Optimizing throughput of babies with infantile hypertrophic pyloric stenosis.

BACKGROUND: Definitive surgical care is often delayed in hypertrophic pyloric stenosis (HPS). Our aim is to evaluate the effect modifiable factors in preoperative HPS management have on efficiency of care. METHODS: A retrospective review of all patients undergoing pyloromyotomy for HPS at two US children's hospitals between 2008 and 2018 was performed. RESULTS: 406 patients were included in the study. The majority (310, 76 â€‹%) were adequately resuscitated and ready for surgery upon diagnosis in the ER. However, only 133 patients (43 â€‹%) had surgery on the day of admission. Patients diagnosed between 12pm and 6pm were more likely to have surgery the next day than those diagnosed before noon (67 â€‹% vs 33 â€‹%, p â€‹< â€‹.001), which correlated with a longer length of stay (32 vs 47 â€‹h, p â€‹< â€‹.001). CONCLUSION: The majority of patients presenting with HPS can safely undergo same day surgery. Delaying surgery due to an afternoon diagnosis is common, and leads to a modifiable increased total length of stay.

Food Insecurity in the Elective Enhanced Recovery After Surgery Colorectal Surgical Population: Prevalence and Implications for Surgical Outcomes.

BACKGROUND: Food insecurity is defined as having limited or uncertain availability of nutritionally adequate food. Approximately 10.5% of U.S. households are food-insecure. Our study aimed to determine the prevalence and postoperative implications of food insecurity in a diverse group of colorectal surgery patients admitted to a hospital in an area with a higher-than-average median income. METHODS: The 6-question Household Food Security Survey was added to the colorectal surgery ERAS program preoperative paperwork. Patient demographics, comorbidities, operative parameters, length of stay, and postoperative outcomes were collected by review of electronic medical records. RESULTS: A total of 294 ERAS patients (88.8%) completed the survey over an 11-month period. Thirty-three patients (11.2%) were identified as food-insecure. Food-insecure patients were more likely to be non-white (P = .003), younger (P = .009), smokers (P = .004), chronic narcotic users (P < .001), unmarried (P = .007), and have more comorbidities (P = .004). The food-insecure population had more frequent postoperative ileus (P = .044). Hospital length of stay was significantly longer in food-insecure patients (8.6 days vs 5.4 days, P < .001). Food-insecure patients also had higher rates of >30-day mortality (P = .049). DISCUSSION: Food insecurity was found to occur in patients that lived in communities deemed both affluent and distressed. These patients had longer hospital stays and higher mortality. A food insecurity questionnaire can easily identify patients at risk. Further investigations to mitigate these complications are warranted.

Indications for a "Surgery-First" Approach for the Treatment of Lower Extremity Arterial Disease.

BACKGROUND: In recent years, there has been a tendency toward an "endovascular-first" approach for the treatment for femoropopliteal arterial disease. The purpose of this study is to determine if there are patients that are better served with an initial femoropopliteal bypass (FPB) rather than an endovascular attempt at revascularization. METHODS: A retrospective analysis of all patients undergoing FPB between June 2006 - December 2014 was performed. Our primary endpoint was primary graft patency, defined as patent using ultrasound or angiography without secondary intervention. Patients with <1-year follow-up were excluded. Univariate analysis of factors significant for 5-year patency was performed using χ2 tests for binary variables. A binary logistic regression analysis incorporating all factors identified as significant by univariate analysis was used to identify independent risk factors for 5-year patency. Event-free graft survival was evaluated using Kaplan-Meier models. RESULTS: We identified 241 patients undergoing FPB on 272 limbs. FPB indication was disabling claudication in 95 limbs, chronic limb-threatening ischemia (CLTI) in 148, and popliteal aneurysm in 29. In total, 134 FPB were saphenous vein grafts (SVG), 126 were prosthetic grafts, 8 were arm vein grafts, and 4 were cadaveric/xenografts. There were 97 bypasses with primary patency at 5 or more years of follow-up. Grafts patent at 5 years by Kaplan-Meier analysis were more likely to have been performed for claudication or popliteal aneurysm (63% 5-year patency) as compared with CLTI (38%, P < 0.001). Statistically significant predictors (using log rank test) of patency over time were use of SVG (P = 0.015), surgical indication of claudication or popliteal aneurysm (P < 0.001), Caucasian race (P = 0.019) and no history of COPD (P = 0.026). Multivariable regression analysis confirmed these 4 factors as significant independent predictors of 5-year patency. Of note, there was no statistical correlation between FPB configuration (above or below knee anastomosis, in-situ versus reversed saphenous vein) and 5-year patency. There were 40 FPBs in Caucasian patients without a history of COPD receiving SVG for claudication or popliteal aneurysm that had a 92% estimated 5-year patency by Kaplan-Meier survival analysis. CONCLUSIONS: Long-term primary patency that was substantial enough to consider open surgery as a first intervention was demonstrated in Caucasian patients without COPD, having good quality saphenous vein, and who underwent FPB for claudication or popliteal artery aneurysm.

