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S Vuckovic

Publications and source records attributed to S Vuckovic.

15 recordsLinked to original sources
PubMed2004

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay.

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUNT beads and the whole blood "Lyse/No-Wash" protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 microl of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 h after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained.

Adult↗
PubMed2002

Growth factors, cytokines and dendritic cell development.

Dendritic cells (DC) initiate tumor specific immune responses in animal studies and initial human trials suggest that certain tumor-antigen loaded DC preparations generate clinical responses. DC may be obtained from blood or generated in vitro from precursor cells. In vitro generation of DC from precursor cells, under the influence of cytokines, has been favoured to date as a source because of the greater numbers of DC produced. However, the different cytokine combinations and serum or plasma component(s) used, differentiate precursor cells into DC with different physiological properties and ultimate immunogenicity. Thus, the quality of in vitro cytokine derived DC may have a profound influence on clinical outcomes. The administration of certain growth factors, which increase the number of circulating blood DC, may provide an alternative source of DC for use in clinical trials. Although clinical trials in prostate cancer, melanoma and metastatic renal carcinoma patients are encouraging, some data suggest certain DC preparations and administration protocols are sub optimal, even potentially tumor enhancing. As basic scientific studies establish how to provide DC with stable phenotype, resistance to tumour inhibitory factors and high migratory capacity, the technology for producing cytokine derived DC in vitro using Good Manufacturing Practise (GMP) conditions needs to be developed. Future DC vaccination protocols will require careful control of the DC used for tumor-antigen loading and repetitive long term DC vaccination may be necessary to maintain effective anti-tumor immune responses.

Animals↗
PubMed2002

Prostate specific antigen inhibits immune responses in vitro: a potential role in prostate cancer.

PURPOSE: Prostate specific antigen (PSA) is found in high concentration in prostate tissue and in semen, in which its physiological function appears to be liquefaction. In prostate cancer the peripheral PSA concentration is elevated, which may be used as a disease marker. Systemic and local immune defects have been demonstrated in prostate cancer and we postulated a role for PSA in this immunosuppression. We explored the effects of PSA on human T-lymphocyte proliferation in vitro. MATERIALS AND METHODS: PSA was purified from normal seminal plasma using a modified chromatographic technique. The effect of PSA or control protein on lymphocyte responses to mitogens, tetanus toxoid and alloantigens was tested. The inhibitory effect observed was further explored by varying the time of PSA addition, denaturing PSA and including interleukin-2 and anti-PSA antibodies. RESULTS: PSA suppressed in vitro phytohemagglutinin and alloantigen stimulated lymphocyte proliferation in a dose dependent manner. This effect was reversed by adding anti-PSA antibodies but not by interleukin-2. CONCLUSIONS: These in vitro PSA effects suggest another T-lymphocyte mediated immunosuppressive mechanism. In vivo high levels of PSA may compromise natural immune responses to cancer and current attempts at immunotherapy for prostate cancer.

Disease Progression↗
PubMed2001

Surgical and physical stress increases circulating blood dendritic cell counts independently of monocyte counts.

Dendritic cells (DCs) are specialized antigen-presenting cells that have the unique ability to initiate a primary immune response. The effect of physiologic stress on circulating blood DCs has thus far not been studied. In this study, we applied a recently developed method of counting blood DCs to test the hypothesis that significant stress to the body such as surgery and exercise might induce measurable changes in the DC numbers, subsets, phenotype, and function. Twenty-six patients scheduled for elective laparoscopic cholecystectomy, 4 for elective hysterectomy, 56 controls, and 5 volunteers who underwent a stress exercise test were enrolled in the study. Absolute DC counts increased acutely (71.7% +/- 11% [SEM], P =.0001) in response to the stress of surgery and dropped below preoperative levels (-25% +/- 14% [SEM], P =.05) on days 2-3. The perioperative DC subset balance remained constant. Interestingly, DC counts changed independently of monocyte counts. Exercise also induced a rise in DC counts but coincidentally with monocyte counts. Surprisingly, no phenotypic or functional activation of DCs was seen in either stress situations in vivo. DCs are rapidly mobilized into the circulation in response to surgical and exercise stress, which may serve to prepare the host's immune defenses against trauma. The independent regulation of the DC and monocyte counts reinforces the distinction between these 2 cell populations.

Adult↗
PubMed2001

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases.

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naïve T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c+ and CD11c- DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin- = CD3-, CD19-, CD14-, CD16-) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86+ and CD40+ lin- cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c+ and CD11c- DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86+ and CD40+ DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44+ antigen was mainly associated with CD11c+ DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86+ and CD40+ DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation.

