The regulation of prostate cancer cell adhesion to human bone marrow endothelial cell monolayers by androgen dihydrotestosterone and cytokines

A previous study from our laboratory suggested that prostate cancer metastasis to bone may be mediated, in part, by preferential adhesion to human bone marrow endothelial (HBME) cells. Tumor cell adhesion to endothelial cells may be modulated by the effect of cytokines on cell adhesion molecules (CAMs). Tumor necrosis factor-alpha (TNF-α) regulates VCAM expression on the endothelium and this effect is enhanced by dihydrotestosterone (DHT). Transforming growth factor-beta (TGF-β) stimulates the expression of α₂β₁integrin on PC-3 cells. The current study investigated the effects of the above cytokines and DHT (singularly and in various combinations) upon HBME and prostate cancer cell expression of VCAM, α₂ integrin subunit, and β₁ integrin subunit by flow cytometry. We also monitored the effects of the above treatments on PC-3 cell adhesion to HBME monolayers. The data demonstrate that none of the treatments significantly altered the expression of selected CAMs on HBME cell and neoplastic prostate cell lines. The treatment of HBME monolayers with various combinations of cytokines and DHT prior to performing adhesion assays with PC-3 demonstrates that treatments containing TGF-β reduced PC-3 cell adhesion to HBME monolayers by 32% or greater (P<0.05). The reduction in PC-3 cell adhesion to TGF-β-treated HBME monolayers was dose dependent. Interestingly, LNCaP cells but not PC-3 cells treated with TGF-β had a reduced ability to adhere to untreated HBME monolayers. These results suggest that TGF-β may reduce tumor cell adhesion to bone marrow microvascular endothelium, in vivo. The biological significance of this observation is discussed.     

Determination of extracellular space and intracellular electrolytes in rat liver in vivo

1. Uptake of a number of isotopically labelled extracellular tracers by rat liver in vivo was studied.

2. A residual intravascular space of 3·0-3·5% following exsanguination was measured by use of [⁵¹Cr]red cells. This space was significantly greater than that of skeletal muscle and was also greater in nephrectomized as compared to intact rats.

3. [¹³¹I]albumin and polyvinylpyrolidine (PVP) were found to distribute initially in a space of 6-7% and this was followed by a delayed rise that was greater for PVP. The extravascular portion of this space is termed the large-molecule accessible space and is believed to correspond to the histological space of Disse.

4. [¹⁴C]sucrose and inulin initially distribute in a space of 10-12% followed by a continued late increase. This initial space is termed the small molecule accessible space and is thought to include most of interstitial space.

5. ³⁶Cl^- and stable Na⁺ and Cl^- spaces were constant at 19-21% and can be used to put an upper limit on extracellular space.

6. Operationally, the 1 hr inulin space in the nephrectomized rat was felt to be the best indicator of extracellular space.

7. Total electrolytes were measured and intracellular values of 22, 165 and 22 m-mole/l. cell water for Na⁺, K⁺ and Cl^-, respectively, were calculated.

8. By use of [¹⁴C]DMO, an intracellular pH of 7·25 and an intracellular HCO₃^- of 13 m-mole/l. cell water were calculated.

     

Glycan bioengineering in immunogen design for tumor T antigen immunotargeting

Bioengineering of Galβ3GalNAcα, known as Thomsen–Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galβ3GalNAcα-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlαTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes.     

Benzo-[<Emphasis Type="Italic">a</Emphasis>]-pyrene increases invasion in MDA-MB-231 breast cancer cells via increased COX-II expression and prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) output

