Absence of "Cytokine Storm" in Hospitalized COVID-19 Patients: A Retrospective Cohort Study.

Background: A rapidly growing number of publications cite "cytokine storm" as a contributing factor in coronavirus disease 2019 (COVID-19) pathology. However, a few recent reports led to questioning of "cytokine storm" theory in COVID-19. This study's primary goal is to determine if exaggerated cytokine response in the range of a "cytokine storm" develops during the initial weeks of hospitalization in COVID-19 patients. Methods: Five proinflammatory cytokines reported to be involved in "cytokine storm" and elevated in COVID-19 (IL-6, IL-8, TNF-α, MCP-1, and IP-10) were analyzed in COVID-19, influenza (with "cytokine storm": CS), and burn injury patients. The effect of dexamethasone use on cytokine response in COVID-19 was also analyzed. Results: None of the five cytokines in COVID-19 patients reached the lower threshold (95% CI) of the influenza (CS) group at any point during the study period. Furthermore, mean concentrations of all five cytokines in the influenza (CS) group and IL-6, IL-8, TNF-α in the burn group were significantly greater than in COVID-19 patients (p < 0.01). Dexamethasone treatment did not significantly alter the concentrations of any of the cytokines analyzed. Conclusions: Exaggerated cytokine response similar to "cytokine storm" was not observed in COVID-19 patients during two weeks of hospitalization.

Factors influencing long-term weight loss after bariatric surgery.

BACKGROUND: Bariatric surgery provides sustained weight loss and improves comorbidities. However, long term data has shown that patients gradually regain weight after 1 year. Several factors have been associated with poor weight loss after bariatric surgery. OBJECTIVE: Our goal is to investigate factors associated with poor weight loss following laparoscopic sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). SETTING: Military academic medical center. METHODS: Retrospective review of 247 patients who underwent laparoscopic SG or RYGB between 2010-2012 at Eisenhower Army Medical Center and followed for 5 years postoperatively. Factors of age, type of surgery, sex, hypertension, depression, and type 2 diabetes (T2D) are analyzed in univariate and multivariate analysis with percent total weight loss (%TWL) and Body Mass Index (BMI) change as primary endpoints measured at 3 and 5 years. RESULTS: Average BMI change are maximized at 1 year and decreased at 3 and 5 years post-surgery. Age, diabetes, hypertension and type of surgery significantly influenced weight loss at 3 and 5 years on univariate analysis. However, patients with diabetes, hypertension and sleeve gastrectomy were significantly older than comparable control group. Multivariable analysis showed that age and type of surgery, not diabetes or hypertension, were associated with poor %TWL and BMI change at 3 and 5 years. CONCLUSION: While presence of hypertension and diabetes initially appeared to be associated with weight recidivism, their impacts were negligible on multivariable analysis. However, age and sleeve gastrectomy are independent risk factors. Our data can be used to counsel patients on expected weight loss after bariatric surgery.

Correlation of intra-operative hamstring autograft size with pre-operative anthropometric and MRI measurements.

The purpose of this study was to compare various pre-operative methods of estimating intra-operative hamstring autograft size. A retrospective review was completed on 74 patients who had an anterior cruciate ligament reconstruction performed using a quadruple-looped hamstring autograft from July 2007-April 2015 at a single institution. A positive correlation was observed between intra-operative graft size and pre-operative imaging using two methods of MRI measurements. Correlation existed between hamstring size and patient height and weight, but not age or BMI. Thus, pre-operative MRI and anthropometric measurements can be used to estimate intra-operative hamstring graft size.

Time Course of Blood Pressure Decrease After Bariatric Surgery in Normotensive and Hypertensive Patients.

