Molecular Epidemiologic Investigation of Mycobacterium intracellulare Subspecies chimaera Lung Infections at an Adult Cystic Fibrosis Program.

Rationale: Outbreaks of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) have been reported at CF centers with conflicting conclusions. The occurrence of NTM at the UVMC (University of Vermont Medical Center) adult CF program was investigated. Objectives: Use the HALT NTM (Healthcare-associated Links in Transmission of NTM) toolkit to investigate the healthcare-associated transmission and/or acquisition of NTM among pwCF having genetically similar NTM isolates. Methods: Whole genome sequencing of NTM isolates from 23 pwCF was conducted to identify genetically similar NTM isolate clusters (30 or fewer single-nucleotide polymorphism differences). The epidemiological investigation, comparison of respiratory and healthcare environmental isolates, and home residence watershed mapping were analyzed. Results: Whole genome sequencing analysis revealed two clusters of NTM isolates (Mycobacterium avium and M. intracellulare ssp. chimaera) among pwCF. The epidemiologic investigation demonstrated opportunities for healthcare-associated transmission within both clusters. Healthcare environmental M. avium isolates revealed no genetic similarity to respiratory isolates. However, M. intracellulare ssp. chimaera respiratory isolates revealed greater genetic similarity to a hospital water biofilm isolate than to each other. Neither cluster had all subjects residing in the same watershed. Conclusions: This study suggests the healthcare-associated transmission of M. avium among pwCF is unlikely at UVMC but supports the healthcare-associated environmental acquisition of M. intracellulare ssp. chimaera. The presence of genetically similar isolates alone is insufficient to confirm healthcare-associated transmission and/or acquisition. The HALT NTM toolkit standardizes outbreak investigation with genetic analysis, epidemiologic investigation, healthcare environmental sampling, and home of residence watershed identification to test the frequency and nature of healthcare-associated NTM transmission among pwCF.

Effect of hypertension and peroxynitrite decomposition with FeTMPyP on CBF and stroke outcome.

We investigated the effect of peroxynitrite decomposition catalyst FeTMPyP treatment on perfusion deficit, vascular function and stroke outcome in Wistar ( n = 26) and spontaneously hypertensive rats stroke-prone (SHRSP; n = 26) that underwent tMCAO for 2 h or Sham operation. Peri-infarct CBF was measured by hydrogen clearance in the absence or presence of FeTMPyP (10 mg/kg, i.v.) or vehicle 10 min before reperfusion. Myogenic tone of parenchymal arterioles (PAs) was measured as an indication of small vessel resistance (SVR). Baseline CBF was similar between Wistar and SHRSP (114 ± 12 vs. 132 ± 9 mL/100 g/min); however, MCAO caused greater perfusion deficit in SHRSP (24 ± 6 vs. 7 ± 1 mL/100 g/min; p < 0.05) and increased infarct volume by TTC (12 ± 6 vs. 32 ± 2%; p < 0.05). Reperfusion CBF was decreased from baseline in both SHRSP and Wistar (54 ± 16 and 46 ± 19 mL/100 g/min; p < 0.05), suggesting increased infarction in SHRSP was related to greater perfusion deficit. PAs from SHRSP had increased tone vs. Wistar that was enhanced after tMCAO. FeTMPyP treatment did not affect CBF during ischemia or reperfusion, or tone of PAs, but decreased the incidence of hemorrhage in SHRSP by 50%. Thus, increased tone in PAs from SHRSP could increase perfusion deficit during MCAO that was not alleviated by FeTMPyP.

Pial Collateral Reactivity During Hypertension and Aging: Understanding the Function of Collaterals for Stroke Therapy.

BACKGROUND AND PURPOSE: We investigated vasoactive properties of leptomeningeal arterioles (LMAs) under normotensive conditions and during hypertension and aging that are known to have poor collateral flow and little salvageable tissue. METHODS: LMAs, identified as distal anastomotic arterioles connecting middle and anterior cerebral arteries, were studied isolated and pressurized from young (18 weeks) or aged (48 weeks) normotensive Wistar Kyoto (WKY18, n=14; WKY48, n=6) rats and spontaneously hypertensive rats (SHR18, n=16; SHR48, n=6). Myogenic tone and vasoactive responses to pressure as well as endothelial function and ion channel activity were measured. RESULTS: LMAs from WKY18 had little myogenic tone at 40 mm Hg (8±3%) that increased in aged WKY48 (30±6%). However, LMAs from both WKY groups dilated to increased pressure and demonstrated little myogenic reactivity, a response that would be conducive to collateral flow. In contrast, LMAs from both SHR18 and SHR48 displayed considerable myogenic tone (56±8% and 43±7%; P<0.01 versus WKY) and constricted to increased pressure. LMAs from both WKY and SHR groups had similar basal endothelial nitric oxide and IK channel activity that opposed tone. However, dilation to sodium nitroprusside, diltiazem and 15 mmol/L KCl was impaired in LMAs from SHR18. CONCLUSIONS: This study shows for the first time that LMAs from young and aged SHR are vasoconstricted and have impaired vasodilatory responses that may contribute to greater perfusion deficit and little penumbral tissue. These results also suggest that therapeutic opening of pial collaterals is possible during middle cerebral artery occlusion to create penumbral tissue and prevent infarct expansion.