Benzo-[a]-pyrene (B[a]P), a carcinogenic component of cigarette smoke, has been shown to increase both COX-II expression and prostaglandin output in vascular smooth muscle and oral epithelial cells. In addition, invasive breast cancer cells have been reported to over express COX-II and PGE₂. Therefore, the objective of this study was to quantify the effect of increasing B[a]P concentrations on COX-II expression, PGE₂ output, and invasion using MDA-MB-231 cells, an invasive estrogen unresponsive breast cancer cell line. B[a]P significantly increased invasion in MDA-MB-231 cells at concentrations greater than 4 × 10⁻⁸ M. Treatment of MDA-MB-231 cells with Vomitoxin (a selective COX-II inducer) enhanced invasion whereas co-treatment with NS398 (a selective COX-II inhibitor) attenuated B[a]P-induced invasion in MDA-MB-231 cells. Immunohistochemical staining and Western blots demonstrated a significant B[a]P treatment-induced increase in both the number of COX-II immunopositive MDA-MB-231 cells and COX-II protein levels. Moreover, B[a]P-treatment induced a profound (46 fold) increase in PGE₂ production by MDA-MB-231 cells. The aryl hydrocarbon receptor (AhR) antagonists resveratrol (RES) and α-naphthaflavone (α-NF) had no effect on their own, whereas B[a]P-induced invasion was significantly inhibited by co-treatment with RES and α-NF. Our data demonstrate that B[a]P-induced changes in invasion are mediated through augmented COX-II expression and PGE₂ production involving an AhR regulated pathway. Moreover, these results suggest a potential role for the AhR signalling pathway in breast cancer invasion.     

Incorporation of [14C] hexoses into the carbohydrate of gamma2-globulin in vitro

A method for the labelling and isolation of specific γ₂-globulin antibody formed in vitro by intact rabbit lymphoid cells has been described. This method has been applied to the study of the synthesis of the carbohydrate portion of γ₂-globulin using [¹⁴C] mannose, [¹⁴C] galactose as labelled precursors. Metabolic alterations of all three sugars led to redistribution of much of the ¹⁴C-label into the amino acids of the protein portion, but a 6- to 18-fold relative enrichment, on a weight basis, was observed in the carbohydrate fraction. Labelling was greatest with mannose and least with galactose under the conditions used in this study.     

The persistence of donor-derived cells in thymus grafts, lymph nodes and spleens of recipient mice

Thymus was labelled in vivo by injecting newborn C3H/Bi mice with three or four doses of 2 μc of [³H]thymidine. The labelled thymus was grafted into the kidney capsule of 22–25-day-old intact and thymectomized syngeneic recipients which were killed 18, 41 or 42 days later. The grafts and lymphoid tissue were sectioned and examined by autoradiography after exposure times of up to 26 weeks.

Small and medium labelled lymphocytes were seen in the thymus-dependent areas of the lymph nodes and spleen of all recipients provided the sections were exposed for a sufficiently long period. More labelled cells were seen in recipients killed at 18 days than at 41 or 42 days after grafting. It was concluded that these cells were the direct descendants of those cells labelled in the graft. There was no evidence of re-utilization of the isotope label.

Most of the cells originally labelled in the graft either lost their label by multiple divisions or were replaced by unlabelled cells; although the epithelial-reticular cells remained heavily labelled. The importance of this last finding is discussed.

     

Increased Expression of Intercellular Adhesion Molecule-1 and Mucosal Adhesion Molecule α4β7 Integrin in Small Intestinal Mucosa of Adult Patients with Food Allergy

