Dose and duration of interferon γ pre-licensing interact with donor characteristics to influence the expression and function of indoleamine-2,3-dioxygenase in mesenchymal stromal cells.

Human mesenchymal stromal cells (MSCs) are a leading cell therapy candidate for the treatment of immune and inflammatory diseases due to their potent regulation of immune cells. MSC expression of indoleamine-2,3-dioxygenase (IDO) upon interferon γ (IFNγ) exposure has been proposed as both a sentinel marker and key mediator of MSC immunomodulatory potency. Rather than wait for in vivo exposure to cytokines, MSCs can be pre-licensed during manufacturing to enhance IDO expression. In this study, we systematically examine the relative role that the dose of IFNγ, the duration of pre-licensing and the donor of origin play in dictating MSC production of functional IDO. We find that across three human MSC donors, MSCs increase their expression of IDO in response to both increased dose of IFNγ and duration of pre-licensing. However, with extended pre-licensing, the expression of IDO no longer predicts MSCs ability to suppress activated peripheral blood mononuclear cells. In addition, pre-licensing dose and duration are revealed to be minor modifiers of MSCs inherent potency, and thus cannot be manipulated to boost poor donors to the levels of high-performing donors. Thus, the dose and duration of pre-licensing should be tailored to optimize performance of specific donors and an emphasis on donor selection is needed to realize significant benefits of pre-licensing.

Aggregation of Human Mesenchymal Stromal Cells Eliminates Their Ability to Suppress Human T Cells.

Mesenchymal stromal cells (MSCs) are administered locally to treat sites of inflammation. Local delivery is known to cause MSCs to aggregate into "spheroids," which alters gene expression and phenotype. While adherent MSCs are highly efficient in their inhibition of T cells, whether or not this property is altered upon MSC aggregation has not been thoroughly determined. In this study, we discovered that aggregation of MSCs into spheroids causes them to lose their T cell-suppressive abilities. Interestingly, adding budesonide, a topical glucocorticoid steroid, alongside spheroids partially restored MSC suppression of T cell proliferation. Through a series of inhibition and add-back studies, we determined budesonide acts synergistically with spheroid MSC-produced PGE2 to suppress T cell proliferation through the PGE2 receptors EP2 and EP4. These findings highlight critical differences between adherent and spheroid MSC interactions with human immune cells that have significant translational consequences. In addition, we uncovered a mechanism through which spheroid MSC suppression of T cells can be partly restored. By understanding the phenotypic changes that occur upon MSC aggregation and the impact of MSC drug interactions, improved immunosuppressive MSC therapies for localized delivery can be designed.

Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge.

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Time for a Time Window Extension: Insights from Late Presenters in the ESCAPE Trial.

BACKGROUND AND PURPOSE: The safety and efficacy of endovascular therapy for large-artery stroke in the extended time window is not yet well-established. We performed a subgroup analysis on subjects enrolled within an extended time window in the Endovascular Treatment for Small Core and Proximal Occlusion Ischemic Stroke (ESCAPE) trial. MATERIALS AND METHODS: Fifty-nine of 315 subjects (33 in the intervention group and 26 in the control group) were randomized in the ESCAPE trial between 5.5 and 12 hours after last seen healthy (likely to have groin puncture administered 6 hours after that). Treatment effect sizes for all relevant outcomes (90-day mRS shift, mRS 0-2, mRS 0-1, and 24-hour NIHSS scores and intracerebral hemorrhage) were reported using unadjusted and adjusted analyses. RESULTS: There was no evidence of treatment heterogeneity between subjects in the early and late windows. Treatment effect favoring intervention was seen across all clinical outcomes in the extended time window (absolute risk difference of 19.3% for mRS 0-2 at 90 days). There were more asymptomatic intracerebral hemorrhage events within the intervention arm (48.5% versus 11.5%, P = .004) but no difference in symptomatic intracerebral hemorrhage. CONCLUSIONS: Patients with an extended time window could potentially benefit from endovascular treatment. Ongoing randomized controlled trials using imaging to identify late presenters with favorable brain physiology will help cement the paradigm of using time windows to select the population for acute imaging and imaging to select individual patients for therapy.

Performance and design considerations for an anaerobic moving bed biofilm reactor treating brewery wastewater: Impact of surface area loading rate and temperature.

