Portal and systemic serum growth factor and acute-phase response after laparotomy or partial hepatectomy in patients with colorectal liver metastases: a prognostic role for C-reactive protein and hepatocyte growth factor.

BACKGROUND: Growth factors play a role in wound healing and tumour growth. The aim of this study was to compare the effect of partial hepatectomy (PH) and laparotomy on serum levels of growth factors and acute-phase proteins in patients with colorectal liver metastases and to correlate these levels with prognosis after PH. METHODS: Epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin like growth factor-I (IGF-I), insulin, interleukin-6 (IL-6), C-reactive protein (CRP) and serum amyloid-A (SAA) were determined in portal and systemic serum in 24 PH patients and 9 laparotomy patients. RESULTS: No differences were found in the clinicopathological characteristics of PH and laparotomy patients with the exception of the number of metastases, blood loss and operation time. The response of SAA, CRP and IGF-I was lower in PH patients than in laparotomy patients (P < 0.02). PH was associated with a higher IL-6 (P = 0.02) and HGF (P = 0.055) response than laparotomy. A higher HGF and CRP response was associated with a poorer prognosis. Total IGF-I was negatively correlated with the resected liver volume (r = -0.48, P < 0.05). CONCLUSIONS: PH is associated with a lower acute-phase and total IGF-I response and a higher HGF and IL-6 response compared with laparotomy. HGF and CRP responses had an influence on the prognosis.

Diagnostic performance of the variant lymphocyte flag of the Abbott Cell-Dyn 4000 haematology analyser.

BACKGROUND: In addition to differential cell counts, modern haematology analysers generate suspect flags if abnormal cells are detected. Reports on validation of suspect flags are scarce. We have routine experience with the Abbott Cell-Dyn 4000 analyser for over 5 years and have previously demonstrated the utility of the blast flag. Here we report a similar study on the performance of the analyser's Variant Lymphocyte (VL) flag. AIM OF THE STUDY: Evaluation of the diagnostic performance of the Cell-Dyn 4000 VL flag, as compared with lymphocyte morphology in blood smears. In addition, we investigated the usefulness of the numerical VL flag confidence index as provided by the analyser. MATERIALS AND METHODS: All samples generating a VL flag were reviewed over a 5-month period. We also reviewed smears from patients with known lymphoid disorders, even if the analyser did not flag the sample. Two experienced investigators assessed lymphocyte morphology independently. RESULTS: In total, 187 samples were included in the study, of which 183 had a VL flag and four had not. Of the 183 flagged samples, 83 appeared to have abnormal lymphocyte morphology and 100, normal lymphocyte morphology. The sensitivity of the VL flag for detecting abnormal lymphocytes was 0.95 and the positive predictive value was 0.44. Using ROC analysis of the VL flag confidence index, the area under the ROC curve was 0.58 (95% confidence interval 0.50-0.65). CONCLUSIONS: The Cell-Dyn VL flag has reasonable sensitivity but a high false-positive rate. In addition, its performance is insufficient for detecting clinically relevant abnormal lymphocytes. As the VL flag appeared to rely mainly on numerical criteria, it has no added value over numerical criteria defined by the laboratory.

Performance characteristics of blast flagging on the Cell Dyn 4000 haematology analyser.

This study investigated the performance characteristics of blast flagging on the Abbott Cell Dyn 4000 (CD4000) haematology analyser. Our special interest was focused on the usability and prognostic value of the confidence fraction (CF), accompanying blast flagging. From our routine patient samples, 100 with blast flagging on the CD4000 were selected and examined microscopically for the presence of blasts. Blast flagging on the CD4000 resulted in a high number of false positive events. The results indicate that the blast flag confidence fraction (CF) of the CD4000 can be useful as a parameter for the prediction of the presence of blasts in samples with normal and increased white blood cell count. A model with criteria for blood smear examination following blast flagging was developed, using the blast flag CF combined with the WBC count. The model was tested and validated in a new set of 100 samples with blast flagging. Implementation of the criteria of the model resulted in a total reduction of the number of microscopic smear examinations of 45%, without losing clinically significant sensitivity.

Hybridization characteristics of biomolecular adaptors, covalent DNA--streptavidin conjugates.