The mechanisms of adverse reactions to foods in the gastrointestinal tract are poorly understood. Previous studies of other atopic diseases and animal models suggest that adhesion molecules and mucosal lymphocytes may be implicated in the pathogenesis of food allergy (FA). The aim of our study was to investigate the expression of adhesion molecules and mucosal lymphocytes in duodena of patients with food allergies and of controls. Ten patients with FA to cereals (wheat, oats, and rye) or cow's milk and 9 control patients were included in the study. Quantitative analysis and immunohistochemical stainings for two pairs of adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), α₄β₇ integrin, and mucosal addressin cell adhesion molecule (MAdCAM-1) and lymphocyte markers on endoscopic duodenal biopsy specimens were performed. The villous structure and density of LFA-1-positive cells were normal in every biopsy specimen, but the patients had significantly more α₄β₇⁺ cells in the intraepithelial space (P = 0.01). The expression of ICAM-1 in the lamina propria of patients with FA was also substantially increased (P = 0.003); however, staining with MAdCAM showed no intergroup difference. Moreover, we found significantly increased CD4⁺ and HLA-DR⁺ cells in the lamina propria of patients, in comparison to the controls, P = 0.05 and P = 0.04, respectively. The densities of CD3, CD8, HLA-DP, T cell receptor αβ⁺ and γδ⁺ cells and IgA-, IgA₁-, and IgA₂-containing cells did not differ in the two groups studied. Our results suggest that the increased expression of ICAM-1 and α₄β₇ integrin may play an important role in the pathogenesis of food hypersensitivity and with the elevation of CD4- and HLA-DR-positive cells reflect a stage of inflammation in the structurally normal intestines.     

Selective migration of lymphocytes within the mouse small intestine

The factors which determine the migration of lymphoid cells to lamina propria or Peyer's patches of mouse small intestine have been investigated by autoradiographic tracing of intravenously injected spleen, thymus and lymph node cells. The numbers of labelled cells found in antigen-free grafts of foetal small intestine were compared with the numbers in normally sited gut. Thymus, normal spleen and B spleen lymphocytes, labelled with [³H]adenosine or [5-³H]uridine, were confined to Peyer's patches in normal and grafted gut. [³H]Thymidine-labelled lymphoblasts from the mesenteric nodes of young (19–22 days) mice and mice infected with Nippostrongylus brasiliensis were found in the lamina propria of both graft and normal small intestine, but [³H]thymidine-labelled lymphoblasts from oxazolone-primed lymph nodes did not migrate to the villi. The possible roles of intraluminal antigens, source of cells and changes in cell surface receptors during differentiation, in determining the selective migration of cells to the lamina propria and Peyer's patches, are discussed.     

Selectivity of a series of clonidine-like drugs for α1 and α2 adrenoceptors in rat brain

The relative selectivity of a series of imidazoline derivatives and substituted guanidines structurally related to clonidine for α₁ and α₂ adrenoceptors has been examined in binding studies using membranes prepared from rat cerebral cortex and the radioligands [³H]prazosin and [³H]clonidine. There was a 150-fold difference between the most and least selective clonidine-like drugs in their relative affinity for α₁ and α₂ adrenoceptors.     

Hyperpolarized ketone body metabolism in the rat heart

The aim of this work was to investigate the use of ¹³C‐labelled acetoacetate and β‐hydroxybutyrate as novel hyperpolarized substrates in the study of cardiac metabolism. [1‐¹³C]Acetoacetate was synthesized by catalysed hydrolysis, and both it and [1‐¹³C]β‐hydroxybutyrate were hyperpolarized by dissolution dynamic nuclear polarization (DNP). Their metabolism was studied in isolated, perfused rat hearts. Hyperpolarized [1‐¹³C]acetoacetate metabolism was also studied in the in vivo rat heart in the fed and fasted states. Hyperpolarization of [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate provided liquid state polarizations of 8 ± 2% and 3 ± 1%, respectively. The hyperpolarized T₁ values for the two substrates were 28 ± 3 s (acetoacetate) and 20 ± 1 s (β‐hydroxybutyrate). Multiple downstream metabolites were observed within the perfused heart, including acetylcarnitine, citrate and glutamate. In the in vivo heart, an increase in acetylcarnitine production from acetoacetate was observed in the fed state, as well as a potential reduction in glutamate. In this work, methods for the generation of hyperpolarized [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate were investigated, and their metabolism was assessed in both isolated, perfused rat hearts and in the in vivo rat heart. These preliminary investigations show that DNP can be used as an effective in vivo probe of ketone body metabolism in the heart.     

