In vivo imaging of protease activity in arthritis: a novel approach for monitoring treatment response

OBJECTIVE: Sensitive noninvasive strategies for monitoring treatment response in rheumatoid arthritis (RA) would be valuable for facilitating appropriate therapy and dosing, evaluating clinical outcome, and developing more effective drugs. Because different proteases are highly up-regulated in RA and contribute significantly to joint destruction, in the present study we investigated whether such enzymes are suitable in vivo imaging biomarkers for early evaluation of treatment response in a murine model of RA. METHODS: Using a protease-activated near-infrared fluorescence (NIRF) imaging "smart" probe, we examined the presence and distribution of fluorescence in arthritic joints of mice with collagen-induced arthritis by both noninvasive fluorescence imaging and histology. Proteases that target the Lys-Lys cleavage site, including cathepsin B, activate probe fluorescence. Treatment monitoring data were obtained following methotrexate (MTX) therapy. RESULTS: Twenty-four hours after intravenous injection of the protease sensor, affected toes and paws of arthritic mice showed significantly higher fluorescence intensity than did toes and paws of healthy mice. Fluorescence from the protease probe and cathepsin B antibody histologic staining were localized in the vast majority of cells in the inflamed synovium. In arthritic animals treated with MTX (35 mg of MTX/kg 48 hours prior to probe injection), a significantly lower fluorescent signal (inflamed paws 50%, inflamed toes 70%) was observed as compared with untreated arthritic animals. CONCLUSION: Protease-activated NIRF probes are sensitive means of imaging the presence of target enzymes in arthritic joints and can be used for early monitoring of treatment response to antirheumatic drugs such as MTX.     

Carbon monoxide, but not endothelin-1, plays a major role for the hepatic microcirculation in a murine model of early systemic inflammation

OBJECTIVE: Endothelin-1 and carbon monoxide play a major role in the regulation of liver microcirculation in numerous disease states. During sepsis and endotoxemia, elevated formation of endothelin-1 results in reduced sinusoidal blood flow. However, the role of carbon monoxide and endothelin-1 and its receptors endothelin receptor A and endothelin receptor B in the deranged liver microcirculation during early systemic inflammation remains unclear. DESIGN: Prospective, randomized, controlled experiment. SETTING: University animal laboratory. SUBJECTS: Male C57/BL6 mice, weighing 23-27 g. INTERVENTIONS: To induce a systemic inflammation, mice were treated with 1 hr of bilateral hind limb ischemia followed by 3 hrs or 6 hrs of reperfusion. Animals were randomly exposed to the nonselective endothelin receptor antagonist Ro-61-6612 (Tezosentan) and/or a continuous endothelin-1 infusion. Different animals were randomized to methylene chloride gavage or carbon monoxide inhalation during the reperfusion period. MEASUREMENTS AND MAIN RESULTS: After ischemia/reperfusion, endothelin-1 plasma concentrations, endothelin-1 messenger RNA expression, and endothelin receptor A and B messenger RNA expression revealed no significant changes when compared with sham animals. After 6 hrs of ischemia/reperfusion, hepatic microcirculatory variables (sinusoidal density, sinusoidal diameter, and red blood cell velocity) deteriorated. Tezosentan after 6 hrs of ischemia/reperfusion did not improve the liver microcirculation, whereas the continuous infusion of endothelin-1 after 6 hrs of ischemia/reperfusion further impaired sinusoidal blood flow. Tezosentan treatment did not produce any alterations in hepatocellular injury or hepatic redox status when compared with the untreated animals receiving 6 hrs of ischemia/reperfusion. Animals receiving 6 hrs of ischemia/reperfusion and exposed to methylene chloride gavage or inhaled carbon monoxide during limb reperfusion showed significantly improved microcirculatory variables, hepatic redox status, and attenuated hepatocellular injury. CONCLUSIONS: These data suggest that endothelin-1 and the endothelin receptors A and B are not responsible for the observed hepatic microcirculatory and cellular dysfunction during early systemic inflammation, but exposure to exogenous carbon monoxide protected the hepatic microcirculation and improved the impaired hepatic cellular integrity and the hepatocellular redox status.     

Hypogonadism in HIV-1-infected men is common and does not resolve during antiretroviral therapy

OBJECTIVES: To assess the prevalence of abnormal testosterone and gonadotropin values in HIV-infected men before and after 2 years of combination antiretroviral therapy (cART). DESIGN: Multicentre cohort of HIV-infected adults. METHODS: We identified 139 Caucasian antiretroviral-naive male patients who started zidovudine/ lamivudine-based cART that was virologically successful over a 2 year period. Ninety-seven were randomly chosen and plasma hormone determinations of free testosterone (fT) and luteinizing hormone (LH) at baseline and after 2 years of cART were evaluated. RESULTS: At baseline 68 patients (70%) had subnormal fT levels. In these, LH levels were low in 44%, normal in 47% and high in 9%. There was a trend for an association between lower CD4+ T-cell counts and hypogonadism. Most participants had normal FSH levels. No significant changes of fT, LH and FSH levels were observed after 2 years of cART. CONCLUSIONS: Low fT levels, mainly with normal or low LH levels and thus indicating secondary hypogonadism, are found in the majority of HIV-infected men and do not resolve during 2 years of successful cART.     

