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R Andreesen

Publications and source records attributed to R Andreesen.

At least 91 records · Page 5Linked to original sources
PubMed1995

Carboxypeptidase M is identical to the MAX.1 antigen and its expression is associated with monocyte to macrophage differentiation.

The two monoclonal antibodies MAX.1 and MAX.11 recognize cell surface antigens that are almost undetectable on monocytes but highly expressed on differentiated macrophages. Biochemical characterization revealed that both antibodies detect the same 58-64-kDa glycoprotein anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified the MAX.1/11 antigen by immunoaffinity chromatography using monoclonal antibody MAX.11. The NH2-terminal amino acid sequence was determined and turned out to be identical to the NH2-terminal sequence of the membrane-bound carboxypeptidase M. By precipitation with antibodies MAX.1 and MAX.11, membrane preparations of macrophages and placental microvilli were almost completely depleted of enzyme activity, indicating that the two antibodies indeed recognize carboxypeptidase M. Immunoreactivity of both antibodies correlates with the reported tissue distribution of enzyme activity. Expression of carboxypeptidase M on mRNA level and enzymatic activity markedly increase during in vitro differentiation of monocytes, according to the described increase in MAX.1 and MAX.11 antigen expression. Moreover, in vitro differentiated macrophages show the highest specific activity yet described in any tissue. In addition, carboxypeptidase M expression could be detected in HL-60, U937, and THP-1 myeloid cell lines. Vitamin D3-induced monocytic differentiation resulted in an increased carboxypeptidase M expression in all three cell lines. Further studies are needed to elucidate the functional role of carboxypeptidase M during monocytic differentiation and activation.

Antigens, Surface↗
PubMed1995

Beta-tubulin and P-glycoprotein: major determinants of vincristine accumulation in B-CLL cells.

Vincristine (VCR) accumulation in chronic lymphatic leukemia of B-cell origin (B-CLL) has recently been shown not to be inversely correlated to P-glycoprotein (PGP) levels. Therefore, we studied, in addition to PGP expression and accumulation of VCR, the cellular beta-tubulin content in quiescent and rhIL-2 activated B-CLL cells. VCR mediates cytotoxicity by binding to tubulin. Constitutive beta-tubulin levels in B-CLL cells varied considerably. Upon activation with rhIL-2, beta-tubulin expression increased significantly. Therefore, tubulin levels could be correlated over a wide range to VCR accumulation. When the PGP-mediated drug efflux was blocked by verapamil (VRP), tubulin levels correlated linearly to VCR accumulation. All B-CLL cases expressed PGP at different levels. There was no linear correlation between PGP expression and VCR accumulation. A modulation factor m was defined as a quotient of VCR accumulation in the presence and absence of VRP to define the extent by which VRP inhibited a steady-state accumulation of VCR. The factor allowed discrimination between B-CLLs expressing low versus high PGP, irrespective of the levels of tubulin. However, PGP and beta-tubulin levels together were predictive for VCR accumulation in steady state. There was no uniform-accumulation defect for VCR in B-cell CLL because beta-tubulin and PGP were expressed independently. Non PGP-mediated VCR transport seems to play a minor role in B-cell CLL. Leukemia-associated varying of cytoskeletal organization in B-cell CLL might be one reason for the diverse cellular responses to receptor-mediated signals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Interferon-alpha does not improve the antineoplastic efficacy of high-dose infusional 5-fluorouracil plus folinic acid in advanced colorectal cancer. First results of a randomized multicenter study by the Association of Medical Oncology of the German Cancer Society (AIO).