Adolescent↗
PubMed2000

Lectin ligands on human dendritic cells and identification of a peanut agglutinin positive subset in blood.

As only a few cell surface markers for dendritic cells (DC) have been identified to date, this study examined the expression of ligands for lectin on different human DC populations. The ability of Concanavalin A (Con A), Wheat Germ Agglutinin (WGA), peanut agglutinin (PNA), and Helix pomatia (HPA) to bind to cell lines and PBMC and DC populations was analyzed by flow cytometry and specificity of binding confirmed using inhibitory and noninhibitory sugars. The cell lines showed non-lineage-restricted binding with Con A and WGA, independent of sialidase treatment. HPA and PNA bound to a restricted number of lines, but showed broad reactivity after sialidase treatment. The peripheral blood mononuclear cells (PBMC) and directly isolated blood DC, activated CD83(+) blood DC, epidermal Langerhans cells (LC), and monocyte-derived DC (Mo-DC) showed strong binding of Con A and WGA, both before and after sialidase treatment. No HPA binding ligands were detected on PBMC populations, including directly isolated blood DC. Following sialidase treatment CD3(+), CD16(+), and a subset of CD19(+) lymphocytes bound HPA. The lectin PNA bound weakly to CD14(+) monocytes and a subpopulation of circulating DC that were HLA-DR(hi)CDw123 Dr(hi)CDw123(dim)/(neg)CD11c(+). The HLA-DR(mod)CDw123(hi)CD11c(neg) subpopulation did not bind PNA. Without sialidase treatment LC expressed both HPA and PNA ligands, but these were either absent on activated CD83(+) blood DC or weakly expressed on Mo-DC. Following sialidase treatment PBMC populations, activated CD83(+) blood DC, and Mo-DC became PNA positive. Thus human DC express several lectin ligands and PNA binding identifies a subset of blood DC. That may reflect discrete changes associated with stages of DC development or functional maturation.

Binding Sites↗
PubMed2000

The role of dendritic cells in the innate immune system.

Dendritic cells (DCs) are bone-marrow-derived leucocytes that are specialised antigen-presenting cells capable of stimulating a primary T-lymphocyte response to specific antigen. In this chapter we discuss the role DCs play in the innate response acting as a critical link with the adaptive response and the influence of the innate response on dendritic cells.

Animals↗
PubMed2000

Expression of multilectin receptors and comparative FITC-dextran uptake by human dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC-dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC). Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR. FITC-dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC-dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC-dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.

Animals↗
PubMed1999

Dendritic cells in chronic myelomonocytic leukaemia.

Blood dendritic cells (DC) differentiate in vitro via two separate pathways: either directly from blood DC precursors (DCp) or from CD14+ monocytes. In chronic myelomonocytic leukaemia (CMML) abnormal bone marrow precursors contribute to blood monocyte development but DC development has not been studied previously. Monocytes comprised 60% of blood MNC in 15 CMML patients studied, compared with 20% in 16 age-matched controls. The increase in blood monocytes was accompanied by a reciprocal decrease in mean blood DC percentage (from 0.42% of MNC in normal individuals to 0.16% of MNC in CMML patients). Absolute blood DC numbers showed a minimal (non-significant) reduction from 9.8 x 10(6)/l in normal individuals to 7.5 x 10(6)/l in CMML patients. The CD14(low) WCD16+ monocyte subpopulation was not found in CMML patients. After culture in GM-CSF/IL-4, CMML CD14+ monocytes acquired the phenotype of immature monocyte derived DC (Mo-DC) with similar yields to normal blood Mo-DC generation. Addition of TNF-alpha or LPS induced both normal and CMML Mo-DC to express prominent dendritic processes, the CMRF44+ and CD83+ antigens and high levels of HLA-DR, CD80 and CD86. Treatment either with TNF-alpha or LPS increased the allostimulatory activity of normal Mo-DC, but had little effect on the allostimulatory activity of CMML Mo-DC, perhaps reflecting the underlying neoplastic changes in monocyte precursors. We conclude that the blood DC numbers are relatively unaffected in CMML, suggesting discrete regulation of monocyte and DC production.

Adult↗
PubMed1999

Expression of the RelB transcription factor correlates with the activation of human dendritic cells.

