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Biomedical subjects

S Vuk-Pavlović

Publications and source records attributed to S Vuk-Pavlović.

At least 19 recordsLinked to original sources
PubMed2006

Testing the safety of clinical-grade mature autologous myeloid DC in a phase I clinical immunotherapy trial of CML.

BACKGROUND: We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype. METHODS: We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance. RESULTS: DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro. DISCUSSION: The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.

Aged↗
PubMed2004

Clinical-grade manufacturing of DC from CD14+ precursors: experience from phase I clinical trials in CML and malignant melanoma.

BACKGROUND: We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14 + precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. METHODS: We isolated CD 14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15' medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-a, IL-lIf, IL-6 and prostaglandin E2 for 3 days. Some cells were electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 andCD14. RESULTS: CD14+ cells constituted 14.4+/-6.2% (mean + SD) of nucleated cells in apheresis products and 98.3+/- 3.6% of isolated cells. Normal DC and CML DC were 77.4+/-7.3% CD83+ and 93.5+/- 7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1 + 7.2% and 94.1 + 7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1 + 7.2% for normal and CML patients and 9.8 + 3.7% for melanoma patients. DC viability was 92.7 + 5.8%; after cryopreservation and thawing it was 77+/-13.5%. DISCUSSION: Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.

Blood Component Removal↗
PubMed2001

Transgenic interleukin 2 secreted by CML dendritic cells stimulates autologous T(H)1 T cells.

BACKGROUND: We investigated if dendritic cells (DC), derived from patients suffering from chronic myeloid leukemia (CML) could be modified by recombinant replication-defective adenoviruses to express functional interleukin 2 (IL-2). Such modification might confer onto antigen-presenting cells the ability to stimulate expansion of effector cells. METHODS: To quantify the infection efficiency of CML dendritic cells (CML-DC) by recombinant adenovirus, we measured the expression of green fluorescent protein (GFP) gene contained in the virus. In CML-DC infected with an adenovirus containing the IL-2 gene, we evaluated their ability to secrete IL-2 and stimulate proliferation of autologous T cells. RESULTS: Uninfected CML-DC and normal DC secreted similar amounts of IL-12 and stimulated similarly efficient autologous mixed leukocyte reaction. Immature CML-DC infected by an adenovirus containing the gene for IL-2 secreted large amounts of IL-2 and stimulated proliferation of autologous T cells more efficiently than the corresponding CML-DC alone. High levels of interferon eta, but not of IL-4, in cell culture supernates indicated that the proliferating cells were T(H)1. Infected mature CML-DC were more effective than infected immature CML-DC, showing that T cell stimulation by mature DC and by IL-2 was additive. DISCUSSION: CML-DC can be modified genetically and functionally by recombinant replication-defective adenoviruses, providing new possibilities for clinical trials in dendritic cell-based immunotherapy.

Adenoviridae↗
PubMed2001

Can there be a meaningful participation of Croat expatriates in Croatian science?

The author discusses the possible role for Croat expatriate scientific community in Croatian science. He argues that proper leadership could engage Croatian expatriate scientific community in supporting the foundation of a multidisciplinary science institute in Split, Croatia. The institute could play a key role in regional economic development focused on health, energy, and environment. Establishment and maintenance of a program of such magnitude requires a diminished and more flexible role of the government and introduction of the private sector in a government-private partnership. Development of necessary attitudes and economic base will take time.

Croatia↗
PubMed2001

Engineering dendritic cell grafts for clinical trials in cellular immunotherapy of cancer: example of chronic myelogenous leukemia.

Dendritic cells are pivotal regulators of immune reactivity and immune tolerance. The observation that dendritic cells can recruit naive T-cells has invigorated cancer immunology and stimulated clinical trials of dendritic cells in immunotherapy. However, variables inherent in preparation and use of dendritic cell grafts remain to be tested. Here we discuss the role of ex vivo dendritic cell processing for in vivo antigen presentation in clinical trials. As an example of the complexity in a clinical trial of dendritic cell vaccines, we present our ongoing trial in immunotherapy of chronic myelogenous leukemia.

