Description: The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity in vivo , is required for efficient infection of human dendritic cells (DCs) but not B and T cells in vitro . We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for ex vivo human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-α1, IFN-β, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity in vivo , suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.

Description: Oxidative stress promotes vascular dysfunction in chronic kidney disease (CKD). We utilized the cutaneous circulation to test the hypothesis that reactive oxygen species derived from NADPH oxidase and xanthine oxidase impair nitric oxide (NO)-dependent cutaneous vasodilation in CKD. Twenty subjects, 10 stage 3 and 4 patients with CKD (61 ± 4 yr; 5 men/5 women; eGFR: 39 ± 4 ml·min –1 ·1.73 m –2 ) and 10 healthy controls (55 ± 2 yr; 4 men/6 women; eGFR: 〉60 ml·min –1 ·1.73 m –2 ) were instrumented with 4 intradermal microdialysis fibers for the delivery of 1 ) Ringer solution (Control), 2 ) 10 μM tempol (scavenge superoxide), 3 ) 100 μM apocynin (NAD(P)H oxidase inhibition), and 4 ) 10 μM allopurinol (xanthine oxidase inhibition). Skin blood flow was measured via laser-Doppler flowmetry during standardized local heating (42°C). N g -nitro- l -arginine methyl ester ( l -NAME; 10 mM) was infused to quantify the NO-dependent portion of the response. Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum CVC achieved during sodium nitroprusside infusion at 43°C. Cutaneous vasodilation was attenuated in patients with CKD (77 ± 3 vs. 88 ± 3%, P = 0.01), but augmented with tempol and apocynin (tempol: 88 ± 2 ( P = 0.03), apocynin: 91 ± 2% ( P = 0.001). The NO-dependent portion of the response was reduced in patients with CKD (41 ± 4 vs. 58 ± 2%, P = 0.04), but improved with tempol and apocynin (tempol: 58 ± 3 ( P = 0.03), apocynin: 58 ± 4% ( P = 0.03). Inhibition of xanthine oxidase did not alter cutaneous vasodilation in either group ( P 〉 0.05). These data suggest that NAD(P)H oxidase is a source of reactive oxygen species and contributes to microvascular dysfunction in patients with CKD.

Description: Background— The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A , and regulatory proteins that reduce or eliminate sodium current ( I Na ) have been linked to BrS. We studied the properties of a BrS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H. Methods and Results— In vitro PKA phosphorylation was detected in the I-II linker peptide of wild-type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels was reduced compared with WT. Whole-cell I Na through all channel variants revealed no significant differences in the steady-state activation, inactivation, and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared with WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity compared with noninfected cells. PKA stimulation significantly increased peak I Na and conduction velocity of WT but not mutant channels. Oxidant stress inhibits cardiac I Na ; WT and mutant I Na decreases with the intracellular application of reduced nicotinamide adenine dinucleotide (NADH), an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels. Conclusions— We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of I Na and a failure of PKA stimulation to augment the current that may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.

Description: Background and Purpose— The National Institutes of Health policy calls for the inclusion of under-represented groups, such as women and minorities, in clinical research. Poor minority recruitment and retention in stroke clinical trials remain a significant challenge limiting safety and efficacy in a general population. Previous research examines participant barriers to clinical trial involvement, but little is known about the investigator perspective. This study addresses this gap and examines researcher-reported barriers and best practices of minority involvement in stroke clinical trials. Methods— Quantitative and qualitative methods, including surveys, focus groups, and key informant interviews were used. Results— In a survey of 93 prominent stroke researchers, 43 (51.2%; 70% response rate) respondents reported proactively setting recruitment goals for minority inclusion, 29 respondents (36.3%) reported requiring cultural competency staff training, and 44 respondents (51.2%) reported using community consultation about trial design. Focus groups and key informant interviews highlighted structural and institutional challenges to recruitment of minorities, including mistrust of the research/medical enterprise, poor communication, and lack of understanding of clinical trials. Researcher-identified best practices included using standardized project management procedures and protocols (eg, realistic budgeting to support challenges in recruitment, such as travel/parking reimbursement for participants), research staff cultural competency and communication training, and developing and fostering community partnerships that guide the research process. Conclusions— This study’s formative evaluation contributes a new dimension to the literature as it highlights researcher-reported barriers and best practices for enhancing participation of minority populations into stroke clinical trials.