BACKGROUND: High-dose 5-FU given weekly as a 24-h infusion in combination with folinic acid (FA) has been associated with low toxicity and a high response rate. Interferon-alpha (IFN) either alone or in combination with FA has also improved treatment results by modulating 5-FU activity. We therefore initiated a randomized multicenter trial comparing the ability of FA or IFN to modulate infusional 5-FU. The statistical design using a sequential analysis allows us to report on the comparison of 5-FU/FA vs. 5-FU/FA/IFN while randomization of patients into 5-FU/FA vs. 5 FU/IFN continues. METHODS: Chemotherapy-naive patients with advanced progressive colorectal cancer and measurable metastatic lesions were randomized to receive 5-FU 2600 mg/m2 i.v. as a 24-h infusion, combined with either FA 500 mg/m2 as a 2-h infusion (A), or IFN 3 x 10(6) U s.c. 3 x/week (B), or the combination of FA plus IFN as in arms A and B (C). Treatment arms were repeated weekly for 6 weeks followed by a 2-week rest period. These 8-week cycles were administered until tumor progression. Because of the occurrence of 2 toxic deaths among the first 17 patients treated in arm C, 5-FU was reduced to 2000 mg/m2 for all patients in arm C. Sequential analysis according to Whitehead for objective response was planned with alpha = 0.05/3 and a power of 80% (beta = 0.2) to detect a difference of > or = 25% (delta = 0.25) or equivalence of response rates. For pairwise comparison of treatment arms a minimum of 30 patients per arm and a maximum of 90 patients per arm were expected in case of equivalence or difference. RESULTS: An interim analysis was performed after the first 93 of 149 randomized patients were evaluable for response and toxicity (A 31 pts, B 33 pts, C 29 pts). Despite the 5-FU dose reduction in arm C, 28% of patients experienced grade 3/4 toxicity (CTC) including diarrhea, mucositis and handfoot syndrome compared to 16% in arm A and 12% in arm B (not significant). No treatment related toxic death occurred in arms A or B, but 3 patients (10%) in arm C died of diarrhea and septicemia. Among patients treated with 5-FU/FA objective tumor response occurred in 12/31 patients (39%) (21%-56%, 95% confidence interval) (3 CR, 9 PR), no change in 13/31 (42%) and PD in 6/31 (19%) patients. Eleven of 29 patients (38%) (20%-56%, 95% confidence interval) receiving 5-FU/FA/IFN achieved complete (3 patients) or partial (8 patients) remissions, 10/29 patients (34%) had stable disease and 8/29 patients (28%) tumor progression. According to the sequential analysis the rates of objective responses observed in patients treated with 5-FU/FA or 5-FU/FA/IFN were equivalent. CONCLUSION: This interim analysis allows the conclusion that infusional 5-FU plus FA/IFN is no more active than infusional 5-FU plus FA alone. However, 5-FU/FA/IFN despite 5-FU dose reduction was associated with unacceptably high toxicity, including 10% deaths. Therefore, further investigation of this regimen is not justified. The study is continued with the comparison of 5-FU/FA vs. 5-FU/IFN.

Adult↗
PubMed1995

111In-oxine labelling of tumour-cytotoxic macrophages generated in vitro from circulating blood monocytes: an in vitro evaluation.

The labelling of ex-vivo activated macrophages with doses of 111In-oxine sufficient for gamma camera imaging does not damage cell viability or the functional competence of cells (secretion of tumour necrosis factor-alpha and interleukin-6). The mean labelling efficiency was about 84%. Release of 111In equivalent to 15% occurred over 24 h under cell culture conditions, indicating good stability of the radioactive cell label. 111In-oxine-labelled macrophages are a suitable tool for biokinetic studies of adoptive immunotherapy.

Cell Survival↗
PubMed1995

[New in vitro test systems].

Experimental in vitro systems are discussed with reference to their use as models für the analysis of basal tumor-host interactions. Multiple functions of macrophages are treated and there is also a special focus on the differentiation of macrophages in vitro and the influence of cocultures with the influence of cocultures with tumour cells (monolayer, spheroids). These experiments have shown that differentiation can be suppressed by tumour cells with three-dimensional growth. These findings are of interest for (a) adoptive immunotherapy and (b) the interpretation of tumour-infiltrating macrophages. These tests are not suitable for in vitro monitoring of clinical immunotherapy.

Carcinoma, Transitional Cell↗
PubMed1994

Chemomodulation of drugs involved in multidrug resistance in chronic lymphatic leukemia of the B-cell type.

