Diffusion weighted imaging of the breast: Performance of standardized breast tumor tissue selection methods in clinical decision making.

OBJECTIVES: In breast diffusion weighted imaging (DWI) protocol standardization, it is recently shown that no breast tumor tissue selection (BTTS) method outperformed the others. The purpose of this study is to analyze the feasibility of three fixed-size breast tumor tissue selection (BTTS) methods based on the reproducibility, accuracy and time-measurement in comparison to the largest oval and manual delineation in breast diffusion weighted imaging data. METHODS: This study is performed with a consecutive dataset of 116 breast lesions (98 malignant) of at least 1.0 cm, scanned in accordance with the EUSOBI breast DWI working group recommendations. Reproducibility of the maximum size manual (BTTS1) and of the maximal size round/oval (BTTS2) methods were compared with three smaller fixed-size circular BTTS methods in the middle of each lesion (BTTS3, 0.12 cm3 volume) and at lowest apparent diffusion coefficient (ADC) (BTTS4, 0.12 cm3; BTTS5, 0.24 cm3). Mean ADC values, intraclass-correlation-coefficients (ICCs), area under the curve (AUC) and measurement times (sec) of the 5 BTTS methods were assessed by two observers. RESULTS: Excellent inter- and intra-observer agreement was found for any BTTS (with ICC 0.88-0.92 and 0.92-0.94, respectively). Significant difference in ADCmean between any pair of BTTS methods was shown (p = <0.001-0.009), except for BTTS2 vs. BTTS3 for observer 1 (p = 0.10). AUCs were comparable between BTTS methods, with highest AUC for BTTS2 (0.89-0.91) and lowest for BTTS4 (0.76-0.85). However, as an indicator of clinical feasibility, BTTS2-3 showed shortest measurement times (10-15 sec) compared to BTTS1, 4-5 (19-39 sec). CONCLUSION: The performance of fixed-size BTTS methods, as a potential tool for clinical decision making, shows equal AUC but shorter ADC measurement time compared to manual or oval whole lesion measurements. The advantage of a fixed size BTTS method is the excellent reproducibility. A central fixed breast tumor tissue volume of 0.12 cm3 is the most feasible method for use in clinical practice.

A Subset of Malignant Mesothelioma Tumors Retain Osteogenic Potential.

Malignant mesothelioma (MM) is an aggressive serosal tumor associated with asbestos exposure. We previously demonstrated that mesothelial cells differentiate into cells of different mesenchymal lineages and hypothesize that osseous tissue observed in a subset of MM patients is due to local differentiation of MM cells. In this study, the capacity of human and mouse MM cells to differentiate into osteoblast-like cells was determined in vitro using a functional model of bone nodule formation and in vivo using an established model of MM. Human and murine MM cell lines cultured in osteogenic medium expressed alkaline phosphatase and formed mineralized bone-like nodules. Several human and mouse MM cell lines also expressed a number of osteoblast phenotype markers, including runt-related transcription factor 2 (RUNX2), osteopontin, osteonectin and bone sialoprotein mRNA and protein. Histological analysis of murine MM tumors identified areas of ossification within the tumor, similar to those observed in human MM biopsies. These data demonstrate the ability of MM to differentiate into another mesenchymal cell type and suggest that MM cells may contribute to the formation of the heterologous elements observed in MM tumors.

The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor.

Malignant mesothelioma (MM) is a solid tumor largely unresponsive to conventional therapies. Immunological gene therapy shows promise in murine models and human clinical trials; however, the role of surgery in combination with gene therapy has not been widely studied. The aim of this study was to determine if debulking surgery improved the effectiveness of gene therapy in a murine MM model. Mice were subcutaneously inoculated with the MM cell line, AC29, at two different sites, 4 days apart, to allow a surgical and distal site tumor to develop. Once tumors were established, the surgical site tumor was debulked and vaccination of syngeneic tumor transfectants encoding genes for IL-4, IL-2, GM-CSF, B7-1 or allogeneic MHC molecules commenced at a site away from both tumors, and tumor growth was measured. Neither debulking surgery nor gene therapy alone delayed tumor growth. However, there was a clear delay of tumor growth when debulking surgery was combined with vaccination of tumor transfectants expressing B7-1 or high levels of GM-CSF. Combinations of these two transfectants did not lead to a synergistic effect. This study demonstrates that debulking surgery can augment the immunostimulatory effects of immunological gene therapy and can delay tumor growth. This has implications for the future design of human gene therapy trials for solid tumors such as MM.

Environmental scanning electron microscopic study of macrophages associated with the tunica vasculosa lentis in the developing rat eye.

