Prognostic value of genomic alterations in minimal residual cancer cells purified from the blood of breast cancer patients.

The prognostic value of disseminated tumour cells derived from 353 breast cancer patients was evaluated. Disseminated tumour cells were purified from blood using a newly established method and nucleic acids were subsequently isolated. We investigated genomic imbalances (GI) such as mutation, amplification and loss of heterozygosity of 13 tumour suppressor genes and 2 proto-oncogenes using DNA from isolated minimal residual cancer cells. Significant correlations were found between genomic alterations of the DCC - and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free survival. Furthermore, increasing numbers of genomic imbalances measured in disseminated tumour cells were significantly associated with worse prognosis of recurrent disease. Logistic regression and Cox multivariate analysis led to the identification of genomic imbalances as an independent prognostic factor. Determination of disseminated tumour cells by genotyping of oncogenes and tumour suppressor genes seems not only to be a useful adjunct in follow up of carcinoma patients but provides also valuable additional individualized prognostic and predictive information in breast cancer patients beyond the TNM system.

Detection of mammaglobin expressing cells in blood of breast cancer patients.

Expression of human mammaglobin (hMAM) was published to be exclusively expressed in mammary tissue, in solid tumors, axillary lymph nodes and disseminated cancer cells in blood of breast cancer patients. A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) test was applied to investigate hMAM expression in blood of breast cancer patients. Mammaglobin mRNA expression was found not only in breast cancer cell lines but also in cell lines of other cancer origin. In our patient cohort hMAM expression in 11/98 (11%) samples of breast cancer and 3/12 (25%) ovarian cancer patients could be detected. hMAM mRNA expression as a candidate marker for the detection of disseminated cancer cells in blood of breast cancer patients showed low sensitivity and reduced tissue specificity. A prognostic significance of hMAM expression could not be demonstrated.

Independent prognostication and therapy monitoring of breast cancer patients by DNA/RNA typing of minimal residual cancer cells.

Clinical relevance, purification techniques and molecular characterization of minimal residual cancer cells (MRCC) is a controversial topic in the literature. An analytical concept including a novel isolation procedure and a panel of tests for DNA and RNA typing of MRCCs is described and clinically evaluated in this paper. The purification procedure exploiting the physical characteristics of MRCCs shows superior performance leading to > 50% pure and viable tumor cells. Proof of the presence and purity of MRCCs in an isolated sample is given by multiparametric DNA typing (amplifications, mutations, losses of heterozygosity). On the basis of the proven presence of MRCCs tumor-relevant mRNAs can be adequately analyzed by normalized quantitative real-time RT-PCR. The molecular characterization of MRCCs isolated from blood of breast cancer patients could have a strong clinical impact on prognostication, drug targeting and therapy monitoring.

IFN-gamma induces the high-affinity Fc receptor I for IgG (CD64) on human glomerular mesangial cells.

The deposition of immune complexes, followed by activation of complement and/or Fc receptors and generation of chemoattractants, is the most common feature of human glomerulonephritis. Recently we have shown that primary cultured human glomerular mesangial cells (HMC), which are normally negative for IgG Fc receptors, can be stimulated to express the low-affinity FcgammaRIII-A receptor isoform. In this study we further demonstrate that activation of HMC through IFN-gamma resulted in the functional expression of the high-affinity Fc receptor for IgG (FcgammaRI, CD64). IFN-gamma-dependent induction of classical FcgammaRIa1 mRNA as well as a2, b2 splice variants were evident after 24 h in proliferating HMC and after 48 h in resting HMC. Transcription of FcgammaRI mRNA was also induced by IL-10 in proliferating HMC, whereas other cytokines such as IL-3, transforming growth factor-beta1 and granulocyte-macrophage colony-stimulating factor were not effective. Cell surface expression of FcgammaRI could be detected by flow cytometric analysis after IFN-gamma stimulation and was accompanied by the augmentation of MHC class II and the up-regulation of intercellular adhesion molecule-1 expression. Triggering of HMC by cross-linking FcgammaRI with F(ab')2 fragments of the anti-CD64 monoclonal antibody 22 led to enhanced synthesis of mRNA for the chemokines IL-8 and monocyte chemoattractant protein-1, indicating that the FcgammaRI of HMC is functionally active. These in vitro data suggest that engagement of both FcgammaRI and FcgammaRIII-A on activated HMC through IgG immune complexes may result in an increased chemoattraction of leukocytes into the glomerulus, contributing to the development of glomerulonephritis.