Spontaneous Pneumomediastinum is Not Associated With Esophageal Perforation: Results From a Retrospective, Case-Control Study in a Pediatric Population.

What is the optimal management of spontaneous pneumomediastinum (SPM) and is there a risk of esophageal perforation in patients with SPM? We performed a retrospective case-control study of children through age 21, diagnosed with SPM in one hospital system over 10 years with the primary aim of describing the diagnostic workup, treatment patterns, and clinical outcomes. We hypothesized that SPM is a self-limited disease and is not associated with esophageal injury. Cases were identified using International Classification of Disease codes and excluded for trauma or severe infections. Median age was 16 years, 66% were male (n = 179). Chest radiography was performed in 97%, chest computed tomography (CT) in 33%, and esophagrams in 26%. Follow-up imaging showed resolution in 83% (mean = 17.2 days). SPM was not associated with esophageal perforation. We recommend avoiding CT scans and esophagrams unless there is discrete esophageal concern. Management of SPM should be guided by symptomatology.

Cervical Endocrine Surgery With a Novel Opioid-Limited Perioperative Protocol.

BACKGROUND: Recent studies have demonstrated that patients undergoing cervical endocrine surgery could be comfortably discharged with minimal opioid analgesia. However, no study to date has examined the efficacy of limiting administration of opioids intraoperatively. We have developed a novel protocol for patients undergoing cervical endocrine surgery that eliminates perioperative opioids. We sought to determine the efficacy of this protocol and its impact on opioid use at discharge. METHODS: We conducted a prospective opt-in opioid-limited surgery program study to opioid-naive patients scheduled for cervical endocrine surgery beginning in August 2019. Postoperatively, nonopioid analgesia was encouraged, but patients were also given a low dose prescription for opioids at discharge. Patients were then matched with 2 retrospective control groups, patients from 2014-2016 and 2017-2018, in order to account for increased public awareness of opioid-prescribing patterns. Primary end points included perioperative opioid use. Secondary end points included postoperative pain scores and complications. RESULTS: 218 patients underwent cervical endocrine surgery with our opioid-limited protocol between August 2019 and February 2020. Nine patients received opioids intraoperatively (4%) and 109 (50%) filled their opioid prescriptions at discharge. Compared to retrospective control groups, the average oral morphine equivalents (OME) administered intraoperatively and prescribed postoperatively were significantly lower (P < .0001). Pain scores and complication rates were similar in all groups (P = .7247). DISCUSSION: Our novel opioid-limited surgery protocol used in conjunction with preoperative counseling is an effective approach for pain control in patients undergoing cervical endocrine surgery and limits opioid exposure throughout the perioperative period.

Incidence of venous thromboembolism in patients with sickle cell disease undergoing noncardiovascular surgery.