AIMS: To demonstrate the value of environmental scanning electron microscopy (ESEM) when used in combination with immunogold/silver enhancement methods as a valuable tool in ocular research, and to determine the phenotype of macrophages associated with the tunica vasculosa lentis while maintaining a topographical view of the lens surface. METHODS: Prenatal and postnatal rat eyes were investigated by conventional scanning electron microscopy and ESEM. In the latter case tissues were prestained with a panel of antileucocyte monoclonal antibodies and visualised with colloidal gold conjugated secondary antibody followed by silver enhancement. RESULTS: The preliminary data demonstrate that ED1(+) ED2(+) macrophages occur in large numbers around the lens and are associated with sectors of both normal vessels and those undergoing regression. CONCLUSION: The present study demonstrates that ESEM is an ideal way to combine scanning electron microscopy with immunohistochemistry and is therefore likely to have multiple other applications in ocular research.

Quantitative investigations of apoptosis of murine mononuclear phagocytes during mild hyperthermia.

DNA fragmentation in murine monocytes and freshly harvested resident peritoneal macrophages (RPM) was assessed. Similar proportions of fragmented DNA were present in both cell types indicating that apoptosis is a significant occurrence in these cells and may provide a mechanism for controlling the numbers of mononuclear phagocytes in the tissues that house them. Culturing both of these cell types at 41 degrees C for 5 hr revealed a significant increase in the proportion of fragmented DNA only in cultures containing RPM. These latter preparations when cytophotometrically assessed also displayed reduced Feulgen DNA and cytoplasmic naphthol yellow S staining, again confirming the presence of apoptosis. It is suggested that the induction of apoptosis by mild hyperthermia may therefore be due to the relative absence of a temperature-sensitive inhibitor of apoptosis in a significant proportion of resident peritoneal macrophages. Lastly, electron microscope assessment indicated that the apoptotic macrophages were engulfed by their nonaffected neighbors. Programmed cell death in macrophage populations followed by phagocytosis of the apoptotic cells may therefore be a mechanism for controlling macrophage numbers in mammalian organisms.

The effect of mild hyperthermia on the morphology and function of murine resident peritoneal macrophages.

During short term culture of murine resident peritoneal macrophages, increasing the temperature from 37 to 39 degrees C resulted in an increased activity of several surface receptors (FcR and receptor for gluteraldehyde-fixed sheep red blood cells), enhanced phagocytosis of yeast particles, improved spreading, and an accelerated reduction of nitroblue tetrazolium. At 41 degrees C, however, significant reduction of several functional properties (endocytosis of colloidal gold and horseradish peroxidase, phagocytosis of yeast particles) and a decrease in the reduction of nitroblue tetrazolium, the incorporation of tritiated uridine, and Fc and C3b surface receptor activity were observed. In addition morphological evidence of apoptosis, observed in a small number of cells cultured at 39 degrees C and in the majority of macrophages maintained at 41 degrees C, was confirmed by DNA electrophoresis. The data indicates that a reduction of several functional activities of macrophages occurs at 41 degrees C and apoptosis may largely account for these effects.

Morphological and functional changes during cytomegalovirus replication in murine macrophages.

Structural and functional changes were studied in murine peritoneal macrophages infected with murine cytomegalovirus by using centrifugal enhancement to achieve a high-level (greater than 90%) pulsed infection. During 3 d of culture the infected cells became enlarged and rounded with smooth surface contours. Transmission electron microscopy demonstrated various stages of viral maturation in the nucleus and cytoplasm. Intracellular organization was generally retained, apart from the development of large, irregular, intracytoplasmic vacuoles, in which enveloped virions and cell debris accumulated. The infected macrophages lost most surface markers tested (F4/80, Mac-1, FcR, and the receptor for gluteraldehyde-fixed sheep red blood cells), but H-2 expression was increased. Moreover, ingestion of colloidal gold or horseradish peroxidase was depressed, and the levels of acid phosphatase activity, lymphocytostatic activity, and interleukin 1 production were also decreased. The latter may explain the observed loss of accessory cell function.

The effects of malnutrition on murine peritoneal macrophages.

Differences were detected between peritoneal macrophages (both resident and elicited) from mice on a low protein diet and from normal animals. The concentration of resident peritoneal macrophages was lower in animals on low protein diets than in normal controls. Although total protein (and therefore cell mass) of resident macrophages from malnourished mice was increased, their contents of thiamine pyrophosphatase, succinate dehydrogenase, and non-specific esterase were disproportionately reduced. In addition they did not ingest as many glutaraldehyde-fixed sheep erythrocytes or attach to as many adherent C3b sensitized sheep red blood cells as those from normal animals, although reduction of nitroblue tetrazolium was unaffected. Initially (24 hr after thioglycollate), elicited macrophages from malnourished mice did not divide as frequently as those from normal mice but by 48 hr the differences were insignificant. The elicited macrophage possessed lower levels of total protein (indicating a reduced cell mass); the levels of acid phosphatase, thiamine pyrophosphatase, succinate dehydrogenase, and nonspecific esterase and nitroblue reducing activity were also proportionately reduced. They ingested fewer glutaraldehyde-fixed erythrocytes and reacted with fewer C3b sensitised sheep red blood cells than those from normal mice; ingestion of IgG-coated sheep erythrocytes, on the other hand, was somewhat increased. These abnormalities may influence adversely the efficiency of early phlogistic responses and favor the establishment of infection in malnourished animals.

