Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

Maltohexaose-indocyanine green (MH-ICG) for near infrared imaging of endocarditis.

Infectious endocarditis is a life-threatening disease, and diagnostics are urgently needed to accurately diagnose this disease especially in the case of prosthetic valve endocarditis. We show here that maltohexaose conjugated to indocyanine green (MH-ICG) can detect Staphylococcus aureus (S. aureus) infection in a rat model of infective endocarditis. The affinity of MH-ICG to S. aureus was determined and had a Km and Vmax of 5.4 µM and 3.0 X 10-6 µmol/minutes/108 CFU, respectively. MH-ICG had no detectable toxicity to mammalian cells at concentrations as high as 100 µM. The in vivo efficiency of MH-ICG in rats was evaluated using a right heart endocarditis model, and the accumulation of MH-ICG in the bacterial vegetations was 2.5 ± 0.2 times higher than that in the control left ventricular wall. The biological half-life of MH-ICG in healthy rats was 14.0 ± 1.3 minutes, and approximately 50% of injected MH-ICG was excreted into the feces after 24 hours. These data demonstrate that MH-ICG was internalized by bacteria with high specificity and that MH-ICG specifically accumulated in bacterial vegetations in a rat model of endocarditis. These results demonstrate the potential efficacy of this agent in the detection of infective endocarditis.

Novel PET and Near Infrared Imaging Probes for the Specific Detection of Bacterial Infections Associated With Cardiac Devices.

OBJECTIVES: The aim of this study was to develop imaging agents to detect early stage infections in implantable cardiac devices. BACKGROUND: Bacteria ingest maltodextrins through the specific maltodextrin transporter. We developed probes conjugated with either a fluorescent dye (maltohexaose fluorescent dye probe [MDP]) or a F-18 (F18 fluoromaltohexaose) and determined their usefulness in a model of infections associated with implanted cardiac devices. METHODS: Stainless steel mock-ups of medical devices were implanted subcutaneously in rats. On post-operative day 4, animals were injected with either Staphylococcus aureus around the mock-ups to induce a relatively mild infection or oil of turpentine to induce noninfectious inflammation. Animals with a sterile implant were used as control subjects. On post-operative day 6, either the MDP or F18 fluoromaltohexaose was injected intravenously, and the animals were scanned with the appropriate imaging device. Additional positron emission tomography imaging studies were performed with F18-fluorodeoxyglucose as a comparison of the specificity of our probes (n = 5 to 9 per group). RESULTS: The accumulation of the MDP in the infected rats was significantly increased at 1 h after injection when compared with the control and noninfectious inflammation groups (intensity ratio 1.54 ± 0.07 vs. 1.26 ± 0.04 and 1.20 ± 0.05, respectively; p < 0.05) and persisted for more than 24 h. In positron emission tomography imaging, both F18 fluoromaltohexaose and F18 fluorodeoxyglucose significantly accumulated in the infected area 30 min after the injection (maximum standard uptake value ratio 4.43 ± 0.30 and 4.87 ± 0.28, respectively). In control rats, there was no accumulation of imaging probes near the device. In the noninfectious inflammation rats, no significant accumulation was observed with F18 fluoromaltohexaose, but F18 fluorodeoxyglucose accumulated in the mock-up area (maximum standard uptake value 2.53 ± 0.39 vs. 4.74 ± 0.46, respectively; p < 0.05). CONCLUSIONS: Our results indicate that maltohexaose-based imaging probes are potentially useful for the specific and sensitive diagnosis of infections associated with implantable cardiac devices.

A Trimethoprim Conjugate of Thiomaltose Has Enhanced Antibacterial Efficacy In Vivo.

Trimethoprim is one of the most widely used antibiotics in the world. However, its efficacy is frequently limited by its poor water solubility and dose limiting toxicity. Prodrug strategies based on conjugation of oligosaccharides to trimethoprim have great potential for increasing the solubility of trimethoprim and lowering its toxicity, but they have been challenging to develop due to the sensitivity of trimethoprim to chemical modifications, and the rapid degradation of oligosaccharides in serum. In this report, we present a trimethoprim conjugate of maltodextrin termed TM-TMP, which increased the water solubility of trimethoprim by over 100 times, was stable to serum enzymes, and was active against urinary tract infections in mice. TM-TMP is composed of thiomaltose conjugated to trimethoprim, via a self-immolative disulfide linkage, and releases 4'-OH-trimethoprim (TMP-OH) after disulfide cleavage, which is a known metabolic product of trimethoprim and is as potent as trimethoprim. TM-TMP also contains a new maltodextrin targeting ligand composed of thiomaltose, which is stable to hydrolysis by serum amylases and therefore has the metabolic stability needed for in vivo use. TM-TMP has the potential to significantly improve the treatment of a wide number of infections given its high water solubility and the widespread use of trimethoprim.

PET imaging of bacterial infections with fluorine-18-labeled maltohexaose.