Semisynthetic, covalent streptavidin-DNA adducts are versatile molecular connectors for the fabrication of both nano- and microstructured protein arrays by use of DNA hybridization. In this study, the hybridization characteristics of six adduct species, each containing a different DNA sequence of 21 or 24 bases, have been compared. First, the adducts were conjugated to biotinylated alkaline phosphatase, and their binding to immobilized oligomer complements of similar lengths was quantified in a microplate assay. The binding efficiency observed varied to a great extent with the specific sequence of the oligonucleotide attached, and could not be predicted from affiliated thermodynamic data of duplex stability. To further elucidate the hybridization properties, the hybridization rate constants of association and dissociation (kassn and kdissn) have been determined for both unconjugated oligonucleotides and protein adducts, using a surface plasmon resonance biosensor. The kassn values observed for the oligonucleotides are in the range of 9 x 10(3) to 2 x 10(5) M[-1] s(-1) and correlate with structural properties of the probe strands. Up to 3-fold decreased kassn values were obtained for the corresponding protein adducts. Likewise, values were observed for kdissn ranging from 1.4 x 10(-4) to 1.9 x 10(-5) s[-1] for the oligonucleotides. The dissociation of the analogous protein conjugates was reduced by up to 5-fold. The extent of this decrease correlates with the formation of homodimeric or intramolecular aggregation of probe strands. A mechanistic model for explaining these data is based on attractive intramolecular interaction between the nucleic acid and protein moiety.

Heterogeneity in secretory responses of rat liver macrophages of different size.

Four subpopulations of hepatic macrophages, differing in size, were isolated from rat liver. The secretion of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E (PGE) and interleukin-1 (IL-1) by freshly isolated as well as cultured cells was studied after in vitro stimulation with the immunomodulator muramyl dipeptide (MDP) in free or liposome-encapsulated form. Freshly isolated liver macrophages could be induced to secrete significant levels of NO, TNF-alpha, PGE and IL-1. The extent of secretion, however, varied substantially between macrophages of different size. The highest levels of secretion of TNF-alpha, PGE and IL-1 were observed in the fraction containing the large-size macrophages, while progressively lower levels of secretion were observed with decreasing size. In contrast, the highest levels of NO secretion were observed by small macrophages and steadily decreased with increasing size. Hepatic macrophages of different size displayed differences in secretory potential during in vitro culture. The ability of small liver macrophages to secrete NO, TNF-alpha, or PGE, following activation with MDP, gradually increased with time in culture. In contrast, large liver macrophages gradually lost their secretory ability after 1-2 days and the intermediate-size cells after 2-3 days in culture. This functional heterogeneity in secretory properties among rat liver macrophages of different size is discussed with reference to their potential role and significance in host defense against metastatic tumor growth in the liver.

Histochemical and electron microscopic characterization of hepatic macrophage subfractions isolated from normal and liposomal muramyl dipeptide treated rats.

Subfractions of the hepatic macrophage population, differing in cell size, were isolated from normal rats and rats treated with liposomal muramyl dipeptide (lipMDP) and analyzed histochemically and by ultrastructural peroxidase cytochemistry. The majority of cells in all subfractions of control rats displayed the ultrastructural endogenous peroxidase pattern of resident liver macrophages and showed positive staining with the general macrophage markers nonspecific esterase (NSE) and monoclonal antibody ED1. Heterogeneity in intensity of NSE and ED1 staining was observed among macrophages of different size. Generally, the intensity of NSE and ED1 staining decreased with decreasing cell size. After injection of lipMDP, we observed the appearance of a discrete subpopulation of cells in the liver in addition to the resident macrophages. These cells, containing a nucleus with a characteristic shape, were predominantly recovered in the small-sized fractions and were characterized by an immature ultrastructural macrophage morphology (no or only a few lysosomes and phagosomes) and a lack of ED1 reactivity, NSE, and endogenous peroxidase. We suggest an important role for these cells in lipMDP induced antitumor capacity of the liver.

Secretion pattern of the rat liver macrophage population following activation with liposomal muramyl dipeptide in vivo and in vitro.

