Immunolymphoscintigraphy for metastatic sentinel nodes: test of a model.

Aim. To develop a method and obtain proof-of-principle for immunolymphoscintigraphy for identification of metastatic sentinel nodes. Methods. We selected one of four tumour-specific antibodies against human breast cancer and investigated (1), in immune-deficient (nude) mice with xenograft human breast cancer expressing the antigen if specific binding of the intratumorally injected, radioactively labelled, monoclonal antibody could be scintigraphically visualized, and (2) transportation to and retention in regional lymph nodes of the radioactively labelled antibody after subcutaneous injection in healthy rabbits. Results and Conclusion. Our paper suggests the theoretical possibility of a model of dual isotope immuno-lymphoscintigraphy for noninvasive, preoperative, malignant sentinel node imaging.

Quantification of in vivo tumor invasion and vascularization by computerized image analysis.

The matrix-inserted surface transplantation model is an in vivo assay used to analyse the kinetics of tumor-vessel interactions during different stages of skin carcinoma progression. This system allows the study of host-tumor interface, i.e. penetration of tumor cells into normal host tissue as well as infiltration of normal host cells into the tumor. In the present study, image analysis algorithms for processing and quantifying the extent of such migratory and tissue remodeling events are presented. The proposed method is non-parametric and its originality lies in its particularity to take into account the specific geometry of tumor-host interface. This methodology is validated by evaluating the contribution of matrix metalloproteases (MMPs) in skin carcinoma invasion and vascularization through pharmacological and genetic approaches.

Systemic administration of anti-urokinase plasminogen activator receptor monoclonal antibodies induces hepatic fibrin deposition in tissue-type plasminogen activator deficient mice.

BACKGROUND: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and hence pro-u-PA activation, is an attractive approach to anti-invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u-PAR (mR1) by immunization of u-PAR-deficient mice. OBJECTIVES: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. METHODS: Wild-type and tissue-type plasminogen activator (t-PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. RESULTS: Systemic administration of mR1 caused significantly increased fibrin signal in anti-u-PAR treated t-PA-deficient mice compared to mock-treated, which mimics the phenotype of u-PAR;t-PA double-deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t-PA-deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u-PAR. CONCLUSION: We show that u-PAR-expressing macrophages are involved in cell-mediated fibrinolysis of liver fibrin deposits, and that the antimouse-u-PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u-PA/u-PAR interaction in mouse cancer models.

Regulation of pancreatic beta-cell mass and proliferation by SOCS-3.

Growth hormone and prolactin are important growth factors for pancreatic beta-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in beta-cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Suppressors of cytokine signaling (SOCS) proteins are specific inhibitors of the JAK/STAT pathway acting through a negative-feedback loop. To investigate in vivo effects of SOCS-3 in growth hormone (GH)/prolactin signaling in beta-cells we generated transgenic mice with beta-cell-specific overexpression of SOCS-3. The relative beta-cell proliferation and volume in the mice were measured by morphometry. Beta-cell volume of transgenic female mice was reduced by over 30% compared with beta-cell volume in wild-type female mice. Stimulation of transgenic islets in vitro with GH showed a reduced tyrosine phosphorylation of STAT-5 when compared with wild-type islets. Transduction of primary islet cultures with adenoviruses expressing various SOCS proteins followed by stimulation with GH or glucagon-like peptide-1 (GLP-1) revealed that SOCS-3 inhibited GH- but not GLP-1-mediated islet cell proliferation, indicating that the decreased beta-cell volume observed in female transgenic mice could be caused by an inhibition of GH-induced beta-cell proliferation by SOCS-3. In spite of the reduced beta-cell volume the transgenic female mice exhibited enhanced glucose tolerance compared with wild-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic beta-cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.

FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be cultured from colonic mucosa of patients with Crohn's disease.