OBJECTIVE: Patients with sickle cell disease (SCD) will have a baseline hypercoagulable state and an increased risk of venous thromboembolism (VTE). Few data are available regarding the efficacy of standard prophylaxis in preventing VTE after noncardiovascular surgery for patients with SCD. Our objective was to investigate the incidence of VTE in patients with SCD who had undergone noncardiovascular surgery. METHODS: We performed a retrospective medical record review of 352 patients with SCD who had undergone noncardiovascular surgery from August 2009 to August 2019 at Beaumont Hospitals. An equal number of control patients without SCD were propensity matched for age, sex, race, body mass index, and specific surgery. The data collected included demographics, comorbidities, VTE prophylaxis used, occurrence of deep vein thrombosis (DVT) and pulmonary embolism (PE), hospital length of stay, and 30-day mortality. RESULTS: We found no differences in age, race, sex, ethnicity, operative time, or hospital length of stay between the SCD and propensity-matched control patients. DVT prophylaxis was used more frequently for the SCD patients than for the controls (96.0% vs 88.6%; P < .001). Four SCD patients (1.1%) had developed DVT vs five control patients (1.4%; P > .999). One patient in each group had developed PE (0.3%; P > .999). No difference was found in 30-day mortality between the SCD group and the control group (1 [0.3%] vs 3 [0.9%]; P = .312). Of those with a diagnosis of VTE ≤30 days postoperatively, no differences were present in age, sex, race, BMI, or procedure type. DVT had been diagnosed significantly later in the SCD patients than in the controls (median, postoperative day 12 vs 5; P = .014). None of the five SCD patients with VTE was a smoker compared with four of the six non-SCD patients with VTE, who were current or former tobacco users (P = .061). All the patients who had developed VTE had received DVT prophylaxis at surgery. CONCLUSIONS: We found no differences in the perioperative rates of DVT, PE, or mortality between the SCD patients and matched control patients after noncardiovascular surgery. Vigilant attention to routine VTE prophylaxis seemed to effectively reduce the VTE risk for these hypercoagulable patients. SCD patients might need VTE prophylaxis for a longer period postoperatively compared with those without SCD.

Intramural hematoma of the thoracic aorta: A single-institution, 12-year experience.

OBJECTIVE: The natural history and management of intramural hematoma (IMH) has varied significantly worldwide. From the present retrospective analysis of our institutional database, we have reported the long-term results from medical and surgical management of types A and B IMH. METHODS: Computed tomography reports completed at our tertiary care hospital from July 2007 to July 2020 were used to identify patients with IMH with a thickness of ≥7 mm. Those with IMH directly related to trauma, previous aortic surgery, penetrating atheromatous ulcer, dissection flap, or an iatrogenic source and those who had never received any treatment of IMH at presentation were excluded. RESULTS: A total of 54 patients with IMH had met the inclusion and exclusion criteria. Of the 54 patients, 24 had presented with Stanford type A. Of these 24 patients, 10 had initially undergone surgery and 14 had initially received medical treatment. Two patients in the medical group had subsequently undergone surgery. In addition, 30 patients had presented with type B IMH and had initially received medical treatment, with 3 eventually requiring surgical intervention. In-hospital survival was 90% for type A IMH treated surgically, 93% for type A IMH treated medically, and 97% for type B IMH treated medically. At the last follow-up imaging study of the medically treated patients, 36% of those with type A IMH and 31% of those with type B IMH had experienced complete resolution of IMH at 3.7 and 31.5 months respectively, without surgical intervention. The development of an aortic aneurysm at the site of a previous IMH had occurred in 18% (2 of 11) and 12% (3 of 26) of the type A medical and type B medical cohorts. The overall rate of aortic aneurysm formation in the region of IMH or in another segment was 50%. No difference was found in long-term survival between the three cohorts at a mean follow-up of 22.8 months. CONCLUSIONS: A role appears to exist for medical treatment with anti-impulse therapy for appropriately selected patients with type A IMH. These patients must be followed up closely clinically and radiographically for signs of deterioration in the short- and long-term phases of their care. They can achieve long-term survival similar to that of surgically treated type A IMH and medically treated type B IMH patients using this algorithm. However, they might require late surgical intervention, especially for aneurysmal disease.

Predictors of mortality in nonagenarians undergoing abdominal aortic aneurysm repair: Analysis of the National Surgical Quality Improvement Program dataset.