T cell antigen receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-alpha.

Activation and translocation of protein kinases C is a key event in the regulation of T lymphocyte activation, proliferation and function. Stimulation of human peripheral blood lymphocytes with the monoclonal antibody BMA 031 raised against the T cell antigen receptor led to a bimodal activation of protein kinases C. The immediate activation and translocation of the protein kinase C isoform PKC-alpha was followed by activation and translocation of the protein kinase C-beta isoenzyme after 90 min of stimulation. Pretreatment of the cells with cholera toxin for 90 min completely abolished activation of protein kinase C-alpha. In sharp contrast, activation and translocation of protein kinase C-beta was not influenced by the bacterial toxin, suggesting that activation and translocation of different protein kinase C isoenzymes are regulated by distinct mechanisms of transmembrane signalling coupled to the T cell antigen receptor/CD3 complex. The expression of high affinity IL-2 receptors was completely inhibited by cholera toxin, while IL-2 synthesis and secretion were not influenced in BMA 031-stimulated human lymphocytes. Extensive control experiments have shown that the effects of cholera toxin were not mediated by its B subunit, and were independent of elevation of intracellular cAMP concentration, suggesting that cholera toxin interfered with a signalling pathway leading to activation of protein kinase C-alpha, which could be responsible for the inhibition of IL-2 receptor expression. This hypothesis was substantiated by the finding that upon introduction of antibodies against protein kinase C-alpha, IL-2 receptor gene expression was completely suppressed. The results suggest, that protein kinase C-alpha might be the major protein kinase C isoenzyme of a signal transduction cascade regulating IL-2 receptor expression in stimulated human lymphocytes.

Lymphocyte-derived cytokines induce sequential expression of monocyte- and T cell-specific chemokines in human mesangial cells.

Chemokines are a family of small related proteins that play an important role in the selective recruitment of different leukocyte populations to the sites of inflammation. Human glomerular mesangial cells are potent producers of a variety of chemokines. Here we examined the kinetics of mesangial cell chemokine expression with focus on the C-C or beta chemokines monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1 alpha (MIP-1 alpha), and the C-X-C or alpha chemokine interleukin-8 (IL-8) in response to lymphocyte- or monocyte-derived cytokines and mesangial cell growth factors. It was found that interferon-gamma (IFN-gamma), a cytokine produced by TH1 lymphocytes, synergized with tumor necrosis factor-alpha (TNF-alpha) in RANTES expression and with IL-1 beta in MCP-1 synthesis. Time course studies revealed an early peak of mRNA expression of monocyte-specific MCP-1 upon activation with TNF-alpha in contrast to T cell-specific RANTES, which reached the highest mRNA level after 18 hours. This sequence of TNF-alpha-induced MCP-1 and RANTES expression was confirmed on the protein level. As another T-lymphocyte specific chemokine, MIP-1 alpha mRNA and protein was expressed only in response to TNF-alpha plus IFN-gamma with kinetics similar to those of RANTES expression. Finally, unlike other mesangial growth factors basic fibroblast growth factor (bFGF) induced MCP-1, RANTES, and IL-8 mRNA expression, suggesting an involvement of autocrine regulation mechanisms in mesangial chemokine expression.

Interleukin 1 activates jun N-terminal kinases JNK1 and JNK2 but not extracellular regulated MAP kinase (ERK) in human glomerular mesangial cells.