Antigen Presentation↗
PubMed2001

Maturation of human dendritic cells as sulfasalazine target.

AIM: Sulfasalazine, a nonsteroidal anti-inflammatory drug, is effective in treating some autoimmune diseases, but its mechanism of action is unclear. To determine whether dendritic cells could be a possible target of the drug, we studied the effects of sulfasalazine and its metabolites, aminosalicylate and sulfapyridine, on in vitro maturation (terminal differentiation) of human myeloid dendritic cells. METHODS: We prepared immature dendritic cells by incubating CD14-positive cells in the presence of granulocyte- macrophage colony-stimulating factor and interleukin (IL)-4. The cells were matured by addition of tumor necrosis factor (TNF)-a, IL-1 beta, and prostaglandin E2 in the presence of sulfasalazine or its metabolites -- aminosalicylate and sulfapyridine, or their combinations. We quantified the effect of drugs on the dendritic cell characteristics, such as stimulation of autologous and allogeneic pan-T cell proliferation, surface marker phenotype, IL-12 p40 subunit secretion, and activation of nuclear transcription factor (NF)-kappa B. RESULTS: Dendritic cells treated with sulfasalazine (1.25 micromol/L or 2.5 micromol/L) could not stimulate T cells (p<0.028, two-sided paired t-test). In distinction to drug-free maturing dendritic cells, 2.5 micromol/L sulfasalazine upregulated the levels of CD14 and CD68 and downregulated the levels of CD40, CD80, and CD83 (for all CD markers, p<0.03 for difference between measurements in the absence and the presence of sulfasalazine). From concentration-dependent changes in CD83 expression, we found an apparent ID50 >>1.5 micromol/L sulfasalazine. The apparent ID50 value for aminosalicylate-inhibited maturation was 4 micromol/L. Sulfapyridine had no effect. At 1.25 micromol/L, sulfasalazine largely inhibited NF-kB activation in dendritic cells. CONCLUSION: Maturing human dendritic cells are hundred-fold more sensitive to sulfasalazine than T cells and NK cells and the most sensitive human cells described so far. Thus, dendritic cell maturation is an important target of sulfasalazine. Because of the role of dendritic cells in (auto)immunity, inhibition of their maturation might provide a target for further optimization of sulfasalazine therapy.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Maturation of human monocyte-derived dendritic cells studied by microarray hybridization.

We compared the transcript profiles of human myeloid immature dendritic (IDC) cells and mature dendritic cells (MDC) by hybridization of cell-derived cDNA to DNA probes immobilized on microarrays. The microarrays contained probes for 4110 known genes. We report maturation-dependent changes in transcription of clusters of differentiation, cytokines, cytokine receptors, chemokines, chemokine receptors, neuropeptides, adhesion molecules, and other genes. We identified 1124 transcripts expressed in IDC and 1556 transcripts expressed in MDC. Maturation increased the levels of 291 transcripts twofold or more and reduced the levels of 78 transcripts to one-half or less than in IDC. We identified a concerted maturation-stage-dependent transcription of the variable chains of the members of the gamma-chain-cytokine receptor family IL-4R, IL-7R, and IL-15R. Also, we found the reversal of the ratio of transcripts for galectin-3 and galectin-9 upon maturation. We identified maturation-dependent changes in the levels of transcripts for numerous genes encoding proteins previously undetected in dendritic cells such as indoleamine 2,3-deoxygenase, Epstein-Barr virus induced protein 3 and kinesin-2. Moreover, MDC transcribed and translated insulin like growth factor-1 receptor, transforming growth factor alpha, and neuropeptide Y.

Antigens, CD↗
PubMed2000

Optimizing preparation of normal dendritic cells and bcr-abl+ mature dendritic cells derived from immunomagnetically purified CD14+ cells.

The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.

Cell Division↗
PubMed2000

Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.

Acid Phosphatase↗
PubMed1999

Dexamethasone inhibits dendritic cell maturation by redirecting differentiation of a subset of cells.