Differential involvement of μ1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of μ₁-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for μ-opioid receptors, while β-endorphin, which is also an endogenous opioid peptide, is non-selective for μ-, δ- and putative -opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6ByJ mice. Endomorphin-1 (0.1–10 μM), endomorphin-2 (0.1–10 μM) and β-endorphin (0.1–10 μM) increased guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by β-endorphin in C57BL/6ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM endomorphin-1 or endomorphin-2 were almost completely blocked by a μ-opioid receptor antagonist β-funaltrexamine (10 μM) in both strains, the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM β-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 μM β-funaltrexamine in both strains. The residual stimulation of [³⁵S]guanosine-5′-o-(3-thio)triphosphate binding by 10 μM β-endorphin in the presence of 10 μM β-funaltrexamine was further attenuated by the addition of putative -opioid receptor partial agonist β-endorphin (1–27) (1 μM) in both strains. Like the endomorphins, the synthetic μ-opioid receptor agonist [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin at 10 μM showed lower increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM of the selective δ₁-opioid receptor agonist [ -Pen^2,5]enkephalin, δ₂-opioid receptor agonist [ -Ala²]deltorphin II or κ-opioid receptor agonist U50,488H.The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both μ₁- and μ₂-opioid receptors. Moreover, β-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by μ₂- and putative -, but not by μ₁-opioid receptors.     

Milky Spots Promote Ovarian Cancer Metastatic Colonization of Peritoneal Adipose in Experimental Models