A positron emission tomography (PET) tracer composed of (18)F-labeled maltohexaose (MH(18)F) can image bacteria in vivo with a sensitivity and specificity that are orders of magnitude higher than those of fluorodeoxyglucose ((18)FDG). MH(18)F can detect early-stage infections composed of as few as 10(5) E. coli colony-forming units (CFUs), and can identify drug resistance in bacteria in vivo. MH(18)F has the potential to improve the diagnosis of bacterial infections given its unique combination of high specificity and sensitivity for bacteria.

Mesenchymal stem cell-based prostacyclin synthase gene therapy for pulmonary hypertension rats.

Mesenchymal stem cells (MSCs) are the pluripotent cells, which enter the circulation and home to sites of tissue injury or inflammation. MSCs are highlighted as a potential cell vector for gene therapy. In this study, we investigated whether transplanted allogeneic MSCs preferentially accumulate in the lung in rats with pulmonary hypertension (PH) and if so to determine the efficacy of MSC-based prostacyclin synthase (PCS) gene therapy for PH. PH was induced in Lewis rats by injecting monocrotaline at 7-weeks-old (week 0). MSCs were obtained by culturing bone marrow mononuclear cells. Allogeneic MSCs were intravenously transplanted at week 2 when moderate PH had been established. PH enhanced indium-111-oxine-labeled MSC accumulation in the lungs, but not in other organs, 2.5-times and 6-times, 1 and 14 days after transplantation, respectively. Transplantation of MSCs transduced with PCS (PSC-MSCs), but not with GFP (GFP-MSCs), reduced PH, pulmonary arterial thickening, and RV hypertrophy at week 4. The lung prostacyclin production was impaired in PH rats, which was restored and maintained for long time by PCS-MSCs, but not by GFP-MSCs. The survival rate at week 7 was 100% in PCS-MSC-transplanted PH rats, whereas they were 38 and 44% in PH rats and GFP-MSC-transplanted PH rats, respectively. In conclusion, the gene-engineered MSCs would be a suitable cell vector for gene delivery specifically to the PH lung. The allogeneic PCS-MSC transplantation attenuated PH and cardiovascular remodeling, and improved the prognosis in PH rats. The MSC-based PCS gene therapy may be a promising strategy for PH treatment.

Stanford type B aortic dissection associated with pregnancy in patients with Marfan syndrome-A case report and review of the literature.

A 36-year-old female patient known to have Marfan syndrome (MFS) presented with Stanford type B aortic dissection (type B-AD) 3 days after delivery although she had taken oral ß-blocker and underwent prophylactic cesarean section at 34 weeks when she showed 42 mm of the ascending aorta. She was successfully treated medically without further progression of the dissection. A review of the literature revealed an additional 19 patients with MFS who suffered from type B-AD associated with pregnancy. Of 20 patients, 1 (5%) died but the remaining 19 patients were successfully treated either medically (n = 9) or surgically (n = 10). Of 13 patients whose aortic diameter was known, 5 showed <40 mm of the ascending aorta. Pregnancy in MFS can be complicated by type B-AD with a peak around term delivery irrespective of the size of ascending aorta and even with ß-blocker.

[Gene therapy and cell therapy for idiopathic pulmonary arterial hypertension].

Idiopathic pulmonary arterial hypertension is a rare, but severe disease with high mortality. The endothelial dysfunction is a major pathogenesis of pulmonary arterial hypertension. Gene therapy, cell therapy and their combination therapy are developed to improve pulmonary hypertension by restoring the impaired endothelial function or regenerating endothelial cells. Although these therapies are still in experimental stage, they might be promising for pulmonary arterial hypertension. Here, we review the characteristics of the gene therapy, the cell therapy and the combination of them.

Pressure overload-induced transient oxidative stress mediates perivascular inflammation and cardiac fibrosis through angiotensin II.

Oxidative stress is implicated in the pathogenesis of various cardiovascular diseases. We have shown that in Wistar rats with a suprarenal aortic constriction (AC), pressure overload-induced transient perivascular inflammation (monocyte chemoattractant protein-1 [MCP-1] induction and macrophage accumulation) in the early phase is the determinant of reactive myocardial fibrosis and resultant diastolic dysfunction in the late phase. Thus, we investigated the role of reactive oxygen species production in cardiac remodeling in AC rats. Superoxide production and the footprint of lipid peroxidation were assessed using dihydroethidium staining and immunohistostaining against 4-hydroxy-2-nonenal (4-HNE), respectively. In sham rats, dihydroethidium and 4-HNE signals were scarcely found in the heart. At day 3, AC rats showed dihydroethidium signals mainly in the intramyocardial arterial wall, whereas modest 4-HNE staining was observed diffusely in the myocardium. These signals declined to lower levels by day 14 despite sustained hypertension. Chronic administration of a subdepressor dose of an angiotensin II type 1 receptor blocker candesartan reduced the pressure overload-induced dihydroethidium and 4-HNE signals at day 3. Moreover, candesartan decreased MCP-1 induction and macrophage infiltration at day 3 and prevented myocardial fibrosis at day 14, without affecting left ventricle and myocyte hypertrophy. In conclusion, acute pressure overload induced self-limited superoxide production mainly in the vascular wall. The reactive oxygen species production would contribute to the perivascular inflammation and subsequent myocardial fibrosis. Angiotensin II was suggested to have a pressure-independent effect on the reactive oxygen species production.