BACKGROUND: The present study used the American College of Surgeons National Surgical Quality Improvement Program dataset to identify the predictors of 30-day mortality for nonagenarians undergoing endovascular aortic aneurysm repair (EVAR) or open surgical repair (OSR). METHODS: Patients aged >90 years who had undergone abdominal aortic aneurysm repair from 2005 to 2017 were identified using procedure codes. Those with operative times <15 minutes were excluded. The demographics, preoperative comorbidities, and postoperative complications of those who had died by 30 days were compared with those of the patients alive at 30 days. RESULTS: A total of 1356 nonagenarians met the criteria: 1229 (90.6%) had undergone EVAR and 127 (9.4%) had undergone OSR. The overall 30-day mortality was 10.4%. The patients who had died within 30 days were significantly more likely to have undergone OSR than EVAR (40.9% vs 7.2%; P < .001). They also had a greater incidence of dependent functional status (22.0% for those who had died vs 8.1% for those alive at 30 days; P < .001), American Society of Anesthesiology (ASA) classification of ≥4 (81.2% vs 18.8%; P < .001), perioperative blood transfusion (59.6% vs 20.3%; P < .001), postoperative pneumonia (12.1% vs 2.9%; P = .001), mechanical ventilation >48 hours (22.7% vs 2.6%; P < .001), and acute renal failure (12.1% vs 0.5%; P < .001). The EVAR group had a 30-day mortality rate of 2.6% in 1008 elective cases and 28.6% in 221 emergent cases. The OSR group had a 30-day mortality rate of 19.1% in 47 elective cases and 53.7% in 80 emergent cases. In the EVAR cohort, the 30-day mortality group had had a significantly greater incidence of dependent functional status (17% for those who had died vs 8% for those alive at 30 days; P = .004), ASA classification of ≥4 (76.4% vs 40.3%; P < .001), perioperative blood transfusion (57% vs 19%; P < .001), emergency surgery (71% vs 14%; P < .001), and longer operative times (150 vs 128 minutes; P = .001). CONCLUSIONS: Nonagenarians had an incrementally increased, but acceptable, risk of 30-day mortality with EVAR in elective and emergent cases compared with that reported for octogenarians and cohorts of patients not selected for age. We found greater mortality for patients with dependent status, a higher ASA classification, emergent repair, and OSR. These preoperative risk factors could help identify the best surgical candidates. Given these results, consideration for EVAR or OSR might be reasonable for highly selected patients, especially for elective patients with a larger abdominal aortic aneurysm diameter for whom the risk of rupture is higher.

Prospective evaluation of an evidence-based decision tool to assess pediatric blunt abdominal trauma (BAT).

PURPOSE: Computed tomography (CT) is currently the standard for evaluation of intra-abdominal injury (IAI) after BAT. Pediatric patients receiving CT scans based on adult clinical protocols are potentially exposed to unnecessary radiation. The purpose of this study is to determine the rate of CT scans before and after implementation of a pediatric BAT decision tool. METHODS: We adapted and implemented an evidence-based decision tool for pediatric BAT based on five clinical variables. We reviewed patient charts 18 months pre- and post-implementation. Demographics and outcomes were compared using Chi-square and Fisher's exact test, accordingly. RESULTS: The pre and post-implementation groups were uniform when comparing age, sex, mechanism, and Injury Severity Score. The decision tool was utilized in 85% of patients post-implementation. Fewer CT scans were obtained in the post-implementation group (28 vs. 21%, p = 0.215) with no missed injuries or late diagnoses. CONCLUSION: Implementation of a pediatric BAT decision tool decreased CT usage and radiation exposure without an obvious compromise to patient care. This experience supports the utilization of these tools for the assessment of IAI after BAT and have resulted in more selective use of CT during pediatric BAT in our program.

Long-term outcomes and interventions of postoperative type 1a endoleak following elective endovascular aortic aneurysm repair.