Effect of hypertension and carotid occlusion on brain parenchymal arteriole structure and reactivity.

We studied the effect of hypertension and chronic hypoperfusion on brain parenchymal arteriole (PA) structure and function. PAs were studied isolated and pressurized from 18-wk-old Wistar-Kyoto (WKY18; n = 8) and spontaneously hypertensive stroke prone (SHRSP18; n = 8) and 5-wk-old prehypertensive (SHRSP5; n = 8) rats. In separate groups, unilateral common carotid artery occlusion (UCCAo) was performed for 4 wk to cause chronic hypoperfusion in 18-wk-old WKY (WKY18-CH; n = 8) and SHRSP (SHRSP18-CH; n = 8). UCCAo caused PAs to have significantly diminished myogenic tone (31 ± 3 vs. 14 ± 6% at 60 mmHg; P < 0.05) and reactivity to pressure from WKY18-CH vs. WKY18 animals. The effect of UCCAo was limited to normotensive animals, as there was little effect of chronic hypoperfusion on vascular reactivity or percent tone in PAs from SHRSP18 vs. SHRSP18-CH animals (53 ± 4 vs. 41 ± 3%; P > 0.05). However, PAs from SHRSP18 and SHRSP5 animals had significantly greater tone compared with WKY18, suggesting an effect of strain and not hypertension per se on PA vasoconstriction. Structurally, PAs from SHRSP18 and SHRSP5 animals had similar sized lumen diameters, but increased wall thickness and distensibility compared with WKY18. Interestingly, chronic hypoperfusion did not affect the structure of PAs from either WKY18-CH or SHRSP18-CH animals. Thus PAs responded to UCCAo with active vasodilation, but not structural remodeling, an effect that was absent in SHRSP. The increased tone of PAs from SHRSP animals, combined with lack of response to chronic hypoperfusion, may contribute to the propensity for ischemic lesions and increased perfusion deficit during hypertension.

In vitro manganese-dependent cross-talk between Streptococcus mutans VicK and GcrR: implications for overlapping stress response pathways.

Streptococcus mutans, a major acidogenic component of the dental plaque biofilm, has a key role in caries etiology. Previously, we demonstrated that the VicRK two-component signal transduction system modulates biofilm formation, oxidative stress and acid tolerance responses in S. mutans. Using in vitro phosphorylation assays, here we demonstrate for the first time, that in addition to activating its cognate response regulator protein, the sensor kinase, VicK can transphosphorylate a non-cognate stress regulatory response regulator, GcrR, in the presence of manganese. Manganese is an important micronutrient that has been previously correlated with caries incidence, and which serves as an effector of SloR-mediated metalloregulation in S. mutans. Our findings supporting regulatory effects of manganese on the VicRK, GcrR and SloR, and the cross-regulatory networks formed by these components are more complex than previously appreciated. Using DNaseI footprinting we observed overlapping DNA binding specificities for VicR and GcrR in native promoters, consistent with these proteins being part of the same transcriptional regulon. Our results also support a role for SloR as a positive regulator of the vicRK two component signaling system, since its transcription was drastically reduced in a SloR-deficient mutant. These findings demonstrate the regulatory complexities observed with the S. mutans manganese-dependent response, which involves cross-talk between non-cognate signal transduction systems (VicRK and GcrR) to modulate stress response pathways.

Mechanisms of enhanced basal tone of brain parenchymal arterioles during early postischemic reperfusion: role of ET-1-induced peroxynitrite generation.

The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO); 2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure ('basal tone') was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small- (SK) and intermediate- (IK) conductance, Ca(2+)-sensitive K(+) channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21 ± 4% versus 3 ± 3% at 1 nmol/L; P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphinato iron (III) chloride) (100 µmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.

Treatment for cerebral small vessel disease: effect of relaxin on the function and structure of cerebral parenchymal arterioles during hypertension.