Pressure-independent effects of angiotensin II on hypertensive myocardial fibrosis.

Angiotensin II (Ang II) is implicated in the proinflammatory process in various disease situations. Thus, we sought to determine the role of Ang II in early inflammation-induced fibrosis of pressure-overloaded (PO) hearts. PO was induced by suprarenal aortic constriction (AC) at day 0 in male Wistar rats, and they were orally administered 0.1 mg/kg per day candesartan every day from day -7. This was the maximum dose of candesartan that did not change arterial pressure in hypertensive rats with AC (AC rats). In AC rats, cardiac angiotensin-converting enzyme (ACE) activity was transiently enhanced after day 1 and peaked at day 3, declining to lower levels by day 14, whereas serum ACE activity was not changed. In AC rats, PO induced early fibroinflammatory changes (monocyte chemoattractant factor [MCP]-1 and transforming growth factor [TGF]-beta expression, perivascular macrophage accumulation, and fibroblast proliferation), and thereafter, left ventricular hypertrophy developed, featuring myocyte hypertrophy, intramyocardial arterial wall thickening, and perivascular and interstitial fibroses. Candesartan suppressed the induction of MCP-1 and TGF-beta and reduced macrophage accumulation and fibroblast proliferation in PO hearts. Candesartan significantly prevented perivascular and interstitial fibrosis. However, candesartan did not affect myocyte hypertrophy and arterial wall thickening. In conclusion, a subdepressor dose of candesartan prevented the MCP-1-mediated inflammatory process and reactive myocardial fibrosis in PO hearts. Ang II might play a key role in reactive fibrosis in hypertensive hearts, independent of arterial pressure changes.

Roles of intercellular adhesion molecule-1 in hypertensive cardiac remodeling.

Recently, we have shown that in rats with a suprarenal abdominal aortic constriction (AC), pressure overload induces early perivascular fibro-inflammatory changes (transforming growth factor [TGF]-beta induction and fibroblast proliferation) within the first week after AC and then causes the development of cardiac remodeling (myocyte hypertrophy and reactive myocardial fibrosis) associated with diastolic dysfunction. Intercellular adhesion molecule (ICAM)-1 is implicated in the recruitment of leukocytes, especially macrophages, in various inflammatory situations. Thus, we sought to investigate the causal relation of ICAM-1 to macrophage recruitment and cardiac remodeling in AC rats. In AC rats, immunoreactive ICAM-1 was observed transiently on endothelial cells of the intramyocardial coronary arterioles after day 1, with a peak at day 3, returning to baseline by day 7. Also, ED1+ macrophage accumulation was found in the area adjacent to the arteries expressing ICAM-1. Chronic treatment with an anti-ICAM-1 neutralizing antibody, but not with control IgG, remarkably reduced the accumulations of macrophages and proliferative fibroblasts and inhibited the upregulation of TGF-beta expression. Furthermore, the neutralizing antibody significantly prevented myocardial fibrosis without affecting arterial pressure and left ventricular and myocyte hypertrophy. In conclusion, ICAM-1 expression was induced by pressure overload in the intramyocardial arterioles, and triggered perivascular macrophage accumulation. In pressure-overloaded hearts, a crucial role in ICAM-1-mediated macrophage accumulation was suggested in the development of myocardial fibrosis, through TGF-beta induction and fibroblast activation.

Rho-kinase inhibition reduces neointima formation after vascular injury by enhancing Bax expression and apoptosis.

Recently, we have shown that a specific Rho-kinase inhibitor, Y27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), prevents neointima formation after vascular injury associated with increased terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL)+ smooth muscle cells. Because the mechanism of the action of Y27632 remains unclear, we investigated the expression changes in Bcl family proteins, apoptosis regulators of smooth muscle cells, in the rat carotid artery after balloon injury (BI). Y27632 (BI + Y group) or saline (BI group) was administered peritoneally from Day 1 to Day 14 after BI. Y27632 markedly prevented neointima formation at Day 14. In the BI group, TUNEL+ smooth muscle cells were transiently increased in the neointima, but not in the media, with a peak at Day 7, returning to a lower level by Day 14. Y27632 significantly increased TUNEL+ smooth muscle cells at Days 7 and 14. Smooth muscle cell apoptosis was confirmed by electron microscopic examination. At Day 14, although proapoptotic Bax was slightly, but not significantly, increased in the BI group, it was significantly upregulated in the BI + Y group. Antiapoptotic Bcl-xL was upregulated in the BI group, and the upregulated Bcl-xL was not affected by Y27632. These findings indicate that Rho-kinase inhibition induces neointimal smooth muscle cell apoptosis through Bax upregulation, resulting in reduced neointima formation.