Three 4 L anaerobic moving bed biofilm reactors (AMBBR) treated brewery wastewater with AC920 media providing 680 m2 protected surface area per m3 of media. Different hydraulic retention times (HRT; 24, 18, 12, 10, 8 and 6 h) at 40% media fill and 35 °C, as well as different temperatures (15, 25 and 35 °C) at 50% media fill and 18 h HRT were examined. Best performance at 35 °C and 40% media fill was observed when HRT was 18 h, which corresponded with 92% removal of soluble COD (sCOD). Organic loading rates (OLR) above 24 kg-COD m-3d-1 decreased performance below 80% sCOD removal at 35 °C and 40% media fill. The reason was confirmed to be that surface area loading rates (SALR) above 50 g-sCOD m-2d-1 caused excessive biofilm thickness that filled up internal channels of the media, leading to mass transfer limitations. Temperature had a very significant impact on process performance with 50% media fill and 18 h HRT. Biomass concentrations were significantly higher at lower temperatures. At 15 °C the mass of volatile solids (VS) was more than three times higher than at 35 °C for the same OLR. Biofilms acclimated to 25 °C and 15 °C achieved removal of 80% sCOD at SALR of 10 g-sCOD m-2d-1 and 1.0 g-sCOD m-2d-1, respectively. Even though biomass concentrations were higher at lower temperature, biofilm acclimated to 25 °C and 15 °C performed significantly slower than that acclimated to 35 °C.

Removal of Soluble Phosphorus from Surface Water Using Iron (Fe-Fe) and Aluminum (Al-Al) Electrodes.

The removal of soluble phosphorus using iron and aluminum electrodes was studied in water samples from the Red River, a hyper-eutrophic stream in Winnipeg, Canada. Four trials were conducted: (I) mixed batch with 150-900 mA applied for 1 min to 1 L, (II) stagnant batch with 600-900 mA applied for 1 min to 1 L, and (III and IV) continuously stirred-tank reactor with 6.25-10 min hydraulic retention times and constant 900 mA. Maximum soluble phosphorus removals of 70-80% were observed in mixed batch, and there was no significant difference between aluminum and iron electrodes (P value of 0.0526-0.9487). Aluminum electrodes performed significantly worse than iron electrodes under higher hydraulic loads, with iron removing >70% soluble phosphorus and aluminum <40% (P values of 0.0035-0.0143). The estimated cost of consumables, reported per million liters of water treated, to remove 70% soluble phosphorus from eutrophic waters with 0.35 g m-3 soluble phosphorus would include 5-17.5 USD electricity costs and material costs of 5.3-12.2 USD for iron and 39.2 USD for aluminum.

Granulation of activated sludge under low hydrodynamic shear and different wastewater characteristics.

Five reactors were operated with low upflow superficial air velocities (0.41cmmin-1) in order to observe granulation on synthetic wastewaters with different characteristics: 1) 340mg-CODL-1; 2) 630mg-CODL-1; and 3) 1300mg-CODL-1. Stable granulation was only observed under low hydrodynamic shear for low-strength wastewater. 55-70% of soluble chemical oxygen demand (COD) was utilized before aeration and 91% COD, 62% total nitrogen (TN), and 96% total phosphorus (TP) were removed from the low-strength wastewater. Although medium-strength wastewater did generate granules they rapidly acquired a filamentous surface layer that resulted in decreased performance and loss of nitrification. 94% COD, 30% TN, and 85% TP were removed from the medium-strength wastewater. The high-strength wastewater did not develop granules and 85% COD was removed. Results demonstrated that high shear force was not required for granulation. Rather, granulation depended on multiple parameters to out-select rapidly growing aerobic microorganisms.

Endovascular mechanical thrombectomy for the treatment of acute ischemic stroke due to arterial dissection.

Arterial dissections account for 2% of strokes in all age groups, and up to 25% in patients aged 45 years or younger. The safety of endovascular intervention in this patient population is not well characterized. We identified all patients in the Merci registry - a prospective, multi-center post-market database enrolling patients treated with the Merci Retriever thrombectomy device - with arterial dissection as the most likely stroke etiology. Stroke presentation and procedural details were obtained prospectively; data regarding procedural complications, intracerebral hemorrhage (ICH), and the use of stenting of the dissected artery were obtained retrospectively. Of 980 patients in the registry, ten were identified with arterial dissection (8/10 ICA; 2/10 vertebrobasilar). The median age was 48 years with a baseline NIH stroke scale score of 16 and median time to treatment of 4.9 h. The procedure resulted in thrombolysis in cerebral ischemia (TICI) scores of 2a or better in eight out of ten and TICI 2b or better in six out of ten patients. Stenting of the dissection was performed in four of nine (44%). The single complication (1/9; 11%) - extension of a dissected carotid artery - was treated effectively with stenting. No symptomatic ICH or stroke in a previously unaffected territory occurred. A favorable functional outcome was observed in eight out of ten patients. Despite severe strokes on presentation, high rates of recanalization (8/10) and favorable functional outcomes (8/10) were observed. These results suggest that mechanical thrombectomy in patients with acute stroke resulting from arterial dissection is feasible, safe, and may be associated with favorable functional outcomes.