We investigated the effect of hypertension on the function and structure of cerebral parenchymal arterioles (PAs), a major target of cerebral small vessel disease (SVD), and determined whether relaxin is a treatment for SVD during hypertension. PAs were isolated from 18-wk-old female normotensive Wistar-Kyoto (WKY) rats, spontaneous hypertensive rats (SHRs), and SHRs treated with human relaxin 2 for 14 d (4 µg/h; n=8/group) and studied using a pressurized arteriograph system. Hypertension reduced PA inner diameter (58±3 vs. 49±3 µm at 60 mmHg in WKY rats, P<0.05), suggesting inward remodeling that was reversed by relaxin (56±4 µm, P<0.05). Relaxin also increased PA distensibility in SHRs (34±2 vs. 10±2% in SHRs, P<0.05). Relaxin was detected in cerebrospinal fluid (110±30 pg/ml) after systemic administration, suggesting that it crosses the blood-brain barrier (BBB). Relaxin receptors (RXFP1/2) were not detected in cerebral vasculature, but relaxin increased vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP-2) expression in brain cortex. Inhibition of VEGF receptor tyrosine kinase (axitinib, 4 mg/kg/d, 14 d) had no effect on increased distensibility with relaxin, but caused outward hypertrophic remodeling of PAs from SHRs. These results suggest that relaxin crosses the BBB and activates MMP-2 in brain cortex, which may interact with PAs to increase distensibility. VEGF appears to be involved in remodeling of PAs, but not relaxin-induced increased distensibility.

Acute rosiglitazone treatment during reperfusion after hyperglycemic stroke is neuroprotective not vascular protective.

Reperfusion therapy for ischemic stroke can cause secondary brain injury, especially under hyperglycemic (HG) conditions. Here we investigated the effect of acute treatment with rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, prior to postischemic reperfusion, on stroke outcome during HG stroke. Male Wistar rats that were either normoglycemic (NG) or HG by STZ (50 mg/kg; for 5-6 days) underwent middle cerebral artery occlusion (MCAO) for 2 hours with 2 hours of reperfusion. Animals were treated i.v. with rosiglitazone (1mg/kg; n=16), rosiglitazone (1mg/kg) + the free radical scavenger Tempol (50mg/kg; n=10) or vehicle (n=16) ten minutes prior to reperfusion and infarct volume, edema formation and cerebral blood flow (CBF) were measured. Compared to NG, HG stroke significantly increased infarct volume from 5.2±3.0% vs. 14.7±3.6% (p<0.05). Rosiglitazone prevented the increased infarct volume induced by HG that was only 6.9±2.0% (p<0.05 vs. HG) but did not have any effect on edema formation that was increased by 3.0% in both HG vehicle and rosiglitazone-treated ipsilateral vs. contralateral hemispheres (p<0.05). Combined treatment of rosiglitazone + Tempol did not significantly change brain water content that remained 2.2% greater than contralateral (p<0.05), but reversed the neuroprotective properties of rosiglitazone in HG MCAO animals such that infarct volume was 14.3±4.4% (p>0.05 vs. vehicle). The lack of an effect of combined treatment of rosiglitazone + Temple may be due to a decrease in reperfusion CBF that was only 60% of baseline (p<0.01) compared to 82% and 89% for HG vehicle and rosiglitazone treated animals (p>0.05). In conclusion, acute rosiglitazone treatment prior reperfusion was neuroprotective but not vascular protective during HG stroke.

Peroxynitrite decomposition with FeTMPyP improves plasma-induced vascular dysfunction and infarction during mild but not severe hyperglycemic stroke.

We investigated mechanisms by which circulating factors during hyperglycemic (HG) stroke affect cerebrovascular function and the role of peroxynitrite in stroke outcome. Middle cerebral arteries (MCAs) were isolated from male Wistar rats and perfused with plasma from rats that were hyperglycemic for 5 to 6 days by streptozotocin and underwent either MCA occlusion (HG MCAO) or Sham surgery (HG Sham) compared with MCA perfused with physiologic saline (No plasma). Myogenic responses and endothelial function were compared in untreated MCA (n=8/group) or with inhibitors of NADPH oxidase (apocynin; n=8), peroxynitrite (FeTMPyP; n=8) or endothelin-1 (ET-1)(A) (BQ-123; n=8). Finally, animals were treated in vivo before reperfusion after mild (<68% cerebral blood flow (CBF) decrease) or severe (>68% CBF decrease) MCAO with FeTMPyP (n=12) or vehicle (n=12) and CBF and infarction measured. The HG MCAO plasma increased tone in MCA versus No plasma (P<0.05) that was reversed by FeTMPyP, but not by apocynin or BQ-123. The HG Sham plasma also increased tone in MCA (P<0.05) that was reversed by BQ-123 only. In vivo, FeTMPyP was neuroprotective during mild, but not severe ischemia. These results show that circulating factors in plasma can affect cerebrovascular function through peroxynitrite generation and ET-1. In addition, peroxynitrite decomposition improves stroke outcome acutely during mild, but not severe HG ischemia.