BACKGROUND: Bariatric surgery leads to remission of several obesity-related comorbidities, including hypertension. Although antihypertensive medication use is decreased after bariatric surgery, the exact time course of decrease in blood pressure after surgery is not known. METHODS: A database of patients undergoing bariatric surgery at our institute was used to study the effect of surgery on time course of blood pressure changes. Data from surgeries performed between January 2010 and December 2012 were used. RESULTS: Maximum blood pressure and body weight decreases were observed at 2 weeks and 1 year after surgery, respectively. Average decrease in the mean arterial pressure (MAP) was 4.46 mmHg (61.5 ± 17.1% of maximal decrease) and 7.17 mmHg (maximum decrease) at 1 and 2 weeks after surgery, when the decrease in body weight is 22.8 ± 1.6 and 28 ± 1.4% of maximal weight loss, respectively. In hypertensive patients, MAP decreased from 98.5 ± 0.78 to 92.3 ± 1.76 and 93.1 ± 0.92 mmHg at 1 and 2 weeks post-surgery, respectively. In normotensive patients, the MAP decreased from 96.2 ± 0.79 to 88.7 ± 1.25, 90.0 ± 0.94, 86.5 ± 1.35, 88.0 ± 1.13, and 86.4 ± 2.13 mmHg at 2 weeks, 3 and 6 months, and 1 and 3 years after surgery, respectively. CONCLUSIONS: These data demonstrate that significant decrease in MAP occurs within 2 weeks after bariatric surgery in hypertensive as well as normotensive patients. Future studies are required to investigate the weight-independent mechanisms of blood pressure decreases after bariatric surgery.

Randomized trial of OFIRMEV versus placebo for pain management after laparoscopic sleeve gastrectomy.

BACKGROUND: OFIRMEV is an intravenous form of acetaminophen approved by the Food and Drug Administration for use as an antipyretic and treatment of mild to moderate pain alone or in conjunction with opioid medications. Intravenous APAP use in postsurgical pain management has been reported to decrease opioid usage, time to rescue dose, and subjective pain. OBJECTIVES: We used a placebo-controlled, randomized double-blind study to test the efficacy of OFIRMEV in decreasing opioid use and subjective pain after laparoscopic sleeve gastrectomy. SETTING: U.S. military training hospital. METHODS: Thirty-four patients who met criteria were enrolled and randomly assigned to 2 separate limbs of the study. The OFIRMEV and placebo groups had similar mean age ranges (48±11 and 50±11 yr) and a female/male ratio of 5:1 and 6:1, respectively. The patients received an intraoperative dose and then postoperative administration of intravenous OFIRMEV 1 g or placebo every 6 hours for 24 hours in addition to fentanyl via patient-controlled analgesia. Subjective pain scores, the total amount of fentanyl used, time to rescue of first narcotic dose, and total postanesthesia care unit (PACU) narcotic use were measured during the first 24 hours after surgery. RESULTS: Subjective pain score was significantly decreased compared with baseline at 12, 16, and 20 hours after surgery in OFIRMEV-treated patients but not in the placebo group. However, total narcotic use, time to rescue of first narcotic dose, and total PACU narcotic dose were not statistically different between the 2 groups. CONCLUSION: Intravenous OFIRMEV use caused a modest but statistically significant decrease in subjective pain without affecting narcotic use after laparoscopic sleeve gastrectomy. (Surg Obes Relat Dis 2015;0:000-00.) © 2015 American Society for Metabolic and Bariatric Surgery. All rights reserved.

Permacol interposition graft as an alternative to vein in contaminated wounds using a rabbit model.

