Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef's influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef's ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.

Role of retroviral restriction factors in the interferon-α-mediated suppression of HIV-1 in vivo

The antiviral potency of the cytokine IFN-α has been long appreciated but remains poorly understood. A number of studies have suggested that induction of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) and bone marrow stromal cell antigen 2 (BST-2/tetherin/CD317) retroviral restriction factors underlies the IFN-α-mediated suppression of HIV-1 replication in vitro. We sought to characterize the as-yet-undefined relationship between IFN-α treatment, retroviral restriction factors, and HIV-1 in vivo. APOBEC3G, APOBEC3F, and BST-2 expression levels were measured in HIV/hepatitis C virus (HCV)-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated IFN-α/ribavirin (IFN-α/riba) combination therapy. IFN-α/riba therapy decreased HIV-1 viral load by -0.921 (±0.858) log(10) copies/mL in HIV/HCV-coinfected patients. APOBEC3G/3F and BST-2 mRNA expression was significantly elevated during IFN-α/riba treatment in patient-derived CD4+ T cells (P < 0.04 and P < 0.008, paired Wilcoxon), and extent of BST-2 induction was correlated with reduction in HIV-1 viral load during treatment (P < 0.05, Pearson's r). APOBEC3 induction during treatment was correlated with degree of viral hypermutation (P < 0.03, Spearman's ρ), and evolution of the HIV-1 accessory protein viral protein U (Vpu) during IFN-α/riba treatment was suggestive of increased BST-2-mediated selection pressure. These data suggest that host restriction factors play a critical role in the antiretroviral capacity of IFN-α in vivo, and warrant investigation into therapeutic strategies that specifically enhance the expression of these intrinsic immune factors in HIV-1-infected individuals.

In vivo particle uptake by airway macrophages in healthy volunteers

We combined two techniques, radiolabeled aerosol inhalation delivery and induced sputum, to examine in vivo the time course of particle uptake by airway macrophages in 10 healthy volunteers. On three separate visits, induced sputum was obtained 40, 100, and 160 min after inhalation of radiolabeled sulfur colloid (SC) aerosol (Tc99 m-SC, 0.2 microm colloid size delivered in 6-microm droplets). On a fourth visit (control) with no SC inhalation, induced sputum was obtained and SC particles were incubated (37 degrees C) in vitro with sputum cells for 40, 100, and 160 min (matching the times associated with in vivo sampling). Total and differential cell counts were recorded for each sputum sample. Compared with 40 min (6 +/- 3%), uptake in vivo was significantly elevated at 100 (31 +/- 5%) and 160 min (27 +/- 4%); both were strongly associated with the number of airway macrophages (R = 0.8 and 0.7, respectively); and the number and proportion of macrophages at 40 min were significantly (P < 0.05) elevated compared with control (1,248 +/- 256 versus 555 +/- 114 cells/mg; 76 +/- 6% versus 60 +/- 5%). Uptake in vitro increased in a linear fashion over time and was maximal at 160 min (40 min, 12 +/- 2%; 100 min, 16 +/- 4%; 160 min, 24 +/- 6%). These data suggest that airway surface macrophages in healthy subjects rapidly engulf insoluble particles. Further, macrophage recruitment and phagocytosis-modifying agents are factors in vivo that likely affect particle uptake and its time course.

GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

Peptide hormone receptors overexpressed in human tumors, such as somatostatin receptors, can be used for in vivo targeting for diagnostic and therapeutic purposes. A novel promising candidate in this field is the GLP-1 receptor, which was recently shown to be massively overexpressed in gut and lung neuroendocrine tumors--in particular, in insulinomas. Anticipating a major development of GLP-1 receptor targeting in nuclear medicine, our aim was to evaluate in vitro the GLP-1 receptor expression in a large variety of other tumors and to compare it with that in nonneoplastic tissues. METHODS: The GLP-1 receptor protein expression was qualitatively and quantitatively investigated in a broad spectrum of human tumors (n=419) and nonneoplastic human tissues (n=209) with receptor autoradiography using (125)I-GLP-1(7-36)amide. Pharmacologic competition experiments were performed to provide proof of specificity of the procedure. RESULTS: GLP-1 receptors were expressed in various endocrine tumors, with particularly high amounts in pheochromocytomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. In nonneoplastic tissues, GLP-1 receptors were present in generally low amounts in specific tissue compartments of several organs--namely, pancreas, intestine, lung, kidney, breast, and brain; no receptors were identified in lymph nodes, spleen, liver, or the adrenal gland. The rank order of potencies for receptor binding--namely, GLP-1(7-36)amide = exendin-4 >> GLP-2 = glucagon(1-29)--provided proof of specific GLP-1 receptor identification. CONCLUSION: The GLP-1 receptors may represent a novel molecular target for in vivo scintigraphy and targeted radiotherapy for a variety of GLP-1 receptor-expressing tumors. For GLP-1 receptor scintigraphy, a low-background signal can be expected, on the basis of the low receptor expression in the normal tissues surrounding tumors.