The goal of controlling ovarian cancer metastasis formation has elicited considerable interest in identifying the tissue microenvironments involved in cancer cell colonization of the omentum. Omental adipose is a site of prodigious metastasis in both ovarian cancer models and clinical disease. This tissue is unusual for its milky spots, comprised of immune cells, stromal cells, and structural elements surrounding glomerulus-like capillary beds. The present study shows the novel finding that milky spots and adipocytes play distinct and complementary roles in omental metastatic colonization. In vivo assays showed that ID8, CaOV3, HeyA8, and SKOV3ip.1 cancer cells preferentially lodge and grow within omental and splenoportal fat, which contain milky spots, rather than in peritoneal fat depots. Similarly, medium conditioned by milky spot–containing adipose tissue caused 75% more cell migration than did medium conditioned by milky spot–deficient adipose. Studies with immunodeficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Finally, consistent with the role of lipids as an energy source for cancer cell growth, in vivo time-course studies revealed an inverse relationship between metastatic burden and omental adipocyte content. Our findings support a two-step model in which both milky spots and adipose have specific roles in colonization of the omentum by ovarian cancer cells.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.It is estimated that 22,240 women will be diagnosed with and 14,030 women will die of cancer of the ovary in 2013 (http://seer.cancer.gov/csr/1975_2009_pops09, last accessed June 18, 2013). The majority of patients present with metastases or eventually die of metastatic disease within the abdominal cavity. After escape from the primary tumor, ovarian cancer cells in the peritoneal fluid have access to and can potentially lodge within a variety of tissues.^1,2 In both clinical disease and experimental models, however, the omentum is the site of prodigious metastasis formation.^3–6 Thus, attachment of ovarian cancer cells to the omentum represents an early step in the development of widespread peritoneal disease.^7,8 Although the importance of the omentum is widely acknowledged, there still is no consensus on its role in metastasis formation. This raises the question of what components of the omental tissue microenvironment participate in, or facilitate, ovarian cancer metastatic colonization.Studies of omental function date back to the early 19th century. Jobert de Lamballe, a 19th-century surgeon in France, was reportedly the first to recognize the curious ability of this organ to fight infection and form adhesions to help control injuries.⁹ After nearly two centuries of investigation, a great deal is known about the physiology and surgical applications of the omentum.^9–12 As the central regulator of peritoneal homeostasis, its functions include regulating fluid and solute transport, sensing and repairing injuries, promoting angiogenesis, fighting infection, serving as a source of stem cells, producing regulatory molecules, and storing and supplying lipids. These diverse functions are conferred by the cellular composition and architecture characteristic of human omenta.Aside from the clear collagenous membrane that acts as a scaffold for the organ, the majority of the omentum is composed of bands of adipose tissue that contain adipocytes, blood and lymph vessels, immune cells, stromal cells, and connective matrix components that lie beneath an irregular mesothelium.¹³ In general, adipocytes have a variety of functions, ranging from lipid storage to production of endocrine molecules, and can serve as an integrating hub for inflammation, metabolism, and immunity.^2,14–23 A distinctive feature of the omental vasculature is the presence of numerous branching blood vessels ending in tortuous glomerulus-like capillary beds near the tissue periphery.^24–28 Immune cells aggregate around and within these capillary beds to form milky spots, which are the major immune structure for host defense of the peritoneal cavity.^24,29–36 In milky spots, both the endothelial lining of the capillaries and the overlying mesothelium are specially adapted to facilitate transmigration of immune cells.^24,37,38 Additional structural elements include plasmocytes, fibroblasts, and mesenchymal cells, as well as collagen and reticular and elastic fibers.^29,34,37,39A comprehensive literature review showed that studies examining the role of the omentum in metastasis focus on the contribution of its individual components, and not on the tissue as a whole. In our view, results from the majority of studies support models in which ovarian cancer metastatic colonization is driven either purely by milky spots or purely by adipocytes. The milky spot–driven model is based on a large body of in vivo data showing that, on intraperitoneal injection, cancer cells rapidly and specifically localize, invade, and proliferate within omental milky spots.^{3,6,24,28,40–44} In contrast, the adipocyte-driven model is based on the observation that, in its resting state, the omentum is composed predominantly of adipose and that cultured adipocytes can produce adipokines capable of promoting ovarian cancer cell migration and invasion in vitro.⁴⁵ Adipocytes can also provide a proliferative advantage by transferring fatty acids to ovarian cancer cells.⁴⁵ Although both models have clear strengths, neither addresses the intimate and dynamic interaction among milky spots, surrounding adipocytes, and other components of omental tissues.Taking tissue architecture and function as a guide, we propose that an alternative, more fully integrated model of metastatic colonization is needed. To test this idea, we identified peritoneal fat depots (omentum, mesentery, and uterine, gonadal, and splenoportal fat) that are accessible to ovarian cancer cells after intraperitoneal injection.² Of these, the omentum and splenoportal fat are reported to contain milky spot structures.^24,46 We reasoned that a comparison of peritoneal adipose that either contains or lacks milky spots could be used to determine the contributions of adipocytes and milky spots to the lodging and progressive growth of ovarian cancer cells in physiologically relevant tissues. In vivo studies using a panel of ovarian cancer cell lines showed that milky spots dramatically enhance early cancer cell lodging on peritoneal adipose tissues. Consistent with this finding, conditioned medium from milky spot–containing adipose tissue had a significantly increased ability to direct cell migration, compared with conditioned medium from milky spot–deficient adipose tissue. Studies using a panel of immunodeficient mice showed that the number and size of omental milky spots is not dependent on the mouse genetic background and, similarly, that ovarian cancer cell colonization does not depend on the immune composition of the milky spot. Finally, consistent with the role for lipids as an energy source for ovarian cancer cell growth, in vivo time-course studies revealed an inverse relationship between metastatic burden and adipocyte content in the omentum. Our findings support a two-step model in which both milky spots and adipose have specific roles in colonization of the omentum by ovarian cancer cells.     

The intracellular localization of two antigens after uptake in vivo by peritoneal macrophages from normal mice

Normal mouse macrophages, which had ingested ferritin labelled with fluorescein isothiocyanate and human serum albumin labelled with tetramethylrhodamine isothiocyanate in vivo, were fixed in formalin and embedded for electron microscopy. The examination of sections 1–2 μ thick and adjacent ultrathin sections showed that the yellow-green fluorescent droplets (due to ferritin-FITC) seen by fluorescence microscopy were in the same position as the ferritin-containing phagolysosomes as seen by electron microscopy.