The RelB gene product is a member of the nuclear factor (NF)-kappaB family of transcription factors. It has been identified recently within mouse antigen-presenting cells and human monocyte-derived dendritic cells (DC). Disruption of the mouse RelB gene is accompanied, amongst other phenotypes, by abnormalities in the antigen-presenting cell lineages. In order to define RelB expression during human DC differentiation, we have analysed RelB mRNA by reverse transcriptase-polymerase chain reaction and RelB protein by intracellular staining in CD34+ precursors and different types of DC preparations. RelB mRNA was not detected in CD34+ precursor populations. Fresh blood DC (lineage-human leucocyte antigen-DR+ (lin-HLA-DR+)) lacked RelB mRNA and cytoplasmic RelB protein but a period of in vitro culture induced RelB expression in blood DC. Purified Langerhans' cells (LC) (CD1a+ HLA-DR+) failed to express RelB mRNA. Immunocytochemical staining identified RelB protein in human skin epithelium. RelB protein was expressed in a very few CD1a+, CD83+ or CMRF-44+ dermal DC but was not present in CD1a+ LC. Tonsil DC (lin-HLA-DR+ CMRF-44+) were positive for RelB mRNA and RelB protein. Intestinal DC (HLA-DR+) also lacked immunoreactive RelB protein. The majority of interdigitating CD83+, CMRF-44+, CMRF-56+ or p55+ DC located in paracortical T-lymphocyte areas of lymph node and tonsil contained RelB protein. The expression of RelB mRNA and RelB protein correlates with the activated phase of blood DC and the postmigration cell (activated) stage of tissue DC development.

Antigens, CD34↗
PubMed1998

Generation of CMRF-44+ monocyte-derived dendritic cells: insights into phenotype and function.

The CMRF-44 monoclonal antibody (MoAb) recognizes an intermediate stage of blood dendritic cell (DC) differentiation as well as mature CD83+ blood DC. Here we describe the use of the CMRF-44 MoAb to monitor the in vitro development of DC-like cells from peripheral blood mononuclear cells. Neither granulocyte-macrophage colony-stimulating factor (GM-CSF) nor GM-CSF plus tumor necrosis factor-alpha (TNF-alpha) supported the development of CMRF-44+ cells. However, GM-CSF plus interleukin (IL)-4 generated a substantial number of CMRF-44+ cells among the heterogeneous CD14- myeloid cell population, produced after 7 or 10 days of culture. The addition of TNF-alpha to GM-CSF+IL-4 on the fifth day of culture enhanced the generation of CMRF-44+ cells from days 7 to 14. A concentration of 50 U/mL of IL-4 was sufficient to allow the development of CMRF-44+ cells. The presence of GM-CSF was essential, but a wide range of concentrations (50-800 U/mL) was effective for supporting IL-4-induced generation of CMRF-44+ cells. TNF-alpha at concentrations of 20 or 50 ng/mL induced a maximal increase in the number of CMRF-44+ cells. The CMRF-44+ DCs generated in the presence of GM-CSF+IL-4 were large, irregularly shaped cells with variable CD1a expression and have CD83 transcripts but no CD83 surface expression. Additional TNF-alpha treatment induced prominent dendritic processes and surface expression of CD83 on CMRF-44+ DCs. The CMRF-44+ DCs generated in GM-CSF+IL-4 showed higher allostimulatory activity than CMRF-44 cells but were less efficient at processing and presenting soluble antigen to T-lymphocyte lines. TNF-alpha treatment reduced antigen uptake but increased the allostimulatory activity of CMRF-44+ DCs. CMRF-44+ DC differentiation from blood CD14+ monocytes was not radiosensitive and thus does not involve cell division. We conclude that the MoAb CMRF-44 identifies both intermediate and fully mature stages of monocyte-DC differentiation and may be a useful marker in establishing the optimal timing for antigen loading of in vitro-generated monocyte-derived DCs.

Antibodies, Monoclonal↗
PubMed1991

Dysmegakaryocytopoiesis and thrombocytosis in a patient with acute myelomonocytic leukemia and long evolution.

A patient with acute myelomonocytic leukemia (M4), dysmegakaryocytopoiesis and thrombocytosis is presented. Immunophenotyping and blast colony assay showed the presence of blasts with IaDr, CD 33 and CD 14 antigens. Cytogenetic analysis and level of thrombopoietin were normal. This atypical case represents yet another example of the transitional zone between atypical myeloproliferative disease and acute leukemia, and the apparent absence of any cytogenetic abnormality is noteworthy.

Humans↗
PubMed1989

[Double-blind study of clobazam (Odipam) and diazepam in non-psychotic anxiety states].

Clobazam (Odipam) and Diazepam were investigated by the double blind study method in patients with nonpsychotic anxiety. It was found out that both drugs had equal anxiolytic and tranquilizing effect with the same adverse effects. Greater frequency of excellent marks concerning the final therapeutic effect of Odipam in respect to other marks pointed to its better clinical tolerance, so that this drug was recommended as daily tranquilizer.

Adolescent↗