Interleukin 1 (IL-1) potently activates human glomerular mesangial cells (HMC). In cytosolic extracts of IL-1-stimulated HMC or in anion exchange chromatography fractions we could not find any change in phosphorylation of myelin basic protein (MBP), a good substrate for extracellular regulated kinase (ERK). In contrast, IL-1 stimulated GST-jun kinase activity at least 10-fold. The jun kinase activity could be characterised as JNK1 and JNK2 at the protein and mRNA level. IL-1, TNF, UV light and osmotic stress, but not PMA, LPS, IL-3, IL-4, IL-6, IL-8, IL-10, IL-13, GM-CSF, PDGF, bFGF, TGF-beta and interferon-gamma were able to stimulate jun kinase activity in HMC, suggesting that jun kinase is selectively mediating signal transduction of the proinflammatory cytokines IL-1 and TNF as well as of cellular stress in HMC.

Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor.

For various forms of human glomerulonephritis a close relationship between inflammatory injury and a local mesangial proliferative response has been described. Herein, we used primary cultures of human glomerular mesangial cells (HMCs) from five different donors to determine the autocrine growth-inducing capacity of their supernatants after stimulation with different cytokines and lipopolysaccharide (LPS) to determine whether this effect is due to basic fibroblast growth factor (bFGF). The basal growth-inducing capacity of supernatants collected from serum-free cultured HMC and concentrated 100-fold above a cut-off size of 10 kd was significantly increased by interleukin (IL)-1 beta, platelet-derived growth factor (PDGF), and LPS up to 15-fold, but not by IL-1 alpha, IL-6, or bFGF. An anti-human bFGF antibody blocked the majority of IL-1 or LPS-induced proliferative effect of supernatants; complete inhibition was achieved by a combination of anti-human bFGF- and anti-human platelet-derived growth factor antibodies. HMCs express different isoforms of bFGF (18, 21.5, and 24 kd) in membrane, cytosolic, and nuclear fractions. All isoforms of bFGF were found in the nuclear fraction of HMC, whether stimulated or not. Immunoblots for bFGF protein of HMC demonstrated that only a approximate to 16 kd bFGF protein was released into HMC supernatants after stimulation with IL-1 beta, platelet-derived growth factor-BB, and LPS. The 18 kd isoform of bFGF accumulated in the membranes but was not released after stimulation with IL-1 alpha, IL-6, and bFGF, suggesting that its release was a prerequisite for autocrine growth stimulation. By means of reverse transcription polymerase chain reaction controlled by Southern blots, bFGF-mRNA expression of HMC was enhanced by IL-1 alpha, IL-1 beta, and LPS. Finally, we were able to show that HMCs are expressing bFGF receptors. In summary, our data demonstrate for the first time that the autocrine proliferative response of HMC to major inflammatory factors may primarily be mediated by bFGF.

[Activation-dependent expression of Fc gamma-receptors on glomerular mesangial cells]. / Aktivierungsabhängige Expression von Fc gamma-Rezeptoren auf glomerulären Mesangialzellen.

Resting, non-cycling mesangial cells (MCs) can be induced by IFN gamma to express the Fc gamma-RIIIa and Fc epsilon RI-gamma subunits of the CD16TM receptor complex. After cell surface expression of CD16TM by IFN gamma induction the binding of immune complexes to MCs induces IL-6 secretion. A Fc epsilon RI-gamma gene knockout has recently been described. It is now possible to study the function of Fc gamma RIIIa and other gamma chain associated Fc receptors in the initiation and progression of chronic glomerular inflammation in the context of the intact immune system in vivo.

Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFc gamma RIII-A) and the associated Fc epsilon RI gamma-chain.