OBJECTIVE: This study evaluated the incidence and long-term outcomes of postoperative type 1a endoleak (PT1a) following endovascular aortic aneurysm repair (EVAR). METHODS: A retrospective review of consecutive aortoiliac EVARs performed at a single institution from June 2006 to June 2012 was conducted. Patients with PT1a were identified by postoperative imaging and compared with those who did not develop a PT1a. Late outcomes were also studied of a subset of patients with PT1a who had persistent intraoperative type 1a endoleak (iT1a) on completion angiogram during EVAR that had resolved on initial follow-up imaging. RESULTS: Three hundred eighty-nine patients underwent EVAR with median follow-up of 87 months (interquartile range, 64-111 months). The incidence of PT1a was 8.2% (n = 32) with a median follow-up of 74 months (interquartile range, 52-138 months). Compared with the total cohort, those who developed PT1a were statistically more likely to be female (32% vs 17%; P = .03) and have a higher all-cause mortality (71% vs 40%; P < .01) and aneurysm-related mortality (15.6% vs 1.7%; P < .01). Median time to presentation was 52 months. Of the 32 patients with PT1a, five (15.6%) presented with aortic rupture, of which three underwent extension cuff placement, one had open graft explant, and one declined intervention. Six patients in total (18.7%) declined intervention; five of these died of nonaneurysmal causes and one remains alive. Of the 26 patients with PT1a who had intervention, 21 (80.7%) showed resolution of PT1a, and five (19.2%) had recurrence. For patients with recurrent PT1a, two had resulting aneurysm-related mortality, two endoleaks resolved after relining with an endograft, and one patient declined intervention but remains alive. Patients with PT1a who had intervention with resolution showed no significant difference in median survival estimates (140.0 months) compared with the remaining EVAR cohort (120.0 months; P = .80). Within the PT1a cohort, 6 (18.7%) had also experienced iT1a with a mean time to presentation of the late PT1a of 45 months. iT1a was associated with a significantly increased likelihood of developing a PT1a (P < .01) and decreased median survival (P < .01), but there was no known aneurysm-related mortality. CONCLUSIONS: Development of PT1a following elective EVAR is associated with increased all-cause and aneurysm-related mortality and presents an average of 52 months postoperatively. This underscores the importance of long-term surveillance. Patients with PT1a who had a successful intervention showed no significant difference in median survival. Those with iT1a had a higher risk for PT1a compared with the EVAR cohort overall and had decreased median survival, without increased aneurysm-related mortality.

Vascular invasion predicts advanced tumor characteristics in papillary thyroid carcinoma.

BACKGROUND: The clinical impact of vascular invasion in Papillary Thyroid Carcinoma (PTC) is not well understood. Our aim was to determine if there was an association between vascular invasion and other tumor characteristics and patient outcomes in PTC. METHODS: A retrospective chart review was performed of 536 patients with PTC between January 2007-December 2011. Patient demographics, comorbidities, tumor characteristics, and outcomes were collected. RESULTS: Vascular invasion was associated with lymphatic invasion, capsular invasion, extrathyroidal extension, and the presence of positive lymph nodes. Logistic regression revealed that tumor size was a predictor of vascular invasion. Vascular invasion in PTC tumors was associated with higher tumor recurrence rates, but there were no differences in mortality. CONCLUSION: This study indicates that vascular invasion in PTC is associated with other aggressive pathologic features and an increased recurrence rate. For these reasons, vascular invasion should be an important tumor characteristic when determining extent of treatment.

Type II endoleak with an enlarging aortic sac after endovascular aneurysm repair predisposes to the development of a type IA endoleak.

OBJECTIVE: The most common endoleak after endovascular aneurysm repair is type II. Although type II endoleaks (TIIEL) are generally considered benign, there are reports that they can lead to aortic rupture. In this study, we reviewed the effect of TIIEL on sac size change to determine if sac expansion owing to a TIIEL could result in the development of a type IA endoleak (TIAEL). METHODS: After internal review board approval, all aortoiliac endovascular aneurysm repairs performed at a single institution between June 2006 and June 2012 were retrospectively reviewed. Patient demographics, comorbidities, aneurysm diameter, graft type, need for reintervention, and complications were collected. Patients with TIIEL diagnosed on follow-up imaging were categorized as those who underwent intervention for their TIIEL and those who did not. Outcomes were tabulated with attention to sac size change, development of TIAEL, rupture, and survival. RESULTS: Six hundred twenty-seven patients underwent aortoiliac stent graft placement at our institution during this time period. Patients with an operative indication other than nonruptured infrarenal abdominal aortic aneurysm and those without preoperative computed tomography angiography or follow-up data available for review were excluded. The total number of patients included was 389 with an average follow-up of 58.8 months (range, 0-194 months). Follow-up imaging diagnosed 124 patients with TIIEL (32%). Patients with TIIEL were significantly older (P < .0001) and more likely to be hypertensive (P < .05) but less likely to be smokers (P = .01). They had a significantly larger sac size increase than patients without TIIEL (9.50 vs -0.78 mm; P < .0001). Those with TIIEL were significantly more likely to develop a TIAEL than patients who did not have TIIEL (14% vs 5%; P = .004), but the rate of rupture was not significantly different (4% vs 2%; P = .33). In those with a TIIEL, the average sac size increase at which TIAEL developed was 13 mm. Patients in the TIIEL group who underwent intervention for their TIIEL survived significantly longer than patients who did not undergo intervention (140 months vs 100 months; P = .004). CONCLUSIONS: Our data suggest that there is an increased incidence of late TIAEL in patients with TIIEL compared with those without a TIIEL. Our study also demonstrates an increased overall survival in TIIEL patients who underwent intervention. Future studies are necessary to better define the association between TIIEL with enlarging sac and the development of TIAEL. However, it is reasonable to conclude that intervention for TIIEL should be undertaken at or before a cumulative sac size increase of 13 mm.