Summary CD4(+)CD25(+) regulatory T cells (T(regs)) are involved in the maintenance of peripheral tolerance and ensure a balanced immune response competent of fighting pathogens and at the same time recognizing commensals as harmless. This feature is lost in Crohn's disease (CD). The forkhead/winged helix transcription factor FoxP3 is a master gene for T(reg) function and defects in the FoxP3 gene lead to a clinical picture similar to inflammatory bowel disease (IBD). Murine colitis can be cured by adoptive transfer of T(regs) and ex vivo-generated gut-specific T(regs) represent an attractive option for therapy in CD. Thus, defective T(regs) could contribute to the development of CD. We cultured biopsies of colonic mucosa in the presence of high concentrations of interleukin (IL)-2 and IL-4 to overcome the anergic nature of naturally occurring CD4(+)CD25(+) T(regs) in the mucosa. We investigated the expression of FoxP3 and regulatory potential of gut-derived CD4(+)CD25(+) T cells cultured from patients with CD and healthy individuals. The FoxP3 expression was analysed by reverse transcriptase polymerase chain reaction (RT-PCR), and the suppressive effect of FoxP3(+)CD4(+)CD25(+) T cells on proliferation and cytokine production of autologous CD4(+) T cells was assessed by flow cytometry. Cultured gut-derived T cells with CD4(+)CD25(+) phenotype expressed FoxP3 and were able as the freshly isolated T(regs) from peripheral blood to suppress proliferation and cytokine production of autologous CD4(+) T cells. Thus, we demonstrate that FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be propagated in vitro from inflamed mucosa of CD patients, which may be of interest in adoptive immunotherapy.

Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo.

Integrin alpha4beta 7 is the principal gut-homing receptor, and it is assumed that expression of this specific integrin directs lymphocytes to the gut in vivo. Adoptive cellular immunotherapy against inflammatory bowel disease (IBD) may depend on the expression of integrin alpha4beta 7 to accomplish local delivery of intravenously injected regulatory T cells in inflamed gut mucosa. The present study aimed to investigate whether in vitro expanded human T cells from the colonic mucosa maintain integrin expression, show in vitro adhesion and retain in vivo gut-homing properties during cultivation. Whole colonic biopsies from healthy subjects were cultured in the presence of interleukin-2 (IL-2) and IL-4. The integrin expression of the cultured T cells was determined by flow cytometry and in vitro adhesion was assessed in a mucosal addressin cell adhesion molecule 1 (MAdCAM-1) adhesion assay. We studied the homing pattern after autologous infusion of 3 x 10(8 111)Indium ((111)In)-labelled T cells in five healthy subjects using scintigraphic imaging. The cultured CD4(+)CD45RO(+) gut-derived T cells express higher levels of integrin alpha4beta 7 than peripheral blood lymphocytes (PBLs) and show strong adhesion to MAdCAM-1 in vitro, even after (111)In-labelling. Scintigraphic imaging, however, showed no gut-homing in vivo. After prolonged transit through the lungs, the T cells migrated preferentially to the spleen, liver and bone marrow. In conclusion, it is feasible to infuse autologous T cells cultured from the gut mucosa, which may be of interest in adoptive immunotherapy. Despite high expression of the gut-homing integrin alpha4beta 7 and adhesion to MAdCAM-1 in vitro, evaluation by (111)In-scintigraphy demonstrated no gut-homing in healthy individuals.

Adipogenic and orexigenic effects of the ghrelin-receptor ligand tabimorelin are diminished in leptin-signalling-deficient ZDF rats.

