MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

EXPRESSION OF TUMOR ASSOCIATED ANTIGENS ON HUMAN COLON CARCINOMA CELLS (HUMAN COLON CANCER, MONOCLONAL ANTIBODY)

Human colon cancer cells, LS180 and 174T, exhibit monoclonal antibody (mAb) 1083-17-1A and 5E113 defined tumor associated antigens. By radioimmunoassay, LS180 cells expressed the highest amount of mAb1083 defined antigens among the cell lines tested. Another mAb, 5E113, competed with mAb1083 for binding to LS180 cells, suggesting that both mAbs might bind onto identical (or adjacent) epitopes. By Scatchard analysis, about one million copies of the epitopes were present on LS180 colon cancer cells. The affinity of mAb1083 binding to the cells was 2.97 x 10('10) M('-1); the Sipsian heteroclonality value of mAb1083 was 0.9, thus approximating a single clone of reactive antibody. The qualitative studies showed that the epitopes were probably not carbohydrate because of their sensitivity to proteinases and not to mixed glucosidases and neuraminidase. The tunicamycin homologue B(,2) inhibited the incoporation of ('3)H-labeled galactose but not uptake of ('35)S-labeled methionine, nor expression of monoclonal antibody defined antigens providing further evidence to exclude the possibility of carbohydrate epitopes. There was evidence that the epitope might be partially masked in its "native" conformation, since short exposure or low dose treatment with proteases increased mAbs binding. The best detergent for antigen extraction, as detected by dot blotting and competitive inhibition assays, was octylglucoside at 30 mM concentration. Three methods, immunoprecipitation, Western blotting and photoaffinity labeling, were used to determine the molecular nature of the antigens. These results demonstrated that the antibody bound both 43 K daltons (KD) and 22 KD proteins.^ An in vitro cell-mediated immune approach was also used to attempt identifying function for the antigens. The strategy was to use mAbs to block cytotoxic effector cell killing. However, instead of blocking, the mAb1083 and 5E113 showed strong antibody-dependent cell-mediated cytotoxicities (ADCCs) in the in vitro xenoimmune assay system. In addition, cytotoxic T lymphocytes (CTLs), natural killer cells, and K cell activity were found. Since even the F(ab')2 fragment of mAbs did not inhibit the cytolytic effect, the mAbs defined antigens may not be major target molecules for CTLs. In summary, two molecular species of tumor antigen(s) were identified by mAbs to be present on colon tumor cell lines, LS180 and LS174T. (Abstract shortened with permission of author.) ^

PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

Cancer and inflammation: differentiation by USPIO-enhanced MR imaging.

PURPOSE To assess ultrasmall superparamagnetic iron oxide particles (USPIO) -enhanced MR imaging for the differentiation of malignant from benign, inflammatory lesions. MATERIALS AND METHODS In this study, approved by the local animal care committee, VX2 carcinoma and intramuscular abscesses were implanted into the hind thighs of New Zealand White rabbits. MR imaging was performed pre contrast and serially for 24 h after the injection of USPIO. MR findings were compared with histopathologic results based on Prussian blue stains for the presence of iron. RESULTS Twenty-four hours after the Ferumoxtran-injection, no changes were observed in VX2 carcinomas, whereas a mean reduction of the contrast-to-noise ratio (CNR) of approximately 90% was noticed in abscesses as well as in necrotic tumors. On histopathologic examination, abscess and necrotic parts of the tumor were found to include iron-containing monocytes demonstrating that the reduction in CNR was caused by USPIO-tagged monocytes. CONCLUSION Our results prove the ability of USPIO-enhanced MRI to differentiate benign, inflammatory from malignant lesions.

Differentiation between active and chronic Crohn's disease using MRI small-bowel motility examinations — Initial experience

AIM: To evaluate the influence of locally active Crohn's disease on systemic small-bowel motility in patients with chronic Crohn's disease compared to healthy individuals. MATERIAL AND METHODS: Fifteen healthy individuals (11 men, four women; mean age 37 years) and 20 patients with histopathologically proven active (n = 15; 10 women, 5 men; mean age 45 years) or chronic (n = 5; four women, one man; mean age 48 years) Crohn's disease were included in this institutional review board-approved, retrospective study. Magnetic resonance imaging (MRI; 1.5 T) was performed after standardized preparation. Two-dimensional (2D) cine sequences for motility acquisition were performed in apnoea (27 s). Motility assessment was performed using dedicated software in three randomly chosen areas of the small-bowel outside known Crohn's disease-affected hotspots. The main quantitative characteristics (frequency, amplitude, occlusion rate) were compared using Student's t-test and one-way analysis of variance (ANOVA). RESULTS: Three randomly chosen segments were analysed in each participant. Patients with active Crohn's disease had significantly (p < 0.05) reduced contraction frequencies (active Crohn's disease: 2.86/min; chronic: 4.14/min; healthy: 4.53/min) and luminal occlusion rates (active: 0.43; chronic: 0.70; healthy: 0.73) compared to healthy individuals and patients with chronic Crohn's disease. Contraction amplitudes were significantly reduced during active Crohn's disease (6.71 mm) compared to healthy participants (10.14 mm), but this only reached borderline significance in comparison to chronic Crohn's disease (8.87 mm). Mean bowel lumen diameter was significantly (p = 0.04) higher in patients with active Crohn's disease (16.91 mm) compared to healthy participants (14.79 mm) but not in comparison to patients with chronic Crohn's disease (13.68). CONCLUSION: The findings of the present study suggest that local inflammatory activity of small-bowel segments in patients with active Crohn's disease alters small-bowel motility in distant, non-affected segments. The motility patterns revealed reduced contraction-wave frequencies, amplitudes, and decreased luminal occlusion rates. Thus evaluation of these characteristics potentially helps to differentiate between chronic and active Crohn's disease.