Bupivacaine-induced muscle injury. The role of extracellular calcium.

To investigate the role of extracellular calcium in bupivacaine-induced muscle injury, the effects of the drug on creatine kinase (CK) release and muscle ultrastructure were studied in the isolated rat soleus in the presence and absence of calcium and of the Ca-channel blockers verapamil and nifedipine. Control muscles maintained a constant CK release rate and normal morphology for at least 3 hours. CK release rates increased markedly after exposure to 1.5 mM and 5 mM bupivacaine and electron microscopy showed evidence of damage to mitochondria, sarcoplasmic reticulum and the plasmalemma of muscle fibres with disruption of the Z-lines, I-bands and M-lines of myofibrils. When calcium was omitted from the incubation medium, there was a 3-fold reduction in CK release rates; the morphological changes were less severe initially but by 120 min were comparable to those in muscles incubated with bupivacaine and calcium. Neither 10(-6) M verapamil nor 10(-6) M nifedipine reduced CK release or muscle fibre damage. Verapamil (10(-5) M) reduced CK release but not the severity of muscle damage. The findings indicate that extracellular calcium plays a part in mediating the muscle damage caused by bupivacaine but that other factors must also be involved, and that Ca-channel blockers do not prevent muscle damage.

Evidence that multinucleate giant cells are examples of mononuclear phagocytic differentiation.

Murine multinucleate giant cells and mononuclear phagocytes were examined with various quantitative cytochemical and autoradiographic techniques. No evidence was found that multinucleate giant cells were metabolically effete, in fact they compared favourably with mononuclear phagocytes. In addition, rat multinucleate giant cells consistently expressed surface Ia antigens and to a lesser degree fibronectin. It is suggested that multinucleate giant cells are a differentiated derivative of the mononuclear phagocytic system.

The kinetics of mononuclear phagocyte infiltration in neoplasms: reactions to non-specific stimuli in a rat fibrosarcoma.

The kinetics of macrophage infiltration into a transplantable rat fibrosarcoma were investigated. Monocytes obtained from normal rats and labelled with 51Cr were injected into normal or fibrosarcoma-bearing rats which had previously been implanted with cotton pellets impregnated with BCG, B. pertussis carrageenan or levan. The subcutaneous tissues of normal and fibrosarcoma-bearing rats as well as the neoplasm itself were the sites for pellet implantation. The various additives induced an enhancement of macrophage infiltration into cotton pellets implanted for 1 week into subcutaneous tissues of normal rats. No significant effect was found in pellets implanted for 3 or 14 days. Macrophage infiltration into pellets in the neoplasm was consistently reduced when compared with controls. This occurred regardless of the length of time of implantation, the type of substance added to the pellet or (in the case of BCG or B. pertussis) whether the rats were pre-sensitised to the pellet additive or not. Pellets removed from the subcutaneous tissues of fibrosarcoma-bearing rats were either similar to or more often higher in radioactivity than those implanted into the neoplasm itself. The results indicate that the capacity of macrophages to respond to inflammatory stimuli is reduced in fibrosarcoma-bearing rats, but especially in the stroma of the neoplasm.

Cytophotometric assessment of phagocytosis and pinocytosis on single resident and exudate murine peritoneal macrophages.

Computerised scanning cytophotometry was used to evaluate the phagocytic and pinocytic performance of single resident and exudate macrophages. The results indicate that both receptor mediated phagocytosis and fluid phase pinocytosis are more efficiently performed by activated than resident murine peritoneal macrophages. Exudate macrophages phagocytise at approximately twice the rate of resident macrophages while pinocytosis is five times more prominent in exudate than in resident macrophages. The results are consistent with other published investigations and in addition reflect individual cellular variation in macrophage populations.

A quantitative cytochemical analysis of resident and exudate macrophages.

Quantitative cytochemical investigations have detected individual variations between murine peritoneal macrophages and have shown distinct difference between resident and exudate populations. The latter generally contain greater amounts of protein, RNA, acid phosphatase, succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and NADH dehydrogenase. On te other hand, no differences were detected in the cellular content of DNA, not-specific esterase, and NADPH dehydrogenase. In many instances they reflect the biochemical findings of other investigators including the stimulation of glycolysis, tricarboxylic acid cycle and hexose monophosphate shunt pathways, which can occur in elicited or activated macrophages. Although cytochemical differences between the two populations exist, it cannot be stated whether they represent distinct cell lines or different functional states of the same cell population.