Reduced drug accumulation may be one reason for intrinsic drug resistance in chronic lymphatic leukemia of the B-cell type (B-CLL). Immunophenotyped leukemic human B-cells from 38 cases of B-CLL were characterized for P-glycoprotein (PGP) content. In all, 30 cases of B-CLL were additionally analyzed for further parameters: accumulation of daunorubicin (DNR, n = 20) and rhodamine 123 (Rh123, n = 30) in both the presence and the absence of verapamil (VRP). Also, 16 cases of B-CLL were analyzed for vincristine (VCR) accumulation with or without VRP. Concerning the relative expression of PGP, these 16 cases of B-CLL were representative for the whole group of 30 cases. Spontaneous accumulation of Rh123 and VCR varied over a wide range: accumulation of Rh123, by a factor of 11.8; accumulation of VCR, by a factor of 9.7; and accumulation of DNR, by a factor of 3.6. VRP modulated the accumulation of RH123 in 16/30 cases (53%), that of VCR in 69% of cases, and that of DNR in 11% of cases. The maximal VRP-mediated increases in accumulation amounted to factors of 1.3 for DNR, 2.3 for Rh123, and 7.8 for VCR. Spontaneous drug accumulation did not correlate with the extent of chemomodulation. The amount of PGP in B-CLL cells (all cases studied were PGP-positive) did not correlate with drug accumulation or with the extent of VRP-mediated chemomodulation. Thus, high expression of PGP is only partially responsible for defective drug accumulation in B-CLL. Only the degree of chemomodulation by VRP is predictive for the extent of the PGP-related functional drug accumulation defect. Thus, in B-CLL, PGP-independent drug accumulation defects seem to be as important as those mediated by PGP. The extent of this drug accumulation defect varies for the different drugs in the following order VCR > Rh123 > DNR. The relevance of PGP-mediated and -independent drug accumulation defects in vivo may depend to a large extent on the drug being used and on the individual cell type. Both types of defect in drug accumulation are of high importance when regimens include VCR a drug commonly used in second-line chemotherapy of B-CLL. Both defects in drug accumulation may be responsible for intrinsic VCR resistance in B-CLL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Monocyte differentiation and accessory function: different effects on the proliferative responses of an autoreactive T cell clone as compared to alloreactive or antigen-specific T cell lines and primary mixed lymphocyte cultures.

An autoreactive T cell clone derived from a patient with reactive arthritis, two alloreactive T cell lines, two antigen-specific T cell lines and allogeneic resting T cells were analyzed for their responses to monocytes and macrophages derived from monocytes by in vitro differentiation. The autoreactive T cell clone strongly proliferated in response to fresh monocytes and to macrophages derived from a 7 day culture, but only poorly to monocytes cultured for 2 days. In contrast, alloreactive and antigen-specific T cell lines proliferated to all stimulatory cells equally well. Finally, primary mixed lymphocyte reactions could be stimulated by both fresh and 2-day cultured monocytes, but not by in vitro derived macrophages. The impaired response of the autoreactive T cell clone to 2-day cultured monocytes could not be attributed to reduced expression of several well-defined surface molecules nor to induction of nonresponsiveness. Neither allogeneic monocytes nor cytokines (IL-1, IL-2, IL-4, IL-6) could correct the defective response of the autoreactive T cell clone. However, preculture of monocytes in the presence of interferon-gamma, IL-1, IL-4 or IL-6 retained their stimulatory capacity. Our interpretation of the selectively impaired response of the autoreactive T cell clone is that it most likely recognizes a differentiation-dependent monocyte/macrophage-specific peptide.

Antigen-Presenting Cells↗
PubMed1993

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages.

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.

24,25-Dihydroxyvitamin D 3↗
PubMed1993

Immunohistochemical comparison of cutaneous histiocytoses and related skin disorders: diagnostic and histogenetic relevance of MS-1 high molecular weight protein expression.