To investigate how corticosteroids affect differentiation of human dendritic cells (DC) in a defined inflammatory environment, we incubated immature DC with dexamethasone in the presence of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and prostaglandin E2. Dexamethasone inhibited differentiation into mature DC, as indicated by the reduced expression of antigen-presenting molecules, costimulatory and adhesion molecules, a marker of mature DC, and IL-12. Dexamethasone increased expression of CD14, CD36, and CD68, molecules characteristic of monocytes/macrophages and induced CD14+CD83- cells, a subset distinct both from immature DC and mature DC. The effects were concentration-dependent, with ID50 values between 2 and 30 nM dexamethasone. Unlike T and B cells, in DC dexamethasone induced no apoptosis, although it suppressed activated nuclear transcription factor NF-kappaB. Dexamethasone reduced the ability of DC to stimulate proliferation of allogeneic T cells in proportion to the level of CD14+CD83- cells in the population. CD83+ cells, isolated from dexamethasone-treated populations, retained the synthesis of IL-12 and the ability to stimulate proliferation of allogeneic T cells. Our data demonstrate that the dominant effect of the drug was redirecting differentiation of a subset of cells despite the presence of inflammatory cytokines. The observed ID50 values indicate that inhibition of DC differentiation might contribute significantly to in vivo immunosuppression by chronic administration of corticosteroids.

Antigen Presentation↗
PubMed1998

High efficiency adenovirus-mediated gene transfer to human dendritic cells.

The interest in the use of human dendritic cells in cancer immunotherapy calls for efficient ex vivo methods of dendritic cell education. To extend the range of methods available, we generated phenotypically characteristic dendritic cells from peripheral blood monocytes incubated with granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected them with an adenovirus containing a humanized version of green fluorescent protein as a marker of gene expression. The levels of expressed protein were high, but they were further increased in combination with cationic liposomes. In comparison to transfection efficiency of the homologous expression plasmid, adenovirus-mediated gene transfer was substantially more efficient. With the aid of liposome-mediated infection, gene transfer into CD83+ dendritic cells was highly effective, resulting in more than 90% of the cells expressing the transgene.

Adenoviridae↗
PubMed1996

Nitrogen-15 NMR chemical shifts in oligopeptides coordinated to cobalt(III).

Dipeptide, tripeptide, and tetrapeptide complexes with cobalt(III) ions were studied as model compounds for evaluation of 15N NMR chemical shifts induced in proteins upon binding transition metal ions. Coordination of oligopeptides to cobalt(III) resulted in large negative 15N NMR shifts for amine nitrogens (-76 to -32 ppm) and deprotonated amide nitrogens (-47 to -10). Coordination-induced shifts were affected by the nature of moiety at the trans position; the shifts were always larger with a carboxylato oxygen than with an amine nitrogen in the trans position. Thus, coordination-induced 15N NMR shifts provided direct and specific information on the stereochemistry of peptide coordination. Two new complexes, [Co(Gly-gly-gly-glyH(-3))(NH3)2] and Ba[Co(Gly-L-hisH(-2))(NO2)3], were synthesized and their structure was determined by NMR spectroscopy.

Amino Acid Sequence↗
PubMed1995

Expression of somatostatin receptor subtypes in breast carcinoma, carcinoid tumor, and renal cell carcinoma.

The presence of transcripts for somatostatin receptor (SSTR) subtypes 1, 2, 3, and 4 was probed by reverse transcription and polymerase chain reaction in ribonucleic acid isolated from 46 malignant and 9 nonmalignant breast tissues, 15 carcinoid tumor tissues, and 13 renal cell carcinoma tissues. The transcripts for SSTR2 were amplified in all but 2 tissue samples, whereas transcripts for SSTR1, SSTR3, and SSTR4 were detected sporadically. In renal cell tumors, SSTR3 transcripts were completely absent. In breast cancer tissue, SSTR subtypes were transcribed independently of patient age, menstrual status, diagnosis, histological grade, and levels of estrogen receptor and progesterone receptor. The probability of finding transcripts for SSTR subtypes, P, was ranked differently for the three types of tumor tissues. For breast cancer, P2 > P3 = P1 > P4; for carcinoid tumors, P2 > P1 > P3 = P4; and for renal cell tumors, P2 > P1 > P4 > P3.