BACKGROUND: Vascular injuries are common in trauma and often involve massive soft tissue injury and segmental arterial loss. Current practice uses either autogenous vein or polytetrafluorethylene (PTFE) for interposition grafting in arterial injuries. Decision making between autogenous or synthetic conduit pivots around the physiological state of the trauma patient. Vein is known to increase operative times in an already physiologically depleted patient, whereas synthetic graft can be simply pulled from the shelf. However, when used in contaminated wounds, PTFE is prone to chronic infection and subsequent graft failure. An alternative synthetic conduit resistant to infection would be ideal for such situations. Permacol (Tissue Science Laboratories, Inc, Andover, MA), a biosynthetic material, has demonstrated resistance to bacterial contamination in contaminated hernia repairs. When fashioned into a tubular structure, this material may be useful as an alternative vascular conduit in contaminated trauma wounds. METHODS: New Zealand white rabbits were randomized to one of 4 groups: Permacol graft (P) without bacterial contamination (n = 9), Permacol graft with bacterial contamination (CP; n = 9), autogenous vein graft without bacterial contamination (V; n = 9), or autogenous vein with bacterial contamination (CV; n = 9). All groups then underwent interposition grafting of the right common carotid artery. Grafts were contaminated by applying Staphylococcus aureus (1 × 10(5) colonies/0.1 mL) directly to the exposed surface of the graft on completion of the arterial repair. Each graft was then excised at day 42, and segments were collected for histologic evaluation, bacterial counts, and real-time polymerase chain reaction. RESULTS: Of the 36 rabbits used in this study, 3 animals in the CV group died within 72 hr of surgery. There was no difference in early mortality between P and V (0% vs. 0%; P = 1.0); however, early mortality was higher in the CV compared with the CP group (33% vs. 0%; P = 0.023). At 42 days, histologic evaluation of graft patency demonstrated no difference between P and V (67% vs. 33%; P = 0.157); however, patency was higher in CP than CV (56% vs. 12%; P = 0.040). In addition, no difference was found between the 2 contaminated groups in regard to the number of bacteria present on each graft material. CONCLUSIONS: Permacol as an interposition graft is a feasible alternative to vein in a contaminated setting and shows resistance to infection in a rabbit model. Future studies are needed to evaluate this material in larger animal models.

Evaluating the molecular basis for acute mountain sickness: hypoxia response gene expression patterns in warfighters and murine populations.

Acute mountain sickness (AMS) is an illness that affects many individuals at altitudes above 2,400 m (8,000 ft) resulting in decreased performance. Models that provide quantitative estimates of AMS risk are expanding, but predictive genetic models for AMS susceptibility are still under investigation. Thirty-four male U.S. Army Soldier volunteers were exposed to baseline, 3,000 m, 3,500 m, or 4,500 m altitude conditions in a hypobaric chamber and evaluated for onset of AMS symptoms. In addition, mice were evaluated at extreme hypoxia conditions equivalent to 7,600 m. Real-time polymerase chain reaction hypoxia response array was used to identify 15 genes that were activated in Soldiers and 46 genes that were activated in mice. We identified angiopoietin-like 4 (ANGPTL4) as a gene that is significantly activated in response to hypoxia (5.8-fold upregulated at 4,500 m in humans). The role of ANGPTL4 in high-altitude response has not been explored. Pretreatment of mice with fenofibrate, an ANGPTL4-activating pharmaceutical, had a considerable effect on overall hypoxia response gene expression and resulted in significantly decreased cerebral edema following exposure to hypoxia. Activation of ANGPTL4 may protect against cerebral edema by inhibiting vascular endothelial growth factor and therefore serve as a potential target for AMS prevention.

Regulation of dopamine transporter expression by neuronal activity.

Actions of extracellular dopamine released in the central nervous system are primarily terminated by the dopamine transporter. This protein is also a target for therapeutic and abused psychostimulant drugs. Different methods used to study dopamine transporter function, its expression, and intracellular signaling in neurons are described in this chapter. Function of the dopamine transporter in mesencephalic primary cultures can be measured by dopamine uptake assay. Expression of the transporter protein and mRNA are analyzed by western blots and quantitative RT-PCR, respectively. Finally, methods to study neuronal activity-dependent changes in Ca(2+)/calmodulin-dependent protein (CaM) kinase activity in dopamine neurons are described.

Moderate systemic hypothermia decreases burn depth progression.