Description: Background and Purpose— To investigate the relationship between chronic kidney disease (CKD) and MRI-defined cerebral microbleeds (CMB), a harbinger of future intracerebral hemorrhage (ICH), among patients with a recent history of primary ICH. Methods— Using data from a predominantly black cohort of patients with a recent ICH-enrolled in an observational study between September 2007 and June 2011, we evaluated the association between CKD (defined as estimated low glomerular filtration rate〈60 mL/min per 1.73 m 2 ) and CMB on gradient-echo MRI. Multivariable models were generated to determine the contribution of CKD to the presence, number, and location of CMB. Results— Of 197 subjects with imaging data, mean age was 59 years, 48% were women, 73% were black, 114 (58%) had ≥1 CMBs, and 52 (26%) had CKD. Overall, CKD was associated with presence of CMB (adjusted odds ratio, 2.70; 95% confidence interval [CI], 1.10–6.59) and number of CMB (adjusted relative risk, 2.04; 95% CI, 1.27–3.27). CKD was associated with CMB presence (adjusted odds ratio, 3.44; 95% CI, 1.64–7.24) and number (adjusted relative risk, 2.46; 95% CI, 1.11–5.42) in black patients, but not CMB presence (adjusted odds ratio, 3.00; 95% CI, 0.61–14.86) or number (adjusted relative risk, 1.03; 95% CI: 0.22–4.89) in non-Hispanic white patients (interactions by race were statistically not significant). Conclusions— CKD is associated with a greater presence and number of CMB in ICH patients, particularly in patients of black race. Future studies should assess whether low estimated glomerular filtration rate may be a CMB risk marker or potential therapeutic target for mitigating the development of CMB.

Description: Resistance to antimicrobials is a growing problem in both developed and developing countries. In nations where AIDS is most prevalent, the human fungal pathogen Cryptococcus neoformans is a significant contributor to mortality, and its growing resistance to current antifungals is an ever-expanding threat. We investigated octapeptin C4, from the cationic cyclic lipopeptide class of antimicrobials, as a potential new antifungal. Octapeptin C4 was a potent, selective inhibitor of this fungal pathogen with an MIC of 1.56 μg/ml. Further testing of octapeptin C4 against 40 clinical isolates of C. neoformans var. grubii or neoformans showed an MIC of 1.56 to 3.13 μg/ml, while 20 clinical isolates of C. neoformans var. gattii had an MIC of 0.78 to 12.5 μg/ml. In each case, the MIC values for octapeptin C4 were equivalent to, or better than, current antifungal drugs fluconazole and amphotericin B. The negatively charged polysaccharide capsule of C. neoformans influences the pathogen's sensitivity to octapeptin C4, whereas the degree of melanization had little effect. Testing synthetic octapeptin C4 derivatives provided insight into the structure activity relationships, revealing that the lipophilic amino acid moieties are more important to the activity than the cationic diaminobutyric acid groups. Octapeptins have promising potential for development as anticryptococcal therapeutic agents.

Description: The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.

Description: Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Description: Distension of peripheral veins in humans elicits a pressor and sympathoexcitatory response that is mediated through group III/IV skeletal muscle afferents. There is some evidence that autonomic reflexes mediated by these sensory fibers are blunted with increasing age, yet to date the venous distension reflex has only been studied in young adults. Therefore, we tested the hypothesis that the venous distension reflex would be attenuated in middle-aged compared with young adults. Nineteen young (14 men/5 women, 25 ± 1 yr) and 13 middle-aged (9 men/4 women, 50 ± 2 yr) healthy normotensive participants underwent venous distension via saline infusion through a retrograde intravenous catheter in an antecubital vein during limb occlusion. Beat-by-beat blood pressure, muscle sympathetic nerve activity (MSNA), and model flow-derived cardiac output (Q), and total peripheral resistance (TPR) were recorded throughout the trial. Mean arterial pressure (MAP) increased during the venous distension in both young (baseline 83 ± 2, peak 94 ± 3 mmHg; P 〈 0.05) and middle-aged adults (baseline 88 ± 2, peak 103 ± 3 mmHg; P 〈 0.05). MSNA also increased in both groups [young: baseline 886 ± 143, peak 1,961 ± 242 arbitrary units (AU)/min; middle-aged: baseline 1,164 ± 225, peak 2,515 ± 404 AU/min; both P 〈 0.05]. TPR ( P 〈 0.001), but not Q ( P = 0.76), increased during the trial. However, the observed increases in blood pressure, MSNA, and TPR were similar between young and middle-aged adults. Additionally, no correlation was found between age and the response to venous distension (all P 〉 0.05). These findings suggest that peripheral venous distension elicits a pressor and sympathetic response in middle-aged adults similar to the response observed in young adults.

Description: The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive SM22α CaSR flox/flox [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca 2+ ) concentrations (1–5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro- l -arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca 2+ concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.