Immune complexes localize to the glomerular mesangium in many forms of human glomerulonephritis. In this investigation, we determined whether primary cultured human glomerular mesangial cells (HMC) express mRNA and functional protein of FcR for IgG. Performing reverse transcription-PCR with subsequent control Southern blots, we showed that in contrast to human monocytes or granulocytes, mRNA for Fc gamma RI and Fc gamma RII is not detectable in either growth-arrested (48 h serum free) or unstimulated proliferating (medium with 10% FCS) HMC. However, IFN-gamma in combination with LPSE.coli (LPS), but not LPS alone elicited a significant transcription of hFc gamma RIII mRNA in resting HMC, whereas cytokines, such as IL-1 beta or TGF-beta, failed to induce any of the three Fc gamma Rs in resting HMC. Proliferating HMC showed a basal transcription of Fc gamma RIII mRNA, which was enhanced by IFN-gamma in combination with LPS. Slot-blot analysis indicated that only the Fc gamma RIII-A gene encoding the transmembrane isoform of Fc gamma RIII was expressed by HMC. For the first time, transcripts for the gamma-chain of Fc epsilon RI were found in HMC. Fc gamma RIII protein expression was detected after LPS/IFN-gamma stimulation both by specific staining of paraformaldehyde-fixed HMC and immunoprecipitation of Fc gamma RIII protein from HMC membranes. Fc gamma RIII-A receptors are functionally active, as HMC IL-6 mRNA synthesis was stimulated by heat-aggregated IgG or F(ab')2 fragments of CLBGran1 mAb only after induction of Fc gamma RIII-A. These in vitro data suggest that HMC that are basically negative for Fc gamma RIII can be stimulated to express low affinity Fc gamma Rs, and thus may participate actively in human glomerulonephritis involving immune complexes.

Activation of autoreactive T-lymphocytes by cultured syngeneic glomerular mesangial cells.

The capacity of intrinsic, glomerular mesangial cells (MC) to cause an autoreactive response of syngeneic lymphocytes in vitro are presented. Initial experiments demonstrated the MHC class II dependent capacity of MC to present exogenous antigen to sensitized lymph node lymphocytes (LN) and to activate naive, allogeneic LN in the absence of a nominal antigen. However, the most striking finding of the present investigation was that mouse MC (C57BL/6 or DBA/2) augmented a significant activation of naive, syngeneic lymphocytes. The extent of the proliferative lymphocyte response was comparable to that observed after stimulation with allogeneic MC. Moreover, during syngeneic coculture substantial amounts of interferon bioactivity were generated. Equipotent concentrations of rm IFN-gamma were sufficient to induce class II MHC expression of mouse MC. In control experiments the macrophage cell line, IC-21 (C57BL/6), or freshly prepared DBA/2 mouse peritoneal macrophages did not elicit a syngeneic LN response. Using MC, which had not been pretreated, the MC-specific LN stimulation occurred after prolonged periods of coculture. The stimulation index (S.I.) was 9.77 after 144 hours compared with LN controls (S.I. = 1). However, a 48 hour pretreatment of MC with either rm IFN-gamma alone or in combination with rh TNF-alpha and/or the continuous presence of rm IL-1 alpha during coculture periods from 72 to 144 hours substantially enhanced the proliferative LN response. Analysis of non-adherent LN by flow cytometry (FACS) after 96 or 120 hours coculture with MC revealed an increased ratio of Thy1.2+ to B220+ cells with a predominant rise of L3T4+ T-helper cells compared to Lyt2+ cytotoxic T-cells. Furthermore, immune fluorescence microscopy showed that a fraction of Thy1.2+ lymphoblasts adhered to MC. FACS analysis of these adherent LN after detachment demonstrated that in comparison to cocultures with untreated MC, cocultures of LN with IFN-gamma/TNF-alpha pre-treated MC resulted in a 24.4% increase of Thy1.2+ cells, with 89% of these being L3T4+ T-helper lymphocytes. In conclusion, autoreactivity of preferentially T-helper cells to cocultured glomerular MC was shown, which may represent a useful model of T-lymphocyte dependent glomerulonephritis.

