion partner in acute myeloid leukemia.

Adult↗

PubMed2000

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo.

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.

Animals↗

PubMed2000

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Adenocarcinoma↗

PubMed2000

Identification and characterization of an activating TrkA deletion mutation in acute myeloid leukemia.

In this study, we utilized retroviral transfer of cDNA libraries in order to identify oncogenes that are expressed in acute myeloid leukemia (AML). From screens using two different cell types as targets for cellular transformation, a single cDNA encoding a variant of the TrkA protooncogene was isolated. The protein product of this protooncogene, TrkA, is a receptor tyrosine kinase for nerve growth factor. The isolated transforming cDNA encoded a TrkA protein that contains a 75-amino-acid deletion in the extracellular domain of the receptor and was named DeltaTrkA. DeltaTrkA readily transformed fibroblast and epithelial cell lines. The deletion resulted in activation of the tyrosine kinase domain leading to constitutive tyrosine phosphorylation of the protein. Expression of DeltaTrkA in cells led to the constitutive activation of intracellular signaling pathways that include Ras, extracellular signal-regulated kinase/mitogen-activated protein kinase, and Akt. Importantly, DeltaTrkA altered the apoptotic and growth properties of 32D myeloid progenitor cells, suggesting DeltaTrkA may have contributed to the development and/or maintenance of the myeloid leukemia from which it was isolated. Unlike Bcr-Abl, expression of DeltaTrkA did not activate Stat5 in these cells. We have detected expression of DeltaTrkA in the original AML sample by reverse transcriptase PCR and by Western blot analysis. While previous TrkA mutations identified from human tumors involved fusion to other proteins, this report is the initial demonstration that deletions within TrkA may play a role in human cancers. Finally, this report is the first to indicate mutations in TrkA may contribute to leukemogenesis.

3T3 Cells↗

PubMed2000

Potential mechanisms of human natural killer cell expansion in vivo during low-dose IL-2 therapy.

The continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4-6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G(2)/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34(+) bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56(+)CD3(-) NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2-dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.

Animals↗

PubMed2000

Molecular and clinical advances in core binding factor primary acute myeloid leukemia: a paradigm for translational research in malignant hematology.

Clonal chromosomal abnormalities are the most important prognostic indicators in acute myeloid leukemia (AML). Recent advances in molecular biology have allowed structural and functional characterization of many of these genomic rearrangements and have provided evidence for their primary role in leukemogenesis. Two of the most prevalent cytogenetic subtypes of adult primary or de novo AML, t(8;21)(q22;q22) and inv(16)(p13q22), are characterized by disruption of the AML1(CBF alpha 2) gene at 21q22 and the CBF beta gene at 16q22, respectively. Both genes encode a subunit of core binding factor (CBF), a regulator of normal hematopoiesis. At the molecular level, t(8;21)(q22;q22) and inv(16)(p13q22) result in the creation of novel fusion genes, AML1/ETO and CBF beta/MYH11, whose structures and functions are being successfully characterized by in vitro studies and transgenic animal models. Detection of t(8;21)(q22;q22) or inv(16)(p13q22) in adult patients with primary AML is a favorable independent prognostic indicator for achievement of cure after intensive chemotherapy or bone marrow transplantation and may serve as a paradigm for risk-adapted treatment in AML. The purpose of this review is to summarize the recent advances in the molecular biology and clinical management of t(8;21)(q22;q22) and inv(16)(p13q22) primary AML, collectively referred to here as CBF AML.

Animals↗

PubMed2000

Cytokine replacement in patients with HIV-1 non-Hodgkin's lymphoma: the rationale for low-dose interleukin-2 therapy.

PURPOSE: The drastic increase in the incidence of non-Hodgkin's lymphoma in patients infected with HIV-1 is testimony to the fact that our immune system is critical for the prevention of certain malignancies. Preclinical and clinical studies were conducted to (1) gain further insight into defects in immunity that can lead to malignant transformation and (2) determine if certain immune deficiencies could be corrected by cytokines delivered at doses that result in near-physiologic concentrations in vivo. METHODS: We have used the severe combined immune deficient mouse engrafted with human peripheral blood leukocytes from healthy individuals who are seropositive for the Epstein-Barr virus to study the spontaneous development of malignant Epstein-Barr virus-positive human B-cell lymphoproliferative disorder. RESULTS: We have demonstrated in this model that, in the absence of CD4+ T cells, cytokine replacement with low-dose interleukin (IL)-2 therapy can prevent Epstein-Barr virus-positive human B-cell lymphoproliferative disorder by interacting with mouse natural killer and human CD8+ T cells. We review our clinical experience with administration of low-dose IL-2 therapy in patients with HIV-1-related cancer, noting minimal toxicity and significant immune modulation. We provide evidence that this therapy can favorably alter the type 1 cytokine profile in vivo in these patients, and improve the cellular response to infectious insults in vitro. CONCLUSION: Early clinical studies with low-dose IL-2 therapy in patients with HIV-1-related lymphoma suggest that this therapy may have a role in the prevention and treatment of this disease.