Incubation of rat liver macrophages in vitro with free or liposome-encapsulated muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP) resulted in a rapid but transient release of tumor necrosis factor-alpha (TNF-alpha), followed by a slow, steady release of nitric oxide (NO) and prostaglandin (PG) E. The secretion pattern induced in situ was determined by isolating the liver macrophages at 2, 12, 24, 48, and 72 h after injection of liposomal MDP and measuring the amounts of products secreted in a 24-h period following isolation. TNF-alpha secretion was detected only in macrophages isolated as early as 2 h after injection of liposomal MDP and not at later time points. Considerable heterogeneity was observed among macrophages of different size: For example, the large-sized cells were far more potent in TNF secretion than the smaller cells. Nitric oxide secretion, on the other hand, was maintained over a full 24-h period following MDP administration and was virtually independent of macrophage size. With regard to PGE release, similar to TNF-alpha secretion, considerable differences in secretory activity between cells of different size were observed. Also in this case the large cells were several times more active than the small cells. In contrast to TNF, however, PGE secretion could be detected up to 24 h after injection of liposomal MDP. These findings support the notion that the development and maintenance of the activated state of liver macrophages, induced by immunomodulators such as liposomal MDP, are under the control of a complex network of regulatory functions and that multiple secretory products play a role in the observed macrophage-mediated cytotoxicity.

Proliferation of rat liver macrophages in vitro: influence of hemopoietic growth factors.

We examined the effects of several hemopoietic growth factors on proliferation of rat liver macrophages in vitro. The proliferative response of liver macrophages to hemopoietic growth factors was assayed on the basis of [methyl-3H]thymidine uptake. Macrophage colony-stimulating factor and recombinant murine granulocyte-macrophage colony-stimulating factor stimulated [methyl-3H]thymidine incorporation in a concentration-dependent manner. With granulocyte-macrophage colony-stimulating factor, maximum incorporation was observed at 50 U/ml, whereas with macrophage colony-stimulating factor no incorporation plateau was observed up to 50% L929-conditioned medium. Incubation of liver macrophages with various concentrations of recombinant human interleukin-2, recombinant murine interleukin-3 and recombinant human interleukin-6 or culture medium alone did not result in significant incorporation of [methyl-3H]thymidine. When liver macrophages were fractionated according to cell size, highest incorporation was observed in the large macrophages. Proliferating cells in cultures of all subfractions were microscopically identified as typical macrophages by the use of macrophage-specific monoclonal antibodies. After 6 days in culture, these macrophages had functional properties similar to those of resident liver macrophages with respect to phagocytosis and in vitro activation with immunomodulators to tumorcytotoxicity and secretion of nitric oxide and tumor necrosis factor-alpha. These results suggest that macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor play important roles among the regulatory factors that support local proliferation of rat liver macrophages.

Functional characteristics of the rat liver macrophage population after a single intravenous injection of liposome-encapsulated muramyl peptides.

In this study, we investigated different functional characteristics of the rat liver macrophage population after a single i.v. injection of liposome-encapsulated muramyl dipeptide (MDP) or its lipophilic derivative muramyl tripeptide-phosphatidylethanolamine (MTP-PE). The in situ induced tumoricidal activity of the liver macrophage population was determined in vitro against C26 colon adenocarcinoma cells. For investigating which cells are responsible for the observed cytotoxic effects, subfractions of the liver macrophage population, differing in cell size, were isolated at different intervals after injection of the liposomal muramyl peptides. From these subfractions, the number of cells, degree of cytotoxicity, and the response to an additional activation with free MDP in vitro were determined. Maximal induction of tumoricidal activity of the liver macrophage population was reached between 12 and 24 hours after injection of liposomal MDP, while no significant differences between the subfractions were observed. Heterogeneity of tumor cytolytic capacity was observed in subfractions of macrophages isolated at 2 and 48 hours after injection. At these time points, highest cytolytic activity was observed for the small to intermediate-size macrophages. No significant cytotoxicity was detectable in any subfraction 72 hours after injection of liposomal MDP. An identical pattern of macrophage tumoricidal activity was observed after injection of liposomal MTP-PE, although slightly lower cytotoxicity levels were found. When isolated during the first 12 hours after injection of liposomal MDP, the macrophage population was unable to respond to a subsequent in vitro exposure to MDP, with respect to tumor cytotoxicity. Twenty-four and 48 hours after injection, the smallest cells could be slightly reactivated, whereas the larger cells still remained unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)