The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers.

During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1–coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM.

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Objective-This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury of salt-sensitive hypertension. Methods and Results-Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2) hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan and pravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endotheliumdependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan prevented vascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin, whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effects between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelial dysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity by olmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS. Conclusions-Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differential pleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB with statin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension. Key Words: eNOS dimers Ⅲ DHFR Ⅲ oxidative stress Ⅲ vascular injury Ⅲ combined ARB and statin A ccumulating evidence indicates that renin-angiotensin system (RAS) plays a crucial role in the pathophysiology of cardiovascular diseases in hypertension, and that RAS blockers, including angiotensin-converting enzyme inhibitors and AT1 receptor blockers (ARB), are the useful therapeutic agents for hypertensive cardiovascular diseases. 1 As hypertension is often accompanied by dyslipidemia in the same patients, their treatment frequently involves the combination of RAS blockers with 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statins), potent inhibitors of cholesterol biosynthesis. Clinical evidence show that statins improve endothelial dysfunction and reduce the incidence of atherosclerotic events, 2-5 and these vascular protective effects by statins are at least partially attributed to their pleiotropic vascular effects beyond lowering of plasma cholesterol. 4 -6 However, the difference in vascular pleiotropic effects between RAS blockers and statins remains to be fully understood. Moreover, the significance and the advantage of their combination therapy in hypertension, particularly salt-sensitive hypertension, are not defined.Clinically, salt-sensitive hypertensive patients are more prone to cardiovascular diseases than their salt-insensitive counterparts. 7,8 Therefore, it is a key clinical is...

Our work provided evidence that the increase in heart rate per se, independent of sympathetic nerve activity, enhances cardiac oxidative stress and activates mitogen-activated protein kinases, which seem to be responsible for cardiac remodelling. Azelnidipine, without causing an increase in heart rate, has the potential to be useful for the treatment of cardiac remodelling.

TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1 and that belongs to a tyrosine kinase-like branch in the kinase tree of the human genome. In the present study we investigated the role of TNNI3K in the cardiac myogenesis process and in the repair of ischemic injury. Pluripotent P19CL6 cells with or without transfection by pcDNA6-TNNI3K plasmid were used to induce differentiation into beating cardiomyocytes. TNNI3K promoted the differentiation process, judging from the increasing beating mass and increased number of alpha-actinin-positive cells. TNNI3K improved cardiac function by enhancing beating frequency and increasing the contractile force and epinephrine response of spontaneous action potentials without an increase of the single-cell size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V(+) cells, Bax protein, and p38/JNK-mediated apoptosis. Intramyocardial administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial infarction improved cardiac performance and attenuated ventricular remodeling compared with injection of wild-type P19CL6 cells. In conclusion, our study clearly indicates that TNNI3K promotes cardiomyogenesis, enhances cardiac performance, and protects the myocardium from ischemic injury by suppressing p38/JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a useful therapeutic approach for ischemic cardiac disease.

oth coronary artery bypass grafting and percutaneous coronary intervention ameliorate angina pectoris, prevent myocardial infarction and improve the long-term survival of patients with atherosclerotic coronary artery disease. However, the nature of coronary arteries significantly impacts the quality of life. Standard therapies for myocardial revascularization are often limited because of diffuse lesions or small-caliber vessels. Angiogenic therapy to induce myocardial neovascularization is not dependent on vessel caliber and provides an alternative treatment alone or in combination with standard revascularization. Basic fibroblast growth factor (bFGF) is a potent angiogenic protein that induces endothelial and smooth muscle cell proliferation in vivo and elicits angiogenesis that includes the migration and proliferation of endothelial cells, vascular tube formation and linkage to the extant vascular network. 1 Intracoronary injections of bFGF reduce infarct size in a canine model of myocardial infarction and improve myocardial function in chronically ischemic porcine hearts. 2,3 We have previously shown that the intramyocardial administration of bFGF increased the number of capillaries and arterioles in the peri-infarct region, increased regional myocardial blood flow and consequently improved ven- Circulation Journal Vol.70, April 2006tricular function in a canine infarction model. 4 However, angiogenesis induced by growth factors has not been always successful. Despite high binding affinity for acidic polysaccharides such as heparin and heparan sulfate in the extracellular matrix, bFGF has a short biological half-life in tissues. 5 The sustained release of bFGF might help to enhance its angiogenic activity in vivo. 6,7 Gelatin is a nontoxic, biodegradable, natural polymer with low antigenicity and gelatin hydrogel is considered to be a preferable matrix for the sustained release of protein drugs, such as growth factors. 8,9 However, the effects of intramyocardial injections of slow-release bFGF on vascular growth, cardiomyocyte apoptosis and cardiac function have not been investigated in detail. The present study evaluates the ability of gelatin hydrogels to enhance the benefits of bFGF on neoangiogenesis, cardiomyocyte apoptosis, myocardial fibrosis, ventricular remodeling and cardiac function in a rat infarction model. Methods Preparation of bFGFHuman recombinant bFGF and gelatin hydrogels were provided by Kaken Pharmaceutical, Tokyo, Japan. Gelatin with an isoelectric point of 4.9 was prepared using alkaline treatment of bovine collagen with Ca(OH)2. To obtain gelatin hydrogels that incorporated bFGF, 0.7 ml of saline containing 350 g of bFGF was impregnated into freezedried hydrogels. The release of biologically active bFGF is sustained as a result of hydrogel degradation in vivo. Empty gelatin hydrogels free of bFGF were similarly prepared. Background Basic fibroblast growth factor (bFGF) stimulates neoangiogenesis. Incorporation into biodegradable gelatin hydrogels provides the sustained release of bFGF....