BACKGROUND: Therapeutic hypothermia has been proposed to be beneficial in an array of human pathologies including cardiac arrest, stroke, traumatic brain and spinal cord injury, and hemorrhagic shock. Burn depth progression is multifactorial but inflammation plays a large role. Because hypothermia is known to reduce inflammation, we hypothesized that moderate hypothermia will decrease burn depth progression. METHODS: We used a second-degree 15% total body surface area thermal injury model in rats. Burn depth was assessed by histology of biopsy sections. Moderate hypothermia in the range of 31-33°C was applied for 4h immediately after burn and in a delayed fashion, starting 2h after burn. In order to gain insight into the beneficial effects of hypothermia, we analyzed global gene expression in the burned skin. RESULTS: Immediate hypothermia decreased burn depth progression at 6h post injury, and this protective effect was sustained for at least 24h. Burn depth was 18% lower in rats subjected to immediate hypothermia compared to control rats at both 6 and 24h post injury. Rats in the delayed hypothermia group did not show any significant decrease in burn depth at 6h, but had 23% lower burn depth than controls at 24h. Increased expression of several skin-protective genes such as CCL4, CCL6 and CXCL13 and decreased expression of tissue remodeling genes such as matrix metalloprotease-9 were discovered in the skin biopsy samples of rats subjected to immediate hypothermia. CONCLUSIONS: Systemic hypothermia decreases burn depth progression in a rodent model and up-regulation of skin-protective genes and down-regulation of detrimental tissue remodeling genes by hypothermia may contribute to its beneficial effects.

Methods to detect cell surface expression and constitutive activity of GPR6.

GPR6 is a constitutively active Gs-coupled receptor that can signal from intracellular compartments. We present different methods used to study cell surface expression of receptors and other membrane proteins. A comparison of these methods shows that methods based on susceptibility to proteolytic enzymes are more efficient at providing estimates of cell surface expression than the commonly used cell surface biotinylation method. We also present different methods that can be used to detect constitutive activity of Gs-coupled receptors. Imaging-based assays to detect intracellular cyclic AMP accumulation are well suited to study signaling at a single cell level. These assays are particularly useful when the cells of interest form a small fraction of the culture such as primary cultures with low transfection efficiency.

Role of growth factors in improved skin flap viability caused by phosphodiesterase-5 inhibitor.

Flap necrosis is one of the major complications of reconstructive surgery and sildenafil citrate has been shown to decrease flap necrosis in preclinical animal models. However, the mechanisms underlying sildenafil's therapeutic efficacy are not known. As with other phosphodiesterase 5 selective inhibitors, sildenafil causes vasodilation and enhanced blood flow. In addition, sildenafil can also alter gene expression. This study is designed to test the hypothesis that increased expression of angiogenic growth factors may be responsible for therapeutic efficacy of sildenafil. A modified McFarlane flap measuring 3 x 10 cm was created on the dorsal skin of male Sprague-Dawley rats. For growth factor expression experiment, rats were administered either vehicle or sildenafil 10 mg/Kg intraperitoneal (IP). Ribonucleic acid (RNA) extracted from skin flap was analyzed to assess the messenger ribonucleic acid (mRNA) levels of different angiogenic growth factors. For skin flap viability experiment, fibrin film impregnated with vehicle, fibroblast growth factor (FGF) (5.0 microg) or vascular endothelial growth factor (VEGF) (2.0 microg) was applied to the wound. The skin flap was then returned to its native position and stapled in place. Total affected area (area of necrosis and blood flow stasis) of each rat on postoperative day 14 was analyzed with orthogonal polarization spectral imaging. Daily systemic treatment with sildenafil significantly (P < 0.05) increased the expression of FGF1 and FGF Receptor 3 on postoperative day 3 by 5.08- and 4.78-fold, respectively. In addition, sildenafil increased the expression of VEGF-A, VEGF-B, and VEGF-C by 2.66-, 2.02-, and 2.00-fold, respectively. Subcutaneous treatment with FGF but not VEGF-A tended to decrease total affected area in rats. These data demonstrate that sildenafil altered the expression of FGF and VEGF. Altered expression of growth factors may be, at least partly, responsible for the beneficial effects of sildenafil citrate on skin viability.

Constitutively active GPR6 is located in the intracellular compartments.