Highly efficient in vivo agonist-induced internalization of sst2 receptors in somatostatin target tissues

The successful peptide receptor imaging of tumors, as exemplified for somatostatin receptors, is based on the overexpression of peptide receptors in selected tumors and the high-affinity binding to these tumors of agonist radioligands that are subsequently internalized into the tumor cells in which they accumulate. Although in vitro studies have shown ample evidence that the ligand-receptor complex is internalized, in vivo evidence of agonist-induced internalization of peptide receptors, such as somatostatin receptors, is missing. METHODS: Rats subcutaneously transplanted with the somatostatin receptor subtype 2 (sst(2))-expressing AR42J tumor cells were treated with intravenous injections of various doses of the sst(2) agonist [Tyr(3), Thr(8)]-octreotide (TATE) or of the sst(2) antagonist 1,4,7,10-tetraazacyclododecane-N,N',N'',N''',-tetraacetic acid (DOTA)-Bass and were sacrificed at various times ranging from 2.5 min to 24 h after injection. The tumors and pancreas were then removed from each animal. All tissue samples were processed for sst(2) immunohistochemistry using sst(2)-specific antibodies. RESULTS: Compared with the sst(2) receptors in untreated animals, which localized at the plasma membrane in pancreatic and AR42J tumor cells, the sst(2) receptors in treated animals are detected intracellularly after an intravenous injection of the agonist TATE. Internalization is fast, as the receptors are already internalizing 2.5 min after TATE injection. The process is extremely efficient, as most of the cell surface receptors internalize into the cell and are found in endosomelike structures after TATE injection. The internalization is most likely reversible, because 24 h after injection the receptors are again found at the cell surface. The process is also agonist-dependent, because internalization is seen with high-affinity sst(2) agonists but not with high-affinity sst(2) antagonists. The same internalization properties are seen in pancreatic and AR42J tumor cells. They can further be confirmed in vitro in human embryonic kidney-sst(2) cells, with an immunofluorescence microscopy-based sst(2) internalization assay. CONCLUSION: These animal data strongly indicate that the process of in vivo sst(2) internalization after agonist stimulation is fast, extremely efficient, and fully functional under in vivo conditions in neoplastic and physiologic sst(2) target tissues. This molecular process is, therefore, likely to be responsible for the high and long-lasting uptake of sst(2) radioligands seen in vivo in sst(2)-expressing tumors.

Effects of Alpha Interferon Treatment on Intrinsic Anti-HIV-1 Immunity In Vivo

Alpha interferon (IFN-α) suppresses human immunodeficiency virus type 1 (HIV-1) replication in vitro by inducing cell-intrinsic retroviral restriction mechanisms. We investigated the effects of IFN-α/ribavirin (IFN-α/riba) treatment on 34 anti-HIV-1 restriction factors in vivo. Expression of several anti-HIV-1 restriction factors was significantly induced by IFN-α/riba in HIV/hepatitis C virus (HCV)-coinfected individuals. Fold induction of cumulative restriction factor expression in CD4+ T cells was significantly correlated with viral load reduction during IFN-α/riba treatment (r2 = 0.649; P < 0.016). Exogenous IFN-α induces supraphysiologic restriction factor expression associated with a pronounced decrease in HIV-1 viremia.

The potential of microfluidic lung epithelial wounding: towards in vivo-like alveolar microinjuries

Idiopathic pulmonary fibrosis (IPF) remains a major clinical challenge to date. Repeated alveolar epithelial microinjuries are considered as the starting point and the key event in both the development and the progression of IPF. Various pro-fibrotic agents have been identified and shown to cause alveolar damage. In IPF, however, no leading cause of alveolar epithelial microinjuries can be identified and the exact etiology remains elusive. New results from epidemiologic studies suggest a causal relation between IPF and frequent episodes of gastric refluxes resulting in gastric microaspirations into the lung. The effect of gastric contents on the alveolar epithelium has not been investigated in detail. Here, we present a microfluidic lung epithelial wounding system that allows for the selective exposure of alveolar epithelial cells to gastric contents. The system is revealed to be robust and highly reproducible. The thereby created epithelial microwounds are of tiny dimensions and best possibly reproduce alveolar damage in the lung. We further demonstrate that exposure to gastric contents, namely hydrochloric acid (HCl) and pepsin, directly damages the alveolar epithelium. Together, this novel in vitro wounding system allows for the creation of in vivo-like alveolar microinjuries with the potential to study lung injury and alveolar wound repair in vitro.

Indium-111 labeled gold nanoparticles for in-vivo molecular targeting.