OBJECTIVE: The aim was to investigate the possible interactions of the two peripheral hormones, leptin and ghrelin, that regulate the energy balance in opposite directions. METHODS: Leptin-receptor mutated Zucker diabetic fatty (ZDF) and lean control rats were treated with the ghrelin-receptor ligand, tabimorelin (50 mg/kg p.o.) for 18 days, and the effects on body weight, food intake and body composition were investigated. The level of expression of anabolic and catabolic neuropeptides and their receptors in the hypothalamic area were analysed by in situ hybridization. RESULTS: Tabimorelin treatment induced hyperphagia and adiposity (increased total fat mass and gain in body weight) in lean control rats, while these parameters were not increased in ZDF rats. Treatment with tabimorelin of lean control rats increased hypothalamic mRNA expression of the anabolic neuropeptide Y (NPY) mRNA and decreased hypothalamic expression of the catabolic peptide pro-opiomelanocortin (POMC) mRNA. In ZDF rats, the expression of POMC mRNA was not affected by treatment with tabimorelin, whereas NPY mRNA expression was increased in the hypothalamic arcuate nucleus. CONCLUSION: This shows that tabimorelin-induced adiposity and hyperphagia in lean control rats are correlated with increased hypothalamic NPY mRNA and decreased POMC mRNA expression. The elimination of tabimorelin-induced adiposity and hyperphagia in ZDF rats may be due to lack of POMC mRNA downregulation. In conclusion, we suggest that ghrelin-receptor ligands exert their adipogenic and orexigenic effects via hypothalamic mechanisms that are dependent on intact leptin-receptor signalling.

Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.

Distribution of 16alpha-[18F]fluoro-estradiol-3,17beta-disulfamate in rats, tumour-bearing mice and piglets.

Based on a high affinity to the enzyme estrone sulfatase (ES), 16alpha-[18F]fluoroestradiol-3,17beta-disulfamate ([18F]FESDS) has been suggested as a potential PET radiotracer for imaging steroid-dependent breast tumours. The distribution of [18F]FESDS was studied in rats, tumour-bearing nude mice and piglets. In all species evidence for binding to a second target, the enzyme carbonic anhydrase (CA), was obtained. ES and CA inhibitors significantly reduced the radiotracer uptake in various organs but not in tumours. It is concluded that [18F]FESDS binds to ES and CA in vivo but this binding is not strong enough to allow tumour imaging with positron emission tomography (PET).

Distribution of estrone sulfatase in rat brain determined by in vitro autoradiography with 16alpha-[18F]fluoroestradiol-3,17beta-disulfamate.

16Alpha-fluoroestradiol-3,17beta-disulfamate (FESDS) strongly inhibits estrone sulfatase (ES), an enzyme which is also present in the brain. The enzyme is probably involved in important regulatory functions of neurosteroids which may be disturbed in certain brain diseases. In the present study, [18F]FESDS was used to measure the amount of ES in various rat brain regions using quantitative in vitro autoradiography. The obtained values vary between 0.29 pmol (mg protein)(-1) (pons) and 11.5 pmol (mg protein)(-1) (striatum). They are positively correlated with the enzyme activity measured in homogenates of the corresponding regions. Because this radiotracer binds also to carbonic anhydrase in the brain it is only of limited use for in vivo imaging studies.

Automated synthesis of 16alpha-[18F]fluoroestradiol-3,17beta-disulphamate.

After 16alpha-[15F]fluoroestradiol ([18F]FES) has been successfully prepared in an automated module, the synthesis of 16alpha-[18F]fluoroestradiol-3,17beta-disulphamate ([18F]FESDS) is described as a module-assisted one-pot procedure which can provide 10GBq [18F]FESDS with a radiochemical purity better than 99%. The procedure is reliable and reproducible and requires a time of about 90 min. Because of its high sulphatase-inhibitory effect [15F]FESDS is thought to be a new PET tracer to image sites of high sulphatase activity.

Effects of growth hormone and its secretagogues on bone.

The growth hormone (GH)/insulin-like growth factor-1 axis is not only of importance for linear body growth during childhood, but it is also one of the major determinants of adult bone mass. Studies show that GH treatment increases bone mass in rodents as well as in adult GH-deficient humans, but the effect of GH treatment on bone mass in healthy humans has so far not been impressive. Recently, a new class of GH secretagogues (GHSs) has been developed. In humans, GHS treatment affects biochemical markers of bone turnover and increases growth velocity in selected short children with or without GH deficiency. In rodents, GHS treatment increase bone mineral content, but it has not yet been shown that GHS treatment can affect bone mass in adult humans.

Phagocytosis and killing of Mycobacterium avium complex by human neutrophils.