Inhibition of protein kinase Cß reverses increased blood-brain barrier permeability during hyperglycemic stroke and prevents edema formation in vivo.

BACKGROUND AND PURPOSE: We investigated the effect of circulating factors and protein kinase Cß on blood-brain barrier permeability and edema during hyperglycemic stroke. METHODS: Male Wistar rats that were hyperglycemic by streptozotocin (50 mg/kg) for 5 to 6 days underwent middle cerebral artery occlusion (MCAO) for 2 hours with 2 hours of reperfusion. Blood-brain barrier permeability was measured in middle cerebral arteries that were ischemic (MCAO) or nonischemic (CTL) and perfused with plasma (20% in buffer) from MCAO or CTL animals. A separate set of MCAO vessels was perfused with the protein kinase Cß inhibitor CGP53353 (0.5 µmol/L) and permeability measured. Lastly, hyperglycemic rats were treated intravenously with CGP53353 (10 or 100 µg/kg or vehicle 15 minutes before reperfusion and edema formation measured by wet:dry weights (n=6/group). RESULTS: MCAO vessels had increased permeability compared with controls regardless of the plasma perfusate. Permeability (water flux, µm(3)×10(8)) of CTL vessel/CTL plasma (n=8), CTL vessel/MCAO plasma (n=7), MCAO vessel/CTL plasma (n=6), and MCAO vessel/MCAO plasma (n=6) was 0.98±0.11, 1.13±0.07, 1.36±0.02, and 1.34±0.06; P<0.01). Inhibition of protein kinase Cß in MCAO vessels (n=6) reversed the increase in permeability (0.92±0.1; P<0.01). In vivo, hyperglycemia increased edema versus normoglycemia after MCAO (water content=78.84%±0.11% versus 81.38%±0.21%; P<0.01). Inhibition of protein kinase Cß with 10 or 100 µg/kg CGP53353 during reperfusion prevented the increased edema in hyperglycemic animals (water content=79.54%±0.56% and 79.99%±0.43%; P<0.01 versus vehicle). CONCLUSIONS: These results suggest that the pronounced vasogenic edema that occurs during hyperglycemic stroke is mediated in large part by activation of protein kinase Cß.

Cerebral vascular adaptation to pregnancy and its role in the neurological complications of eclampsia.

The cerebral circulation has a central role in mediating the neurological complications of eclampsia, yet our understanding of how pregnancy and preeclampsia affect this circulation is severely limited. Here, we show that pregnancy causes outward remodeling of penetrating arterioles and increased capillary density in the brain due to activation of peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor involved in cerebrovascular remodeling and highly activated in pregnancy. Pregnancy-induced PPARγ activation also significantly affected cerebral hemodynamics, decreasing vascular resistance and increasing cerebral blood flow by ∼40% in response to acute hypertension that caused breakthrough of autoregulation. These structural and hemodynamic changes in the brain during pregnancy were associated with substantially increased blood-brain barrier permeability, an effect that could promote passage of damaging proteins into the brain and cause the neurological complications of eclampsia, including seizure.

Effect of PPARγ inhibition during pregnancy on posterior cerebral artery function and structure.

Peroxisome proliferator-activated receptor-γ (PPARγ), a ligand-activated transcription factor, has protective roles in the cerebral circulation and is highly activated during pregnancy. Thus, we hypothesized that PPARγ is involved in the adaptation of cerebral vasculature to pregnancy. Non-pregnant (NP) and late-pregnant (LP) rats were treated with a specific PPARγ inhibitor GW9662 (10 ]mg/kg/day, in food) or vehicle for 10 days and vascular function and structural remodeling were determined in isolated and pressurized posterior cerebral arteries (PCA). Expression of PPARγ and angiotensin type 1 receptor (AT1R) in cerebral (pial) vessels was determined by real-time RT-PCR. PPARγ inhibition decreased blood pressure and increased blood glucose in NP rats, but not in LP rats. PPARγ inhibition reduced dilation to acetylcholine and sodium nitroprusside in PCA from NP (p < 0.05 vs. LP-GW), but not LP rats. PPARγ inhibition tended to increase basal tone and myogenic activity in PCA from NP rats, but not LP rats. Structurally, PPARγ inhibition increased wall thickness in PCA from both NP and LP rats (p < 0.05), but increased distensibility only in PCA from NP rats. Pregnancy decreased expression of PPARγ and AT1R (p < 0.05) in cerebral arteries that was not affected by GW9662 treatment. These results suggest that PPARγ inhibition had significant effects on the function and structure of PCA in the NP state, but appeared to have less influence during pregnancy. Down-regulation of PPARγ and AT1R in cerebral arteries may be responsible for the lack of effect of PPARγ in cerebral vasculature and may be part of the vascular adaptation to pregnancy.