The present report describes the synthesis and biological evaluation of a molecular imaging platform based on gold nanoparticles directly labeled with indium-111. The direct labeling approach facilitated radiolabeling with high activities while maintaining excellent stability within the biological environment. The resulting imaging platform exhibited low interference of the radiolabel with targeting molecules, which is highly desirable for in-vivo probe tracking and molecular targeted tumor imaging. The indium-111 labeled gold nanoparticles were synthesized using a simple procedure that allowed stable labeling of the nanoparticle core with various indium-111 activities. Subsequent surface modification of the particle cores with RGD-based ligands at various densities allowed for molecular targeting of the αvß3 integrin in-vitro and for molecular targeted imaging in human melanoma and glioblastoma models in-vivo. The results demonstrate the vast potential of direct labeling with radioisotopes for tracking gold nanoparticles within biological systems.

Potassium and the excitability properties of normal human motor axons in vivo.

Hyperkalemia is an important cause of membrane depolarization in renal failure. A recent theoretical model of axonal excitability explains the effects of potassium on threshold electrotonus, but predicts changes in superexcitability in the opposite direction to those observed. To resolve this contradiction we assessed the relationship between serum potassium and motor axon excitability properties in 38 volunteers with normal potassium levels. Most threshold electrotonus measures were strongly correlated with potassium, and superexcitability decreased at higher potassium levels (P = 0.016), contrary to the existing model. Improved modelling of potassium effects was achieved by making the potassium currents obey the constant-field theory, and by making the potassium permeabilities proportional to external potassium, as has been observed in vitro. This new model also accounted well for the changes in superexcitability and other excitability measures previously reported in renal failure. These results demonstrate the importance of taking potassium levels into account when assessing axonal membrane dysfunction by excitability testing, and provide evidence that potassium currents are activated by external potassium in vivo.

Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo.

Eosinophils natively inhabit the small intestine, but a functional role for them there has remained elusive. Here, we show that eosinophil-deficient mice were protected from induction of Th2-mediated peanut food allergy and anaphylaxis, and Th2 priming was restored by reconstitution with il4(+/+) or il4(-/-) eosinophils. Eosinophils controlled CD103(+) dendritic cell (DC) activation and migration from the intestine to draining lymph nodes, events necessary for Th2 priming. Eosinophil activation in vitro and in vivo led to degranulation of eosinophil peroxidase, a granule protein whose enzymatic activity promoted DC activation in mice and humans in vitro, and intestinal and extraintestinal mouse DC activation and mobilization to lymph nodes in vivo. Further, eosinophil peroxidase enhanced responses to ovalbumin seen after immunization. Thus, eosinophils can be critical contributors to the intestinal immune system, and granule-mediated shaping of DC responses can promote both intestinal and extraintestinal adaptive immunity.

Evidence for a role of sterol 27-hydroxylase in glucocorticoid metabolism in vivo

The intracellular availability of glucocorticoids is regulated by the enzymes 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11β-hydroxysteroid dehydrogenase 2 (HSD11B2). The activity of HSD11B1 is measured in the urine based on the (tetrahydrocortisol+5α-tetrahydrocortisol)/tetrahydrocortisone ((THF+5α-THF)/THE) ratio in humans and the (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) ratio in mice. The cortisol/cortisone (F/E) ratio in humans and the corticosterone/11-dehydrocorticosterone (B/A) ratio in mice are markers of the activity of HSD11B2. In vitro agonist treatment of liver X receptor (LXR) down-regulates the activity of HSD11B1. Sterol 27-hydroxylase (CYP27A1) catalyses the first step in the alternative pathway of bile acid synthesis by hydroxylating cholesterol to 27-hydroxycholesterol (27-OHC). Since 27-OHC is a natural ligand for LXR, we hypothesised that CYP27A1 deficiency may up-regulate the activity of HSD11B1. In a patient with cerebrotendinous xanthomatosis carrying a loss-of-function mutation in CYP27A1, the plasma concentrations of 27-OHC were dramatically reduced (3.8 vs 90-140 ng/ml in healthy controls) and the urinary ratios of (THF+5α-THF)/THE and F/E were increased, demonstrating enhanced HSD11B1 and diminished HSD11B2 activities. Similarly, in Cyp27a1 knockout (KO) mice, the plasma concentrations of 27-OHC were undetectable (<1 vs 25-120 ng/ml in Cyp27a1 WT mice). The urinary ratio of (THB+5α-THB)/THA was fourfold and that of B/A was twofold higher in KO mice than in their WT littermates. The (THB+5α-THB)/THA ratio was also significantly increased in the plasma, liver and kidney of KO mice. In the liver of these mice, the increase in the concentrations of active glucocorticoids was due to increased liver weight as a consequence of Cyp27a1 deficiency. In vitro, 27-OHC acts as an inhibitor of the activity of HSD11B1. Our studies suggest that the expression of CYP27A1 modulates the concentrations of active glucocorticoids in both humans and mice and in vitro.

In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.