The Merci Retrieval System for acute stroke: the Southeast Regional Stroke Center experience.

INTRODUCTION: The Merci Retrieval System was cleared for use in patients with stroke in August 2004. However, there are few published results of "real world experience" with the device. METHODS: We captured single-center data on 25 consecutive patients with acute ischemic stroke treated with the Merci Retrieval System according to the MERCI trial except that we treated some patients with tandem proximal carotid and intracranial lesions with carotid angioplasty and stenting and some patients were treated within the 3-hour window. RESULTS: Median patient age was 63 years and median initial National Institute of Health Stroke Scale (NIHSS) score was 18. Isolated M1 or M2 middle cerebral artery lesions occurred in 52%, "carotid T" lesions in 8%, and vertebrobasilar lesions in 8%. Tandem lesions involving proximal carotid and proximal intracranial vessel occurred in 32%, necessitating emergent multilevel treatment including carotid stenting. Median duration from symptom onset to Merci device utilization was 5.2 hours. Successful reperfusion (> or = thrombolysis in myocardial infarction [TIMI] 2 flow) in the target vessel was obtained in 56% of cases. Statistical analysis revealed a strong correlation between ability to achieve greater than or equal to TIMI 2 flow and good clinical outcome as measured by 3-month NIHSS score, modified Rankin Scale (mRS), and mortality (nine out of the 12 without successful reperfusion died compared to none of the 13 with > or =TIMI 2 flow, p < 0.001). Younger age and lower NIHSS score on presentation were also predictors of good clinical outcome at 3 months. CONCLUSION: These "real world data" demonstrate that the results of the previous MERCI trial can be "independently replicated" at a regional stroke center. Although the results of placebo-controlled trials are still pending, mechanical revascularization has become a critical component of our acute stroke protocol, particularly for severe strokes. Issues still remain regarding recalcitrant lesions and operator experience, which necessitate further clinical testing and device optimization.

Cooling for Acute Ischemic Brain Damage (COOL AID): a feasibility trial of endovascular cooling.

OBJECTIVE: To report results of a randomized pilot clinical feasibility trial of endovascular cooling in patients with ischemic stroke. METHODS: Forty patients with ischemic stroke presenting within 12 hours of symptom onset were enrolled in the study. An endovascular cooling device was inserted into the inferior vena cava of those randomized to hypothermia. A core body temperature of 33 degrees C was targeted for 24 hours. All patients underwent clinical assessment and MRI initially, at days 3 to 5 and days 30 to 37. RESULTS: Eighteen patients were randomized to hypothermia and 22 to receive standard medical management. Thirteen patients reached target temperature in a mean of 77 +/- 44 minutes. Most tolerated hypothermia well. Clinical outcomes were similar in both groups. Mean diffusion-weighted imaging (DWI) lesion growth in the hypothermia group (n = 12) was 90.0 +/- 83.5% compared with 108.4 +/- 142.4% in the control group (n = 11) (NS). Mean DWI lesion growth in patients who cooled well (n = 8) was 72.9 +/- 95.2% (NS). CONCLUSIONS: Induced moderate hypothermia is feasible using an endovascular cooling device in most patients with acute ischemic stroke. Further studies are needed to determine if hypothermia improves outcome.

Possible short-term amelioration of basilar plaque by high-dose atorvastatin: use of reductase inhibitors for intracranial plaque stabilization.

Regression of symptomatic intracranial atherostenosis is not known to be a common occurrence. In this case, delay of basilar reconstruction by endovascular means permitted serial angiographic assessment of plaque change. The use of high-dose atorvastatin over a 2-week period was associated with marked angiographic improvement. Medical programs of plaque stabilization may provide adjunctive benefit in patients with symptomatic intracranial disease.

Effect of hypoxia and reoxygenation on metabolic pathways in rat hepatocytes.