Twenty-nine cases of Langerhans cell histiocytosis (LCH), non-Langerhans cell histiocytoses (N-LCH), non-infectious granulomas, and fibroblast-related lesions were examined with a panel of monoclonal and polyclonal antibodies on freshly frozen tissue sections to characterize the macrophage phenotype of N-LCH syndromes. MS-1 high molecular weight extracellular protein, specific for sinusoidal endothelial cells and dendritic perivascular macrophages in normal human organs, was expressed by N-LCH cells but was not found in LCH cells, epithelioid cells in sarcoidosis, or palisading histiocytes in granuloma annulare. The subcellular location of MS-1 protein, i.e., cytoplasmic vs. peripheral/extracellular, allowed discrimination of small and large (foamy or multinucleated) N-LCH cells. MS-1-positive cells, which were found intermingled in cellular dermatofibromas but not in fibrous dermatofibromas, differed from MS-1-positive N-LCH cells by their dendritic morphology, and thus rather resembled their normal dermal counterparts. A preserved functional relationship of these two MS-1-positive cell types was indicated by the fact that N-LCH and cellular dermatofibromas were the only lesions found to be highly vascularized. As expected, CD1a showed high specificity for LCH, while CD34 was predominantly expressed by fibroblast-related lesions; in cellular dermatofibromas, CD34 and MS-1 expression partially overlapped. The other antigens tested showed non-specific or overlapping patterns of expression. In conclusion, assessment of MS-1 protein expression (in addition to assessment of CD1a and CD34) promises to be of diagnostic value in the discrimination of N-LCH from related skin disorders, and it may indicate a common differentiative pathway for most N-LCH disease entities.

Antibodies, Monoclonal↗
PubMed1993

Acute alveolitis induced by hydroxyurea in a patient with myeloproliferative syndrome.

Hydroxyurea is increasingly being used to control myeloproliferative disorders, in part because of its relative lack of side effects. We present a case of life-threatening alveolitis in a patient treated with hydroxyurea for myeloproliferative syndrome. Absence of exposure to other drugs and the clinical course suggest that the alveolitis was induced by hydroxyurea.

Acute Disease↗
PubMed1993

Allostimulatory cells in fresh human blood: heterogeneity in antigen-presenting cell populations.

The relative ability of unmanipulated monocytes, B cells, and dendritic cells (DC) from peripheral blood to stimulate an allogeneic MLR has not been clearly established. We studied the allostimulatory ability of these cell types from minimally manipulated PBMC populations to exclude the induction of stimulatory properties by the complex isolation procedures commonly used to isolate blood DC. Highly purified cell populations were obtained from volunteer donors by immunolabeling PBMC with mAb directed against known lineage-associated markers and separating the positive and negative population on a FACS. These cells were used as stimulators in an allogeneic MLR. The major allostimulatory activity resides in the CD14, CD11b, and CD19 negative fractions. A mixture of antibodies to T, B, NK, monocyte, and FcRIII positive cells was then used to isolate a minor cell population that contained a markedly superstimulatory population of (CD3, CD14, CD16, CD19, and CD57) negative cells. We demonstrate that this activity is constitutive, and is not an artifact of the adherence and in vitro culture steps used in conventional DC purification procedures. We also show by rigorous depletion of the T cell responders that endogenous HLA class II positive cells in the responder population have little role in presenting processed allogeneic antigens during the primary MLR. Monocytes and B cells are stimulators of the allogeneic MLR, but are considerably less potent on a cell for cell basis than the putative DC population. Finally, because human blood and tonsil DC lack detectable CD43 by immunoperoxidase staining, in contrast to monocytes and activated B cells, we examined the ability of CD43 negative and positive cells to stimulate an allogeneic MLR. Similar allostimulatory activity for the human MLR was shown to reside in both the CD43 positive and negative fractions, suggesting that there may be some heterogeneity in the APC population.

Antigens, CD↗
PubMed1992

Developmental regulation of granulocyte-macrophage colony-stimulating factor production during human monocyte-to-macrophage maturation.