Characterization and differentiation of body fluids, putrefaction fluid, and blood using Hounsfield unit in postmortem CT

The purpose of the present study was to evaluate the ranges of Hounsfield unit (HU) found in body fluids, putrefaction fluids, and blood on postmortem CT and how these ranges are affected by postmortem interval, temperatures, and CT beam energy. Body fluids, putrefaction fluids, and blood from a total of 53 corpses were analyzed to determine the ranges of HU values from postmortem CT images that were taken prior to autopsy. The fluids measured in CT images were obtained at autopsy and examined in terms of macroscopic and microscopic appearances. Body fluids and blood were also collected in plastic bottles, which were subjected to CT scans at different beam energies (80-130 kV) and at various fluid temperatures (4 to 40 °C). At a postmortem interval of 1 to 4 days, the ranges of HU values of the serous fluids (13-38 HU) and the nonsedimented blood (40-88 HU) did not overlap. In the sedimented blood, the upper serum layer exhibited HU value ranges that overlapped with those of the serous fluids. The putrefaction fluids exhibited a range of HU values between 80 and -130 HU. Elevated HU values were observed in fluids with accretive cell impurities. HU values decreased slightly with increasing temperature and CT beam energy. We concluded that serous fluids and blood in fresh corpses can be characterized and differentiated from each other based on HU value ranges. In contrast, body fluids in decomposed corpses cannot be differentiated by their HU value ranges. Different beam energies and corpse temperatures had only minor influences on HU value ranges and therefore should not be obstacles to the differentiation and characterization of body fluids and blood.

[Computer tomographic differentiation of intrathoracic neoplasms and inflammation in the dog].

The purpose of this study was to elaborate on the advantages and limits of computed tomography (CT) in the differentiation of thoracic lesions, in particular neoplasias. In the course of the investigation CT-scans of the thorax of 31 dogs with lesions in the area of the lungs or mediastinum were evaluated. The lesions were rated by morphology, distribution pattern, attenuation values and contrast-enhancement. Biopsies or the whole body underwent a pathohistological examination. Of the 31 dogs 17 had neoplastic and 14 had inflammatory lesions in the thoracic region. With help of the CT, the exact localisations of the different lesions was possible in most cases. Due to their characteristic morphologies, distribution patterns and attenuation values the differentiation between inflammatory and neoplastic lesions was possible in most cases (n=25/31) on the basis of the CT-scans. Mean non-enhanced CT attenuation values of the neoplastic lesions ranged between 31 and 50 HU, of the inflammatory lesions between -251 and 9 HU. Both neoplastic and inflammatory lesions showed contrast enhancement (between 14 and 38 HU and between 2 and 95 HU respectively). The mediastinal abcesses enhanced mainly on the periphery of the lesion. A differentiation of the various types of neoplastic lesions based on the non-enhanced attenuation values was not successful. Only metastasis could be differentiated because of their distribution pattern.

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

Postmortem MR quantification of the heart for characterization and differentiation of ischaemic myocardial lesions.

OBJECTIVES Recently, an MRI quantification sequence has been developed which can be used to acquire T1- and T2-relaxation times as well as proton density (PD) values. Those three quantitative values can be used to describe soft tissue in an objective manner. The purpose of this study was to investigate the applicability of quantitative cardiac MRI for characterization and differentiation of ischaemic myocardial lesions of different age. MATERIALS AND METHODS Fifty post-mortem short axis cardiac 3 T MR examinations have been quantified using a quantification sequence. Myocardial lesions were identified according to histology and appearance in MRI images. Ischaemic lesions were assessed for mean T1-, T2- and proton density values. Quantitative values were plotted in a 3D-coordinate system to investigate the clustering of ischaemic myocardial lesions. RESULTS A total of 16 myocardial lesions detected in MRI images were histologically characterized as acute lesions (n = 8) with perifocal oedema (n = 8), subacute lesions (n = 6) and chronic lesions (n = 2). In a 3D plot comprising the combined quantitative values of T1, T2 and PD, the clusters of all investigated lesions could be well differentiated from each other. CONCLUSION Post-mortem quantitative cardiac MRI is feasible for characterization and discrimination of different age stages of myocardial infarction. KEY POINTS • MR quantification is feasible for characterization of different stages of myocardial infarction. • The results provide the base for computer-aided MRI cardiac infarction diagnosis. • Diagnostic criteria may also be applied for living patients.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.