BACKGROUND: The mechanisms whereby rat hepatocytes undergo irreversible injury due to a lack of oxygen have not been established. METHODS: Liver cells were used for reperfusion injury, and four compartmentalized pathways were evaluated during hypoxia (N2/CO2, 19:1) for 30 min followed by oxygen (O2/CO2, 19:1) for 30 min. RESULTS: Cell viability decreased during the hypoxic, but not during the reoxygenation, phase. Glycogenolysis, as measured by glucose release, was significantly increased during hypoxia as compared to controls in oxygen (205 +/- 15 vs. 155 +/- 10 nmol glucose/mg protein/h, respectively), and did not return to normal levels by reoxygenation. Gluconeogenesis was importantly decreased during hypoxia (102 +/- 10 vs. 8 +/- 2 nmol glucose/mg protein/h) with partial recovery during reoxygenation. Ureagenesis diminished in hypoxia, but recovered during reoxygenation. Additionally, 3-hydroxybutyrate formation was augmented by hypoxia, with some recovery when oxygen was present. CONCLUSIONS: These results suggest that compartmentalized pathways are protected from hypoxic injury in isolated hepatocytes, and also suggest it as a model to test the idea that enzymes of those pathways are organized into multienzyme complexes in vivo.

Intravascular malignant lymphomatosis with neurologic presentation: factors facilitating antemortem diagnosis.

Intravascular malignant lymphomatosis (IML) is a rare disorder of small and medium size vessels that frequently goes undiagnosed until the time of autopsy. The clinical courses of two such patients were examined to determine factors that would facilitate antemortem diagnosis. Both patients had mental status changes, pyramidal tract signs, and peripheral neuropathy. Despite postmortem evidence of widespread lymphocytic invasion of vessels throughout the body including peripheral and central nervous systems, neuroimaging studies, cerebrospinal fluid analysis, peripheral blood studies, and bone marrow biopsy failed to reveal diagnostic evidence of the underlying neoplastic process. Although markedly abnormal, nerve conduction studies were nonspecific. Familiarity with IML and its consideration in the differential diagnosis when central and peripheral nervous system dysfunction occur concurrently may guide the physician to tissue biopsy facilitating antemortem diagnosis and institution of appropriate therapy.

Axillary nerve injury after anterior glenohumeral dislocation: MR findings in three patients.

OBJECTIVE: We performed this study to demonstrate the MR imaging findings that indicate an anterior dislocation has caused an axillary nerve injury. CONCLUSION: MR images of the shoulder can show findings indicating that an axillary nerve injury has been caused by an anterior shoulder dislocation. All MR examinations should be evaluated for these findings, particularly if the patient has a history of anterior shoulder dislocation.

Transcranial doppler sonography to monitor cerebral vasculitis.

We describe a patient with cerebral vasculitis treated with prednisone and cyclophosphamide and followed by serial transcranial Doppler sonography and arteriography. Proximal cerebral angiographic abnormalities correlated with transcranial Doppler abnormalities. The abnormalities gradually normalized with treatment and correlated with the findings of followup arteriography, which also showed improvement in the vascular morphology. Transcranial Doppler sonography is useful in following proximal cerebral vascular abnormalities in some cases of cerebral vasculitis.

Neuro-Behçet's disease: factors hampering proper diagnosis.

We reviewed the clinical course of nine patients with neuro-Behçet's disease to assess difficulties in making this diagnosis. Factors delaying proper diagnosis included lack of accurate history and physical examination, lack of recognition of an underlying systemic syndrome and its relationship to the neurologic symptoms, presence of intermittently normal CSF studies, and use of noncontrasted neuroimaging techniques.

Extracellular ATP inhibits agonist-induced mobilization of internal calcium in human platelets.

Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.

Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.

Effects of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1) on glucose metabolism in liver cells.

Phosphorylase activity of isolated rat liver cells was increased about 2-fold on addition of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1), epinephrine or the Ca2+ ionophore A23187, in all cases presumably due to an increase in cytosolic Ca2+. Extracellular Ca2+ was required with A23187, but not with either tri-Calciphor or epinephrine. Tri-Calciphor, however, did not stimulate a sustained release of glucose from hepatocytes as compared to the other Ca2+ mobilizing agents, even at concentrations 10-fold higher than that required to stimulate the phosphorylase activity. Tri-Calciphor did not alter the glucose release by epinephrine. It is concluded that tri-Calciphor can alter cytosolic Ca2+, but that its mechanism of action is more complex than that of a simple Ca2+ ionophore.