Evaluation of optimal RNA extraction method from human carotid atherosclerotic plaque.

Investigating molecular mechanisms involved in the formation of carotid atherosclerotic plaques has been challenging. Isolating high-quality RNA from plaque tissue can be difficult because of acellularity, calcification, and degradation. It is essential that the mRNA isolated from this tissue preserves and reflects the actual relative gene expression. Two common methods for RNA preservation, snap-freezing and stabilizing reagent, were compared using surgically resected human carotid atherosclerotic tissue. In addition, isolation methods were compared for integrity and quantity: column-based extraction, phenol-based extraction, and a combination of the two. We found that using a stabilizing reagent with column filtration resulted in the lowest yield and quality. Phenol-based extraction resulted in higher yields but also increased fragmentation. Snap-frozen tissue coupled with column-based extraction yielded the highest quality. The higher quality and quantity RNA obtained when processing snap-frozen tissue with column-based extraction make it possible to use difficult sample types for molecular downstream applications.

In vitro response of human chondrocytes to a combination of growth factors and a proteinase inhibitor.

Degenerative disk disease is an accelerating cascade of tissue degeneration in the intervertebral disk. A harsh catabolic environment perpetuates the degeneration of the intervertebral disk. Tissue engineering-based techniques offer effective treatment to slow the progression of degenerative disk disease and regenerate intervertebral disk tissue. The purpose of this study was to assess the efficacy of a regenerative therapy for degenerative disk disease by treating human chondrocytes with anabolic growth factors and a proteinase inhibitor. The use of both proved effective in upregulating important extracellular matrix markers of human chondrocytes. These successful in vitro results have implications for the regeneration of the intervertebral disk.

Cytokines regulate neuronal gene expression: differential effects of Th1, Th2 and monocyte/macrophage cytokines.

Inflammatory mediators, including cytokines, contribute to neuronal and axonal dysfunction and cell death. To examine the roles of cytokines in pathogenesis and regeneration in the central nervous system (CNS), we analyzed effects of cytokines on early gene regulation (6h) in neuronal cultures, employing gene arrays. Our hypothesis is that neuronal gene expression is differentially regulated in vitro by cytokine mixtures typical of Th1 and Th2 T cells and monocytes/macrophages (M/M). Th1 and M/M cytokines showed similar patterns for regulation of numerous pathways including cytokine-receptor interactions, MAP kinase, toll like receptors, apoptosis, PPAR signaling, cell adhesion molecules (CAMS), antigen processing, adipocytokine, and JAK-STAT signaling. M/M cytokines uniquely regulated genes in T cell, B cell and ECM receptor signaling pathways. Th2 cytokines had few effects on pathways regulated by Th1 and MM cytokines, but uniquely regulated genes related to neuroactive ligand-receptors and calcium. Th1 and MM cytokines markedly upregulated a wide array of cytokine-related genes. Notably, M/M cytokines uniquely upregulated G-CSF, GM-CSF, CXCL5 and lymphotactin (Xcl1). Th2 cytokines did not upregulate cytokine-related genes, with the exception of CCL11 and FMS-like tyrosine kinase 1, a VEGF receptor. In neuroactive ligand-receptor pathways, Th1 and M/M cytokines upregulated gene expression for tryptophan hydroxylase. Th1 cytokines upregulated gene expression for GABA A receptor, delta, while Th2 cytokines downregulated GABA A receptor, gamma 3. Significant changes occurred in several genes in the wnt and Notch signaling pathways, which are highly conserved and play critical roles in neuronal and glial differentiation. In the ubiquitin-proteasome pathway, proinflammatory cytokine mixtures induced upregulation of several genes, notably ubiquitin D (Ubd/FAT10), ubiquitin ligase and several proteasomal proteins. In agreement with microarray results, QRT-PCR showed marked upregulation of gene expression for Ubd with Th1 and M/M, for transglutaminase 2 with M/M, and for arginase 1 with Th2 cytokines. Expression of Ubd in the nervous system has not been previously reported. Both message and protein for Ubd are expressed in neurons, and upregulated by pro-inflammatory cytokines. Transglutaminase 2 has been implicated in neurodegenerative diseases, and proposed as a therapeutic target. Upregulation of arginase by Th2 cytokines could be potentially neuroprotective by decreasing NO generation and enhancing neurite outgrowth. Our analysis of changes in neuronal gene expression at the time of initial exposure to an abnormal cytokine milieu provides the opportunity to identify early changes that could be reversed to prevent later irreversible neuronal damage and death in multiple sclerosis and other CNS diseases.