A cytogenetic and cytophotometric analysis of resident and exudate murine macrophages.

Cytogenetic analysis and Feulgen--DNA cytophotometry were performed on murine resident and exudate peritoneal macrophages. A large proportion (14--36 per cent.) of metaphase profiles from cell populations rich in resident macrophages displayed chromosomal aberrations compared with 0--8 per cent. seen in metaphases from cell populations rich in exudate macrophages. The most common aberration observed was the presence of chromosomal gaps. It is suggested that should the resident macrophage represent a later, and perhaps terminal stage of macrophage development, the high frequency of aberrations found may be a result of cell ageing.

Effects of pre-irradiation on isogeneic and semi-isogeneic CFU growth: a study on genetic resistance.

The genetic resistance to a parental bone marrow transplant as demonstrated, when transplantation was performed early after irradiation, failed to occur if the interval between irradiation and transplantation was increased to 4 days. A similar radiation induced weakening of genetic resistance to a parental bone marrow graft in spleen and bone marrow could be demonstrated in mice, which had been irradiated with a sublethal dose at 7 days prior to the lethal irradiation and transplantation. The pre-irradiation of the recipient with a sublethal dose induced an enhancement of the growth in spleen and bone marrow of isogeneic transplanted CFU. The pre-irradiation of a single tibia also resulted in a significant weakening of the resistance in the spleen. The experiments with partial body pre-irradiation suggested a local effect of the pre-irradiation, but it could be shown that the enhanced CFU growth is not caused by an enhanced seeding of CFU in pre-irradiated bone marrow. The role of microenvironment in the phenomenon of genetic resistance is discussed.

Weakening of genetic resistance. II. The effect of injection of the macrophage toxic agents silica and carragheenan and the cytostatic drugs cyclophosphamide, busulphan and vinblastin.

Silica (4 mg/i.v.) and Carragheenan (20 mg/i.p.) were shown to be capable of weakening hybrid resistance in the spleen whereas no improved growth was observed in the femoral bone marrow. Histological data did not indicate that the agents in the doses used caused significant macrophage mortality. It was concluded that the weakening effect of the agents was not due to death of the effector cells but possibly to a stimulation of the mononuclear phagocytic system in the spleen. Cyclophosphamide (250 mg/kg) induced a weakening of hybrid resistance which was observed both in the spleen and the femoral bone marrow. Experiments with the alkylating agent Busulphan and with Vinblastin showed that both agents were only capable of inducing a moderate stimulation of growth of the transplant in the spleen. A direct specific influence of cyclophosphamide on the microenvironment was proposed. Xenogeneic resistance was studied by transplantation of rat bone marrow to mice. Silica and cyclophosphamide were shown to be capable of inducing a weakening of the resistance in the spleen. The development of the graft in the femoral bone marrow did not appear to be affected by the administration of silica or cyclophosphamide.

Weakening of genetic resistance. I. The effect of injection of endotoxin, Freund's complete adjuvant and alloantiserum.

A parent to F1 transplantation combination was used to study the weakening effect of endotoxin, Freund's complete adjuvant and alloantiserum on genetic resistance. The relationship between time of treatment with endotoxin, Salmonella typhosa, and Freund's complete adjuvant, and their weakening effect was assessed by use of the spleen colony technique. CFU growth studies revealed that both endotoxin and alloantiserum were capable of weakening genetic resistance in the spleen but were unable to induce weakening of the resistance in the femoral marrow cavity. These results led us to the conclusion that the agents might not have a direct effect on the effector cells of the resistance. The weakening induced by endotoxin and alloantiserum seemed to be related to a certain immunological phenomenon in the spleen. In this phenomenon macrophages are likely to play a role since a number of agents capable of weakening resistance were known for their capacity to influence the mononuclear phagocytic system.

The influence of genetic resistance on CFU growth kinetics in spleen and femur.

An impaired colony formation of C57BL marrow cells transplanted into F1 (C57BL TIMES CBA) mice was observed. In accordance with the literature this phenomenon has been designated as "genetic resistance". Studies to elucidate the mechanism of the genetic resistance demonstrated that the multiplication phase of the CFU growth curve started in the semi-isogeneic combination about 48 hr later than in the isogeneic combination. In the spleen this resulted in a lower "dip". For the spleen as well as for the femur similar CFU doubling times were found during the multiplication phase when both transplantation combinations were compared. Furthermore the percentage of CFU in S-phase (assessed with the 3-H-TdR suicide technique) during the first days after transplantation were similar in both combinations. When the spleen was removed 5-6 months before irradiation and bone marrow transplantation was performed the growth curve of parental CFU in the femur was identical with the growth curve of isogeneic CFU (no delay was observed). These results are discussed and a few theories explaining the observations are proposed.