Normal mouse macrophages, which had ingested ¹²⁵I-labelled human serum albumin ([¹²⁵I]HSA) and unlabelled ferritin, were investigated by electron microscopic autoradiography. Both antigens were found to be situated within the same lysosomes.

     

Synthesis,storage and release of [14C]acetylcholine in isolated rat diaphragm muscles

1. Segments of rat diaphragms were kept in choline-free media for 4 hr and were then exposed to a physiological concentration of [¹⁴C]-choline (30 μM) at 37° C. The synthesis, storage and subsequent release of [¹⁴C]acetylcholine by the muscles was assessed by isotopic- and bio-assays after isolation of the transmitter by paper electrophoresis.2. Replacement of endogenous acetylcholine (0·92 μ-mole/kg) with labelled acetylcholine proceeded slowly at rest, but rapidly during nerve stimulation. [¹⁴C]Acetylcholine accumulated most rapidly when hydrolysis of the released transmitter, and thus the re-use of endogenous choline, was prevented by an esterase inhibitor. Fully replaced stores were maintained during nerve stimulation by synthesis rates sufficient to replenish at least 35% of the store size in 5 min.3 In the presence of hemicholinium-3, which inhibits choline uptake, acetylcholine stores declined rapidly during stimulation, and residual synthesis was slight, indicating little intraneural choline. Net choline uptake into nerve terminals was estimated from the highest observed synthesis rate and from previous measurements of the number and size of terminals, as 3-6 p-mole/cm² sec.4. Transmitter synthesis was localized in the region of end-plates, and was reduced to a few per cent of normal 6 weeks after phrenic nerve section. Release experiments suggested that at least half of the acetylcholine in phrenic nerves is in their terminals; from this content and the morphology of the terminals, the average concentration of transmitter in the whole endings would appear to be about 50 m-mole/l. Homogenization of the muscles freed choline acetyltransferase into solution, but left some [¹⁴C]acetylcholine associated with small particles, presumably synaptic vesicles.5. Resting transmitter release was about 0·013% of stores/sec. With 360 nerve impulses at 1-20/sec, release increased up to 0·43% of stores/sec, and amounted to 3·5-7 × 10^-18 moles per end-plate per impulse. The release rate was unaffected by the doubling of store size which occurred with eserine, but the extra transmitter did help to maintain releasable stores during prolonged stimulation. Experiments with fractional store labelling indicated that newly synthesized acetylcholine was preferentially released.6. Preformed [³H]acetylcholine was not taken up and retained by muscle or nerve cells in the absence of an esterase inhibitor. With eserine present, labelled acetylcholine was taken up uniformly by muscle segments; when eserine was then removed, radioactive acetylcholine remained only near neuromuscular junctions.     

Inhibition of protein synthesis,pulmonary localization and pulmonary tumour formation by drug-treated tumour cells as a means of predicting their chemosensitivity

Mouse mammary carcinoma cells were exposedin vitro to increasing concentrations of doxorubicin hydrochloride [adriamycin (ADR)] or 5-fluorouracil (5-FU). Uptake of [⁷⁵Se]selenomethionine (⁷⁵SeM) in a methionine-deficient medium measured the resulting inhibition of protein synthesis by the tumour cells. This was compared with the ability of the⁷⁵SeM labelled tumour cells to localize in mouse lungs and to form pulmonary tumours following intravenous (i.v.) injection into isogenic hosts. These parameters were also related to the ability of the drugs to inhibit pulmonary tumour formationin vivo when injected into mice which had received tumour cells i.v. Results from five different tumours were pooled for analysis. At the highest drug concentration (10g/ml ADR, 100g/ml 5-FU) inhibition of protein synthesis was significantly related to thein vivo action of the drugs in limiting formation of pulmonary tumours (P<0·02 using the rank difference coefficient). There was also a direct relationship between pulmonary localization of tumour cells following exposure to drugs, their ability to form tumour nodules (P<0·025) and thein vivo action of the drugs in inhibiting tumour formation (P<0·s05). Thus inhibition of protein synthesisin vitro and pulmonary localization following i.v. injection may be of value in predicting thein vivo effect of cytotoxic drugs.     