Cytokines decrease expression of interleukin-6 signal transducer and leptin receptor in central nervous system glia.

Multiple sclerosis (MS) lesion formation is modulated by cytokines secreted within the central nervous system (CNS). Th1 lymphocytes and monocyte/macrophages (MM) likely induce lesion formation, whereas Th2 lymphocytes may inhibit formation. To explore the role of cytokines in MS lesions, we used gene arrays to investigate effects of cytokines representative of Th1 and Th2 cells and M/M on gene expression in cultured CNS glia; at 6 hr, all three increased expression of the interleukin-6 (IL-6) gene and decreased expression of the leptin receptor gene (obr), which mediates IL-6 production and other inflammatory responses. However, expression of a closely related gene, the interleukin-6 signal transducer or gp130 (IL-6st), showed no changes at 6 hr. IL-6st is an essential component of receptor complexes for IL-6 and other cytokines and growth factors that play critical roles in CNS inflammation, protection, and/or regeneration. To analyze expression of IL-6st and leptin receptor over time, we incubated rat CNS glial cultures for 6 hr to 5 days with the cytokines. All three cytokine mixtures down-regulated both IL-6st and leptin receptor mRNA and protein for up to 5 days. Immunocytochemical staining showed expression of both IL-6st and leptin receptor in all three types of glia, with lower IL-6st expression by 3 days. Down-regulation of IL-6st and leptin receptor in glia by cytokines could lead to decreased signaling by the proinflammatory IL-6 and reduced responses to regenerative/protective growth factors such as leukemia inhibitory factor and ciliary neurotrophic factor, potentially affecting the disease course in MS.

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for molecules associated with metabolism, signaling and regulation in central nervous system mixed glial cell cultures.

BACKGROUND: Cytokines secreted by immune cells and activated glia play central roles in both the pathogenesis of and protection from damage to the central nervous system (CNS) in multiple sclerosis (MS). METHODS: We have used gene array analysis to identify the initial direct effects of cytokines on CNS glia by comparing changes in early gene expression in CNS glial cultures treated for 6 hours with cytokines typical of those secreted by Th1 and Th2 lymphocytes and monocyte/macrophages (M/M). RESULTS: In two previous papers, we summarized effects of these cytokines on immune-related molecules, and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. In this paper, we present the effects of the cytokines on molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. Many of the changes in gene expression were similar to those seen in ischemic preconditioning and in early inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), related to ion homeostasis, mitochondrial function, neurotransmission, vitamin D metabolism and a variety of transcription factors and signaling pathways. Among the most prominent changes, all three cytokine mixtures markedly downregulated the dopamine D3 receptor, while Th1 and Th2 cytokines downregulated neuropeptide Y receptor 5. An unexpected finding was the large number of changes related to lipid metabolism, including several suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Using QRT-PCR we validated the results for regulation of genes for iNOS, arginase and P glycoprotein/multi-drug resistance protein 1 (MDR1) seen at 6 hours with microarray. CONCLUSION: Each of the three cytokine mixtures differentially regulated gene expression related to metabolism and signaling that may play roles in the pathogenesis of MS, most notably with regard to mitochondrial function and neurotransmitter signaling in glia.