Analysis of CD16+dim and CD16+bright lymphocytes--comparison of peripheral and clonal non-MHC-restricted T cells and NK cells.

The Fc gamma RIII receptor (CD16) has been described on natural killer cells and a small subset of T lymphocytes. CD16+bright lymphocytes represent the typical population of peripheral blood CD3- NK cells. In these studies in addition to CD16+bright NK cells Fc gamma RIII expressing cytotoxic T lymphocytes in peripheral blood from one healthy individual are characterized as CD16+dim non-MHC-restricted CTLs either expressing the alpha/beta (80%) or the gamma/delta T cell receptor (20%). Both CD16+ subsets are clearly distinct in their functional capacity performing NK and ADCC activity. Freshly isolated CD16+dim T cells exert higher ADCC, CD16+bright NK cells higher NK activity. They are also differentially activated by interleukin-2 since CD16+bright NK cells reveal a bright expression of the p75 IL-2 receptor beta-chain in contrast to the very low p75 expression on CD16+dim T cells. This activation leads to a gradual increase of ADCC by NK cells. Finally the CD16 expression pattern with low and bright intensity represents a stable phenotype expressed by clones generated from these different subpopulations. On a clonal level CD16+dim non-MHC-restricted T cells can be distinguished from CD16+bright NK cells by their lower capacity in NK killing, but they are equally potent in ADCC. Finally these CD3+CD16+dim clones provide the basis for studies of Fc gamma RIII and TcR interaction.

Fc gamma RIII activation is different in CD16+ cytotoxic T lymphocytes and natural killer cells.

The transmembrane protein CD16 (Fc gamma RIII) is detected on activated macrophages, natural killer (NK) cells and a small subset of T lymphocytes. From CD3-CD56+ CD16+bright NK cells and CD3+ CD56+ CD16+dim non-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) clones were generated reflecting the stable, but different, CD16 expression of the respective peripheral blood subpopulations. To compare the role of CD16 on NK cells and non-MHC-restricted CTL, Fc gamma RIII activation and its mechanisms were investigated using monoclonal antibodies (mAb). Cross-linking of CD16 induced Ca2+ influx in CD16+bright NK clones. In contrast, there was no Ca2+ mobilization after CD16 activation in CD16+dim CTL, which revealed a good response to cross-linking of CD3 antigen. Pretreatment with CD16 mAb alone or cross-linked CD16 mAb did not block the CD3 response of CD16+dim CTL. Again, CD16 cross-linking induced more interferon-gamma transcription in NK cell clones than in non-MHC-restricted CTL clones. Also a higher tumor necrosis factor-alpha production of NK clones after CD16 cross-linking compared to CD16+dim CTL could be observed. These data suggest that after CD16 activation CD16+dim CTL and CD16+bright NK cells use different second messengers. In addition, signal transduction via CD3 and CD16 appears to function independently in CD16+dim non-MHC-restricted CTL.

Differences in activation of normal and glycosylphosphatidylinositol-negative lymphocytes derived from patients with paroxysmal nocturnal hemoglobinuria.

In these studies, the role of glycosylphosphatidylinositol (GPI)-anchored surface molecules during T cell activation was investigated in fresh T cells and T cell lines obtained from patients with paroxysmal nocturnal hemoglobinuria. For control, GPI-expressing T cells of the same patients were used. Unstimulated GPI- T cells exhibited significantly reduced surface expression of the activation Ag CD45R0, compared with GPI+ T cells. In addition, in measurements of proliferation, IFN-gamma production, and induction of second messengers such as cytoplasmic Ca2+, CD48- lymphocytes showed a similar response to TCR-specific stimulation, compared with CD48+ lymphocytes. In contrast, stimulation with the lectin PHA produced a decreased response of CD48- lymphocytes in these functions. In addition, stimulation with cross-linked CD59 mAb increased the proliferation of GPI-molecule expressing CD48+ T cell lines only. From these data, it can be concluded that GPI-anchored surface molecules play an important role in T lymphocyte activation.