We used multiple imaging assays to test the hypothesis that GPR6, a constitutively active Gs-coupled receptor, is present on the cell surface. A pHluorin tag at the N-terminus of rat GPR6 expressed in human embryonic kidney 293 (HEK293) cells was not accessible to protons, chymotrypsin or anti-green fluorescent protein antibody, demonstrating that GPR6 is primarily located in intracellular compartments. Similar intracellular localization of pHluorin-tagged GPR6 was found in striatal neurons, where endogenous GPR6 is expressed. Confirmation of Gs-mediated constitutive activity in HEK293 cells and striatal neurons led us to conclude that GPR6 can signal from intracellular compartments.

Activity-dependent regulation of the dopamine transporter is mediated by Ca(2+)/calmodulin-dependent protein kinase signaling.

The mechanisms regulating expression of the dopamine transporter are poorly understood. We tested the hypothesis that neuronal activity is one of the non-genetic determinants of dopamine transporter abundance. Sustained changes in neuronal activity caused by tetrodotoxin and 4-aminopyridine altered the dopamine uptake and abundance of dopamine transporter and its mRNA in rat mesencephalic cultures. The altered neuronal activity caused by these two drugs is accompanied by changes in intracellular calcium concentrations and Ca(2+)/calmodulin-dependent protein (CaM) kinase II activity in dopamine neurons. Chronic treatment with an L-type calcium channel blocker (nifedipine) or CaM kinase inhibitor (KN93) decreased dopamine transporter-mediated uptake and occluded the effects of tetrodotoxin and 4-aminopyridine. These data suggest that neuronal activity can regulate dopamine transporter function and abundance via calcium/CaM kinase II signaling.

Contributions of nucleus accumbens core and shell GluR1 containing AMPA receptors in AMPA- and cocaine-primed reinstatement of cocaine-seeking behavior.

Glutamate signaling in the nucleus accumbens influences reinstatement of previously extinguished cocaine-seeking behavior in rats. Whether or not region specific glutamate signaling in the nucleus accumbens contributes to reinstatement of cocaine-seeking behavior is not known. We investigated whether directly stimulating ionotropic glutamate receptors (GluRs) within the nucleus accumbens core or shell would differentially influence renewed cocaine-seeking behavior following extinction training. We also tested the hypothesis that GluR1 subunit (GluR1) containing alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors in the nucleus accumbens core and not the shell regulate reinstatement of previously extinguished cocaine-seeking behavior. Microinjection of AMPA into the nucleus accumbens shell and the nucleus accumbens core dose-dependently elicited significant cocaine-seeking behavior. Administration of antisense oligonucleotides (AS) directed against GluR1 subunit mRNA into the core and shell disrupted AMPA- and cocaine-primed reinstatement--with the most pronounced effects seen in the nucleus accumbens shell. These results demonstrate that GluRs in the nucleus accumbens core and shell influence AMPA- and cocaine-primed reinstatement, yet the nucleus accumbens shell exerts a prepotency over the nucleus accumbens core.

Genetic complementation screen identifies a mitogen-activated protein kinase phosphatase, MKP3, as a regulator of dopamine transporter trafficking.

The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.

Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain.

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.

Dopamine transporter-mediated conductances increase excitability of midbrain dopamine neurons.

Uptake by Na(+)/Cl(-)-dependent neurotransmitter transporters is the principal mechanism by which extracellular biogenic amine concentrations are regulated. In addition to uptake, the cloned transporter proteins also elicit ion channel-like currents, but the physiological consequences of these currents are unknown. Here, whole-cell patch clamp and perforated-patch recordings show that substrates of the dopamine transporter (DAT), such as dopamine (DA) and amphetamine, increase the firing activity of rat DA neurons in culture. We found that these substrates elicit inward currents that are Na(+)-dependent and blocked by cocaine. These currents are primarily comprised of anions and result in an excitatory response in DA neurons at lower DA concentrations than are required for D2 autoreceptor activation. Thus, in addition to clearing extracellular DA, our results suggest that the currents associated with DAT modulate excitability and may regulate release of neurotransmitter from midbrain DA neurons.