Organisms belonging to the Mycobacterium avium complex (MAC) cause life-threatening bacteremia in immunocompromised patients. Monocytes and macrophages are thought to be responsible for ingestion and killing of MAC. However, it has been suggested that neutrophils may play a role in the early immune response to MAC infection. Here, neutrophils in autologous plasma were incubated (at 0 and 37 degrees C) with M. avium labeled with Auramine O, a potent fluorochrome. Neutrophil phagocytosis was measured by flow cytometry. Neutrophils incubated at 37 degrees C showed an increase in fluorescence over time with a maximum at 15 min, whereas neutrophils on ice showed no time-dependent increase in FL1. At 15 min Fl 1 at 37 degrees C was twice as high as FL1 at 0 degrees C. Examination under the fluorescent microscope showed multiple intracellular fluorescent mycobacteria. Results in nine independent experiments showed time-dependent decrease of colony-forming units in neutrophil-associated live M. avium. Significant killing was observed within 30 min and was complete by 120 min. Observation by electron microscopy clearly confirmed the presence of intraphagosomal MAC, both intact and with evidence of degradation. These data demonstrate that MAC is rapidly phagocytized and killed by human neutrophils. The newly established flow cytometry method should be useful in further studies of neutrophil function and of the role of G-CSF and other cytokines in MAC disease.

Cancer cell expression of urokinase-type plasminogen activator receptor mRNA in squamous cell carcinomas of the skin.

In this study we have used in situ hybridization with radiolabeled antisense RNA probes to examine the expression of mRNA for urokinase-type plasminogen activator and its receptor in histologic samples of squamous cell (n = 7) and basal cell (n = 7) carcinomas of the skin. Messenger RNA for both urokinase-type plasminogen activator and its receptor were expressed in all of the squamous cell carcinomas, but could not be detected in the basal cell carcinomas. In all of the seven squamous cell carcinomas a signal for urokinase-type plasminogen activator receptor mRNA was detected focally in well-differentiated cancer cells surrounding keratinized pearls, and in four specimens urokinase-type plasminogen activator receptor mRNA was in addition expressed by cancer cells at the edge of invasively growing strands of tumor. Urokinase-type plasminogen activator mRNA expression was found in virtually all the cancer cells of the squamous cell carcinomas, and importantly we found, by hybridizations for urokinase-type plasminogen activator and its receptor mRNA on adjacent sections of squamous cell carcinomas, that it was exactly the invading cancer cells that simultaneously expressed both these components required for plasmin-mediated proteolysis at the cell surface. We have previously shown that both urokinase-type plasminogen activator and its receptor mRNA are expressed by the leading-edge keratinocytes in regenerating epidermis during mouse skin wound healing, and that wound healing is impaired in mice made deficient in plasminogen by targeted gene disruption. We propose that there are similarities between the mechanisms of generation and regulation of extracellular proteolysis during skin re-epithelialization and squamous cell carcinoma invasion. The ability of the squamous carcinoma cells to mimic the "invasive" phenotype of re-epithelializing keratinocytes may be one of the factors that make squamous cell carcinomas more aggressive tumors than basal cell carcinomas.

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake.

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9-39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.

Functional overlap between two classes of matrix-degrading proteases in wound healing.

Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild-type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen-deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix-degrading proteases, probably in the dissection of the fibrin-rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases.

Cancer invasion and tissue remodeling--cooperation of protease systems and cell types.

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non-neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI-1 and MMPs) can be expressed by either the non-neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non-neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.

Analysis of extracellular matrix degradation systems has led to the insight that in cancer invasion there is often crucial interplay between cancer cells and several types of surrounding non-neoplastic stromal cells. Likewise, in normal tissue remodeling processes, the synthesis of proteolytic components is often distributed between several cell types, and there are strong similarities between neoplastic and non-neoplastic processes in the same tissue. Thus, tissue remodeling events are excellent models for studies of extracellular proteolysis in cancer. This has become even clearer by recent analyses of genetically modified mice.