Diagnosis of paroxysmal nocturnal haemoglobinuria using immunophenotyping of peripheral blood cells.

Paroxysmal nocturnal haemoglobinuria (PNH) is now generally accepted as a disease in which bone marrow derived cells are deficient in phosphatidylinositolglycan (PIG)-anchored surface molecules. A series of new monoclonal antibodies detecting PIG-anchored surface structures on human leucocytes (CD48, CD55, CD59) has recently been described. In the present study 12 patients with the diagnosis PNH and a positive Ham test were examined for PIG-anchored surface antigen expression on various cell lineages using immunofluorescence. In all patients deficient cells were detected in erythrocyte, granulocyte and monocyte analysis. A deficient lymphocyte subset was also observed in all but one of these patients. Using two-colour analysis, all lymphocyte subpopulations such as T, B and NK cells were found to be affected. In addition, peripheral blood cells of 22 patients with severe aplastic anaemia (SAA) were tested for the PIG-anchoring defect. In five of these patients the defect was detected, and in four of the five the lack of PIG-anchored molecules was confined to the granulocyte and monocyte lineages apparently without affecting the erythrocytes. The results of these studies demonstrate that cytofluorographic testing of peripheral blood cells provides a simple and reliable method for establishing the diagnosis of PNH. Furthermore, especially in the case of aplastic anaemia patients, the sensitivity of immunophenotyping might be superior to conventional laboratory tests.

The PIG-anchoring defect in NK lymphocytes of PNH patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is clinically characterized by intravascular hemolysis, hemoglobinuria, iron deficiency anemia, and venous thrombosis. Pathophysiologically the disease has now been generally accepted as an acquired defect of phosphatidylinositol glycan (PIG)-anchored molecules on the cell surface of bone marrow-derived cells. This defect is functionally characterized by an abnormal susceptibility to complement-mediated lysis and has been described on erythrocytes, granulocytes, monocytes, and platelets. In contrast, contradictory data exist so far on the involvement of lymphocytes and natural killer (NK) cells. Using monoclonal antibodies (MoAbs) against newly defined PIG-linked surface structures such as CD48, CD55, and CD59, which are homogeneously expressed on lymphocytes of normal donors, we analyzed lymphocytes and their subpopulations in nine PNH patients by two color immunofluorescence. Our results showed that CD3+ T cells as well as CD16+ NK cells are at least partially involved in the deficient PIG-molecule surface expression. To more clearly define the defect in PNH, we generated NK clones from a PNH patient. Phenotypic analysis of these NK clones showed that they either were positive (n = 3) for PIG-linked surface structures such as CD48, CD55, and CD59 (eg, NKP1) or were completely negative (n = 7) for all of them (eg, NKP1). In functional tests the PIG-molecule negative clone NKP2 showed increased susceptibility to human complement compared with the PIG molecule positive clone NKP1. When analyzing the mRNA levels of the PIG-linked molecules CD55 and CD59 there was no difference at all between the two clones. We conclude from our data that NK cells as well as other lymphocyte subpopulations are involved in the PIG-linkage defect of PNH. These NK clones with differential expression of PIG-linked surface structures present for the first time ex vivo mutant cell lymphocyte lines that carry the defect leading to PIG deficiency in PNH.

[The PIG-anchoring defect of lymphocyte populations in paroxysmal nocturnal hemoglobinuria]. / Der PIG-Verankerungsdefekt auf Lymphozytenpopulationen bei paroxysmaler nächtlicher Hämoglobinurie.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a defect expression of phosphatidylinositolglycan-(PIG-)linked surface molecules. This deficiency has been demonstrated for cells of myeloid origin so far, but not for lymphocytes. In these studies we examined the defect on lymphocyte subpopulations.

Activation of cloned human natural killer cells via Fc gamma RIII.

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.