Adult↗
PubMed1994

Somatostatin analogue octreotide modulates metabolism and effects of 5-fluorouracil and 5-fluorouridine in human colon cancer spheroids.

To examine if preferential retention of somatostatin analogues observed in some tumors might be used for modulation of effects of cancer drugs by co-treatment with long acting somatostatin analogues, the effects of somatostatin analogue octreotide on the kinetics of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism were studied by 19F NMR spectroscopy in multicell tumor spheroids comprised of human colon HT-29 adenocarcinoma cells. Octreotide stimulated the rate of formation of fluorouridinephosphates in FUra-treated cells, but inhibited this rate in FUrd-treated cells. Other elements of fluoropyrimidine metabolism were also altered by co-incubation with octreotide. A flow cytometric analysis indicated that FUra and FUrd arrested cells in the S phase, but co-treatment with octreotide almost eliminated the S-phase cells and induced the appearance of DNA fragments. These observations raise the possibility that somatostatin analogues can be used for specific modulation of fluoropyrimidine effects in tumors bearing somatostatin receptors.

Adenocarcinoma↗
PubMed1994

Tumor growth in vivo and as multicellular spheroids compared by mathematical models.

In vivo volume growth of two murine tumor cell lines was compared by mathematical modeling to their volume growth as multicellular spheroids. Fourteen deterministic mathematical models were studied. For one cell line, spheroid growth could be described by a model simpler than needed for description of growth in vivo. A model that explicitly included the stimulatory role for cell-cell interactions in regulation of growth was always superior to a model that did not include such a role. The von Bertalanffy model and the logistic model could not fit the data; this result contradicted some previous literature and was found to depend on the applied least squares fitting method. By the use of a particularly designed mathematical method, qualitative differences were discriminated from quantitative differences in growth dynamics of the same cells cultivated in two different three-dimensional systems.

Animals↗
PubMed1994

Analysis of growth of multicellular tumour spheroids by mathematical models.

We wished to determine the applicability of previously proposed deterministic mathematical models to description of growth of multicellular tumour spheroids. The models were placed into three general classes: empirical, functional and structural. From these classes, 17 models were applied systematically to growth curves of multicellular tumour spheroids used as paradigms of prevascular and microregional tumour growth. The spheroid growth curves were determined with uniquely high density of measurements and high precision. The theoretical growth curves obtained from the models were fitted by the weighted least-squares method to the 15 measured growth curves, each corresponding to a different cell line. The classical growth models such as von Bertalanffy, logistic and Gompertz were considered as nested within more general models. Our results demonstrate that most models fitted the data fairly well and that criteria other than statistical had to be used for final selection. The Gompertz, the autostimulation and the simple spheroid models were the most appropriate for spheroid growth in the empirical, functional and structural classes of models, respectively. We also showed that some models (e.g. logistic, von Bertalanffy) were clearly inadequate. Thus, contrary to the widely held belief, the sigmoid character of a three or more parameter growth function is not sufficient for adequate fits.

Cell Division↗
PubMed1993

Insulin-like growth factor-I is a serum component stimulating growth of human neuroblastoma.

Human non-autocrine neuroblastoma cells SK-N-SH and LF require serum for proliferation in vitro. We wished to determine the role of serum-borne insulin-like growth factor I (IGF-I) as mitogen for these cells. Introduction of the monoclonal antibody alpha-IR3 against human IGF-I receptor reduced proliferation in the presence of fetal bovine serum (FBS). IGF-I (5 nM) was as effective as FBS (10%) in stimulating proliferation. Porcine insulin mimicked the effects of IGF-I, but at a 1000-fold higher concentration. The antibody alpha-IR3 reduced growth stimulated by IGF-I more effectively than growth stimulated by insulin. Thus, proliferation of human non-autocrine neuroblastoma cells can be effectively manipulated by exogenous IGF-I.

Cell Division↗