Workshop I: Dendritic Cells and Costimulation, Abstract I1 bis I24

I. 1 Visualization of dendritic cells in the mouse by a chemokine-driven eGFP-reporterI. 2 Identification and cellular localization of two new genes expressed specifically in dendritic cellsI. 3 IgG induces maturation of human monocyte-derived dendritic cells by cross-linking of FcγRI. 4 Virus-induced IFN-α production by a subset of CD11c⁺CD11b^– dendritic cells in the absence of feedback signaling in vivoI. 5 Comparative analysis of the capacities of different heat shock proteins to stimulate human monocytes and dendritic cellsI. 6 CpG-ODN in vivo potently enhance dendritic cell based tumor therapy in a murine model of colon carcinomaI. 7 Cytokine and chemokine production by dendritic cells in culture is dependent on stimulation with estradiol, progesterone and β-hCGI. 8 The role of Rac-1 in dendritic cell function in vivoI. 9 ICOS-ligand is expressed by human endothelial cells and co-stimulates cytokine secretion by memory CD4⁺ T cellsI. 10 Cytokine release of L. monocytogenes-infected dendritic cellsI. 11 Differentiation and apoptosis in cultures of monocyte-derived dendritic cells is influenced by type I interferons, bacterial stimuli, and interleukin-10I. 12 Targeting of antigens to dendritic cells in vivo via DEC-205 induces short-lived T cell expansion and ultimately leads to unresponsiveness of T cellsI. 13 An analysis of mouse dendritic cell subsets as defined by CD4 and CD8 expressionI. 14 Native human blood dendritic cells as potent effectors in antibody-dependent cellular cytotoxicityI. 15 A Toxoplasma gondii factor that triggers functional maturation of dendritic cells from mouse brainI. 16 Influence of an aqueous mistletoe extract on the generation and activation of dendritic cells (in vitro)I. 17 IL-12/IL-18-dependent interferon-γ release by murine dendritic cellsI. 18 Retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34-derived dendritic cells specifically activates cytotoxic CD8⁺ T cellsI. 19 Impact of rIL-4 on the stimulatory capacity of murine bone marrow-derived dendritic cellsI. 20 Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40L to induce high amounts of IL-12I. 21 Dendritic cells and B cells enrich select MHC II-peptide complexes in tetraspan microdomains distinct from lipid raftsI. 22 C5a receptor expression on human monocyte-derived dendritic cells is functionally upregulated by prostaglandin E2I. 23 ICOS-ligand and CD80/CD86 on dendritic cells induce differential cytokine secretion profiles in CD4⁺ T cellsI. 24 Interferon-γ in synergy with inflammatory stimuli induces high expression of RelB and CCR7 in human monocyte derived dendritic cells in vitro     

Continuous real time ex vivo epifluorescent video microscopy for the study of metastatic cancer cell interactions with microvascular endothelium

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2×10³ μm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2×10³ to 0 μm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human. This revised version was published online in July 2006 with corrections to the Cover Date.     

Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B₁₂). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [⁵⁷Co]-AdoCbl and MeCbl analogs consisting of [⁵⁷Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [⁵⁷Co]-AdoCbl was converted to [⁵⁷Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5′-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [⁵⁷Co]-propylcobalamin to [⁵⁷Co]-AdoCbl and [⁵⁷Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (α4β2-like) and bungarotoxin-sensitive (α7-like) receptors were visualized using [³H]pirenzepine, [3H]AF-DX 384, [³H]cytisine and [¹²⁵I]α-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [³H]vesamicol (vesicular acetylcholine transport sites) and [³H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [³H]vesamicol and [³H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [¹²⁵I]α-BTX binding sites were mostly concentrated in the superficial bulbar layers, [³H]cytisine binding was found in the glomerular layer, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [³H]vesamicol and [³H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [³H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/α4β2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.