Animals↗

PubMed2000

Acquired immunodeficiency syndrome-related lymphomas: future directions.

Despite some exciting new leads, human immunodeficiency virus-I (HIV-I)-related non-Hodgkin's lymphoma (HIV-NHL) remains a fatal malignancy for the vast majority of patients. The use of highly active antiretroviral therapy (HAART) has not produced a fall in the incidence of HIV-NHL and conventional cytotoxic chemotherapy is associated with a negligible cure rate. New treatment options are needed. Future therapeutic directions in HIV-NHL should be based on a better understanding of three fundamental aspects of lymphomagenesis in the setting of acquired immunodeficiency. (I) New information on the molecular and pathological heterogeneity of HIV-NHL should be applied to the development of risk-adapted therapy. The identification of patient subsets with different susceptibility to cytotoxic chemotherapy, immunomodulation, or antiviral strategies is essential for the design of clinical trials of investigational new agents in HIV-NHL. (2) Known viral pathogens need to be better understood. The presence and role of new viruses should be investigated. Key biological interactions between virus and host, mediated by oncogenic, immunomodulatory, and antiapoptotic viral proteins, should become the main target for new drug development. (3) Immune reconstitution with HAART and immunostimulatory cytokines such as interleukin-2 (IL-2) and IL-12, combined with drugs that downregulate the replication or gene expression of tumor-associated viruses such as Epstein-Barr virus (EBV) and human herpes virus-8 (HHV-8), possibly in combination, should remain a primary goal in the treatment of HIV-NHL. Preventive immunization, using autologous tumor cell vaccines or antigen-specific cellular adoptive immunotherapy should be explored.

Anti-HIV Agents↗

PubMed1999

Molecular classification of cancer: class discovery and class prediction by gene expression monitoring.

Although cancer classification has improved over the past 30 years, there has been no general approach for identifying new cancer classes (class discovery) or for assigning tumors to known classes (class prediction). Here, a generic approach to cancer classification based on gene expression monitoring by DNA microarrays is described and applied to human acute leukemias as a test case. A class discovery procedure automatically discovered the distinction between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) without previous knowledge of these classes. An automatically derived class predictor was able to determine the class of new leukemia cases. The results demonstrate the feasibility of cancer classification based solely on gene expression monitoring and suggest a general strategy for discovering and predicting cancer classes for other types of cancer, independent of previous biological knowledge.

Acute Disease↗

PubMed1999

Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder.

There are experimental data which suggest that the primary immune effector cell responsible for maintaining immune surveillance against the outgrowth of EBV-transformed B cells in humans is the CTL, but in vivo proof of this is lacking. In this study we perform a series of cellular and molecular assays to characterize an autologous, endogenous immune response against a transplantation-associated, monoclonal, EBV+ posttransplant lymphoproliferative disorder (PTLD). Following allogeneic bone marrow transplantation, a patient developed a monoclonal PTLD of donor B cell origin. With a decrease in immune suppression, we document the emergence of endogenous, donor-derived CD3+CD8+ CTLs, followed by regression of the PTLD. The TCR Vbeta repertoire went from a polyclonal pattern prior to the development of PTLD to a restricted TCR Vbeta pattern during the outgrowth and regression of PTLD. Donor-derived CD3+CD8+ T lymphocytes displayed MHC class I-restricted cytolytic activity against the autologous EBV+ B cells ex vivo without additional in vitro sensitization. The striking temporal relationship between the endogenous expansion of a TCR Vbeta-restricted, CD3+CD8+ population of MHC class I-restricted CTL, and the regression of an autologous monoclonal PTLD, provides direct evidence in humans that endogenous CD3+CD8+ CTLs can be responsible for effective immune surveillance against malignant transformation of EBV+ B cells.

Adult↗