Cells of the macrophage lineage are a major source of various cytokines and hematopoietic growth factors. With regard to the growth factors acting on cells of their own lineage, macrophage colony-stimulating factor (M-CSF) has been proven to be secreted by monocytes (MO) and macrophages (MAC), whereas the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by human MO/MAC is under debate. Here we report that in elutriation-purified MO, as well as in MAC derived from cultured MO, GM-CSF m-RNA was regularly induced by LPS. In MO the GM-CSF message was still detectable 18 h after stimulation under serum-free conditions, but in contrast was already lost at this time point in MAC. Secreted GM-CSF protein was detected in the culture medium using a sandwich ELISA. Furthermore, a factor-dependent cell line (M-07) was used for a biological assay. Here, a neutralizing anti GM-CSF antibody specifically blocked the proliferation-inducing activity of MO/MAC supernatants. Whereas only small amounts of GM-CSF were detected in MO, its secretion increased severalfold upon MO-to-MAC differentiation in vitro. A similar increase upon in vitro maturation of MO was observed for the production of granulocyte colony-stimulating factor. The highest amounts of GM-CSF (up to 2.8 ng/10(6) cells) were produced by MAC that had been derived from MO cultured under serum-free conditions in the presence of 0.5 mg/ml albumin as the only medium supplement.

Blotting, Northern↗
PubMed1992

The interleukin-2 receptor in human monocytes and macrophages: regulation of expression and release of the alpha and beta chains (p55 and p75).

Human monocytes constitutively express the intermediate-affinity interleukin-2 receptor (IL2R) p75, while freshly isolated monocytes lack the low-affinity IL2R p55 (Tac antigen, CD25). Lipopolysaccharide (LPS) upregulates expression of p75 and effectively induces surface expression of CD25 on human monocytes within 18 h, as detected by two-colour FACS analysis on 59.5 +/- 7.6% of cells. IL2-binding studies using biotinylated IL2 reveal the presence of a functional high-affinity receptor on LPS-activated monocytes. Soluble CD25 (sCD25) is not released into supernatants of elutriation-purified monocytes cultured for 24 h with 100 ng/ml LPS. When these monocytes are cultured for up to 7 days in the presence of serum to induce differentiation into macrophages, increasing amounts of sCD25 can be measured in the 24-h supernatants induced with LPS (173 +/- 86 U/ml at day 7), whereas the percentage of CD25+ cells (14.8 +/- 14.1% at day 7) is significantly lower than in monocytes. Thus, the cell surface expression and release of CD25 is differentially regulated in activated monocytes and macrophages.

Humans↗
PubMed1992

Human macrophages secrete a tumoricidal activity distinct from tumour necrosis factor-alpha and reactive nitrogen intermediates.

Human macrophages, differentiated in vitro from blood monocytes, can be induced to secrete tumouricidal activity when activated by combined treatment with recombinant interferon gamma and bacterial lipopolysaccharide. We have analysed conditioned culture supernatants of activated human monocytes and in vitro differentiated macrophages cultivated under serum-free conditions for cytolytic activity against a TNF alpha-insensitive human tumour cell line and characterized this activity with respect to its relationship to TNF alpha and reactive nitrogen intermediates. Cytolytic activity was recovered in the high molecular weight fraction of culture supernatants conditioned by terminally differentiated macrophages, whereas conditioned culture supernatants of freshly isolated blood monocytes, processed under identical conditions, were devoid of significant cytolytic activity. This activity was tumour-specific, strongly affecting the human lymphoma cell line JMP, whereas freshly isolated human peripheral blood lymphocytes were not affected to a significant extent. It was inactivated by heat or trypsin treatment, but only partially inhibited by a monoclonal antibody against recombinant human TNF alpha, which completely neutralized all of the TNF alpha activity detectable in the supernatants tested. Cytolytic activity could not be reduced further even by a 1000-fold excess of anti-TNF alpha antibody, suggesting that TNF alpha has some synergistic effect on the tumouricidal activity observed, rather than being the central effector molecule. This notion was supported by enhancement of low levels of cytolytic activity by addition of recombinant human TNF alpha at concentrations not having any direct cytotoxic effect on the tumour target cells used.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