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins.

BACKGROUND: In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells. METHODS: To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment. RESULTS: In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells. CONCLUSION: Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system.

Expression of P0 glycoprotein in CNS glia: effects of overexpression in N20.1 cells.

To examine effects of expression of the PNS myelin P0 glycoprotein in glial cells of CNS lineage, we transfected murine N20.1 glial cells with a rat P0 cDNA. A stably transfected cell line expressing high levels of P0 message showed P0 immunostaining, along with changes in morphology. Polymerase chain reaction (PCR) identified the predicted rat P0 sequence in the transfected N20.1 cells and further revealed low levels of mouse P0 message in the nontransfected cells and in primary mouse astrocytes. This is the first evidence of endogenous expression of message for P0 glycoprotein in CNS glia. Quantitative RT-PCR confirmed the expression of rat P0 mRNA in the transfected N20.1 cells, at levels about 400 times greater than murine P0 in nontransfected cells. A 27-kD band was detected in the transfected cells by Western blot with P0 antibody, but not in mock-transfected or nontransfected N20.1 cells. Immunocytochemistry following permeabilization showed intracellular vesicular localization of P0 in the cytoplasm and perinuclear rings in transfected cells, with a similar pattern but much lower levels in nontransfected cells. Faint surface staining for P0 protein without permeabilization was seen only on the transfected cells. A few transfected cells with membrane sheets stained more intensely for surface P0. Quantitative RT-PCR was used to determine if P0 overexpression altered expression of other myelin-related genes compared with glial fibrillary acidic protein (GFAP); the ratios of myelin basic protein (MBP)/GFAP and proteolipid protein (PLP)/GFAP were increased 2- to 3-fold in the P0-transfected cells. We conclude that P0 overexpression alters N20.1 gene expression and cell morphology, and shifts the cells from astroglial to oligodendroglial phenotype.

Regulation of CNS glial phenotypes in N20.1 cells.

The N20.1 oligodendroglial cell line, immortalized with SV40 T antigen, simultaneously expresses oligodendroglial markers and glial fibrillary acidic protein (GFAP), an astroglial marker. This study examines the plasticity of N20.1 cells with regard to GFAP expression, and its relationship to expression of SV40 T antigen, p53, and a novel nuclear antigen detected by the A007 monoclonal antibody. Marked changes occur in GFAP levels and cell morphology when N20.1 cells are switched from the permissive temperature (34 degrees C) to the non-permissive temperature (39 degrees C), and with cyclic AMP elevation at 39 degrees C. At 34 degrees C, levels of GFAP are high; when cells are switched to 39 degrees C, GFAP levels decrease significantly, then increase slightly when forskolin is added. At both temperatures, the cells display feathery GFAP immunostaining. When forskolin is added at 39 degrees C, however, cells display bright fibrous GFAP staining in elongated processes. The changes in GFAP were compared to changes in T antigen and p53. As expected, the decrease in T antigen at 39 degrees C was accompanied by movement of p53 from the nucleus to cytoplasm. Total p53 levels did not change, however, and forskolin did not alter the respective distribution or levels of p53 at either temperature. At both temperatures, the cell bodies and processes show internal expression of sulfatide, as demonstrated with the O4, Sulph I, and A007 antibodies. We show, for the first time, abundant nuclear immunoreactivity with the A007 monoclonal antibody in the N20.1 cells. This nuclear reactivity is seen at 34 degrees C, but not at 39 degrees C, similar to p53, and is not detected with the other sulfatide antibodies. Double-label immunostaining shows that the nuclear A007 immunoreactivity is co-localized in nuclear structures with T antigen and p53 at 34 degrees C, but is not found in every nucleus containing these antigens. We conclude that regulation of GFAP expression and morphology in N20.1 cells is dependent on a combination of T antigen expression and level of cAMP and may be related to regulation of p53 and the A007 nuclear antigen.