Optical imaging of vascular pathophysiology

Pathophysiological processes in the vascular system are the major cause of mortality and disease. Atherosclerosis, an inflammatory process in arterial walls, can lead to formation of plaques, whose rupture can lead to thrombus formation, obstruction of vessels (thrombosis), reduction of the blood flow (ischemia), cell death in the tissue fed by the occluded vessel, and depending on the affected vessel, to myocardial infarction or stroke. Imaging techniques enabling visualization of the biological processes involved in this scenario are therefore highly desirable. In recent years, a number of reporter agents and reporter systems have been developed to visualize these processes using different imaging modalities including nuclear imaging techniques, such as positron emission tomography or single photon emission computed tomography, magnetic resonance imaging, and ultrasound. This article comprises a brief overview of optical imaging techniques, such as fluorescence imaging and bioluminescence imaging for the visualization of vascular pathophysiology.     

Studies concerning the glucosyltransferase of Streptococcus sanguis

We have shown in previous studies that the glucosyltransferase (Gtf) enzymes of Streptococcus mutans have distinct properties when adsorbed to a surface. In the present study, we compared the activity of Gtf from Streptococcus sanguis, designated GtfSs, in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads, and determined the ability of its product glucan to support the adherence of oral microorganisms. Gtf from S. sanguis 804 NCTC 10904 was purified from culture supernatant fluids by means of hydroxyapatite chromatography. Enzyme and the substrate were prepared in buffers at pH values from 3.5 to 7.5. Maximum activity of GtfSs occurred between pH 5.5 and pH 6.5, whether in solution or adsorbed onto a surface. The solubilized and insolubilized enzymes showed highest activity at 40 degrees C; activity was reduced by 50(+/-2)% at 20 and 30 degrees C. The enzyme did not form glucans in either phase at 10 or 60 degrees C. The K(m), determined from Lineweaver-Burk plots, for the enzyme in solution was 4.3(+/-0.4) mmol/l sucrose, and the K(m) for the enzyme on sHA beads was 5.0(+/-1.0) mmol/l sucrose. The ability of the GtfSs glucan synthesized on the surface of sHA beads to support the adherence of oral bacteria was investigated. (3)H-thymidine-labeled bacteria (S. mutans GS-5, S. sobrinus 6715, S. sobrinus 6716, S. sanguis 10904, Actinomyces viscosus OMZ105E, A. viscosus 2085, and A. viscosus 2086) were incubated with sHA beads coated with GtfSs glucan. S. mutans GS-5 displayed the highest level of binding numerically. These results show that the GtfSs of S. sanguis is active on sHA beads, that the pH optimum for activity on a surface differs slightly from that in solution, and that its product glucan can support the adherence of oral microorganisms.     

The role of endogenous heme oxygenase in the initiation of liver injury following limb ischemia/reperfusion

BACKGROUND/AIMS: Heme oxygenase (HO) derived liver protection was tested in mice following 1 h bilateral hindlimb ischemia and either 1.5 or 3 h reperfusion. METHODS: Groups consisted of limb ischemia/reperfusion (I/R), sham (no I/R), I/R+chromium mesoporphyrin (I/R+CrMP;40 micromol/kg, i.p.), or I/R+hemin (10 mg/kg, i.p.). The vital dye propidium iodide (PI), was used to measure hepatocellular death (#/0.1 mm(3)), while the number of sinusoids perfused by red blood cells (SP(RBC)) were measured from the periportal (Pp) and pericentral (Pc) zones of liver acini using intravital microscopy. Whole organ injury was estimated from serum alanine aminotransferase (ALT). RESULTS: SP(RBC) reduced within 1.5 h with no further decline following 3 h. CrMP resulted in a dramatic loss of SP(RBC) following 3 h only. Hemin restored perfusion in both zones. Hepatocellular death and organ injury increased at 1.5 and 3 h. At 1.5 h, CrMP further increased cell death in the Pc zone, as well as whole organ injury, while hemin restored cell viability. Increased HO mRNA, protein and activity suggested induction within 3 h. CONCLUSIONS: HO does not protect perfusion during the early stage (1.5 h), but becomes increasingly important in preserving liver perfusion and cell viability during the later stage (3 h) of liver injury.     

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of ¹²C¹⁸O¹⁶O from a red cell suspension as the ¹⁸O exchanges between labeled CO₂ + HCO₃⁻ and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO₃⁻ (P_m,HCO⁻3). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO₃⁻/Cl⁻ transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced P_m,HCO⁻3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in P_m,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO₂ and HCO₃⁻, we found that DIDS inhibited both P_m,HCO⁻3 and P_m,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO₂ permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO₂ exchange.     

Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2

Tumors have been thought to initiate as avascular aggregates of malignant cells that only later induce vascularization. Recently, this classic concept of tumor angiogenesis has been challenged by the suggestion that tumor cells grow by co-opting preexisting host vessels and thus initiate as well-vascularized tumors without triggering angiogenesis. To discriminate between these two mechanisms, we have used intravital epifluorescence microscopy and multi-photon laser scanning confocal microscopy to visualize C6 microglioma vascularization and tumor cell behavior. To address the mechanisms underlying tumor initiation, we assessed the expression of VEGF, VEGF receptor-2 (VEGFR-2), and angiopoietin-2 (Ang-2), as well as endothelial cell proliferation. We show that multicellular aggregates (< 1 mm(3)) initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Host blood vessels are not co-opted by tumor cells but rather are used as trails for tumor cell invasion of the host tissue. Our data further suggest that the established microvasculature of growing tumors is characterized by a continuous vascular remodeling, putatively mediated by the expression of VEGF and Ang-2. The results of this study suggest a new concept of vascular tumor initiation that may have important implications for the clinical application of antiangiogenic strategies.