Leukemic Optic Neuropathy in Pediatric Patients: A Case Series.

PURPOSE: To characterize the presentation, clinical course, and treatment of a series of children with leukemic optic neuropathy. METHODS: Patients with leukemia who were treated at a tertiary children's hospital for optic nerve infiltration were included (n = 11). Demographic information, cancer history, ophthalmologic examination findings, treatment, and outcomes were retrospectively collected. RESULTS: Mean age was 10.0 ± 4.8 years, and 63.6% were male and 36.4% were female. The most common underlying oncologic diagnosis was B-precursor acute lymphoblastic leukemia (n = 7, 63.6%). Notably, the majority presented with optic nerve infiltration during presumed remission (n = 9, 81.8%), but 2 patients (18.2%) presented with optic nerve infiltration at their initial leukemia diagnosis. Cerebrospinal fluid was positive for leukemic cells in 36.4% of patients. Magnetic resonance imaging demonstrated optic nerve enhancement and/or enlargement in only 8 patients (72.7%). In addition to other leukemia-directed treatment, 8 patients (72.7%) received emergent local radiation within 1.5 ± 1.2 days of initial ophthalmology examination. CONCLUSIONS: The largely negative cerebrospinal fluid results and variable magnetic resonance imaging findings in this study emphasize the importance of clinical context for this diagnosis. Clinicians should consider optic nerve infiltration in patients with leukemia and visual or ocular complaints, because urgent treatment is required to preserve vision and manage systemic disease. [J Pediatr Ophthalmol Strabismus. 2024;61(1):67-72.].

A case of blepharophimosis: Freeman Sheldon syndrome.

PURPOSE: Important implications exist for ophthalmologists when considering possible early surgical intervention for potential amblyogenic anatomical abnormalities. The authors discuss the risks and benefits from an ophthalmological perspective of different interventions and review the genetic testing that confirmed the diagnosis. OBSERVATIONS: The authors describe the findings and management of an infant with Freeman Sheldon syndrome presenting with blepharophimosis of both eyelids resulting in inability to open both eyes during the first several days of life. Although the mode of inheritance for Freeman Sheldon syndrome (formerly known as Whistling Face Syndrome) is often autosomal dominant, our patient had no known family history of congenital abnormalities or consanguinity. However, genetic testing confirmed a heterozygous variant in MYH3, consistent with autosomal dominant Freeman Sheldon Syndrome. When our patient required gastrostomy (G-tube_placement, we performed an exam under anesthesia (EUA)). As is typical for Freeman Sheldon syndrome patients, intubation was difficult and complicated by pneumothorax. Eye-opening improved slightly after several weeks of life; however, the decision was made to proceed with eyelid surgery to prevent deprivation amblyopia. Surgery is scheduled for a future date. Additionally, the patient had congenital nasolacrimal duct obstruction of the left eye; however, a probing and irrigation failed because of obstruction from the abnormal facial anatomy. CONCLUSIONS AND IMPORTANCE: Patients with Freeman Sheldon syndrome are at increased risk for complications from anesthesia and surgery. Risks and benefits should be strongly considered and discussed with parent(s)/guardian(s) prior to any surgical intervention. Genetic testing of the MYH3 gene can confirm the diagnosis.

Microbiology of Pediatric Orbital Cellulitis and Trends in Methicillin-Resistant Staphylococcus aureus Cases.

We reviewed medical records of children with orbital cellulitis with positive cultures at a tertiary institution from 2005 to 2018 to identify microbiology trends and features associated with methicillin-resistant Staphylococcus aureus (MRSA) cases. Cultures obtained from the orbits (n = 33), sinuses (n = 31), and dural cavities (n = 4) had yields of 66.7%, 61.3%, and 75%, respectively, compared with 17.6% of blood cultures (n = 69). Fifty-five patients had positive culture results. Staphylococcus aureus was the most common pathogen isolated (n = 19), followed by Streptococcus species, most commonly Streptococcus anginosus (n = 8). The most frequently prescribed antibiotic combination regimen was ampicillin-sulbactam followed by amoxicillin-clavulanate. There were 8 cases of MRSA. MRSA was associated with an age of presentation <1 year old (P = .034). Other clinical features were similar between MRSA and non-MRSA cases. In infants and neonates, or those with epidemiologic risk factors, MRSA should also be considered.

Tropicamide has limited clinical effect on cycloplegia and mydriasis when combined with cyclopentolate and phenylephrine.

PURPOSE: To examine the cycloplegic and mydriatic effect of tropicamide omission from a common pediatric eye drop combination. METHODS: Consecutive children examined at the Ann & Robert H. Lurie Children's Hospital of Chicago from June 8, 2017 to September 6, 2017 were enrolled prospectively. Tropicamide, cyclopentolate, and phenylephrine (TCP) was instilled in one eye; cyclopentolate and phenylephrine (CP), in the other. Spherical equivalent, maximum pupil size, and pupillary constriction in response to photostimulation were measured before and 30 minutes after instillation using an autorefractor and pupillometer. Iris pigmentation was examined as a between-subjects variable. RESULTS: A total of 75 children 4-11 years of age were included. Mean differences in spherical equivalent between TCP and CP were not statistically significant (P = 0.95). Significant interactions between eye drop regimen and iris pigmentation were observed for pupil size (P = 0.001) and constriction percentage (P = 0.02). Among only patients with dark irides, TCP yielded slightly larger pupils (7.70 vs 7.31 mm [P < 0.001]) that were less responsive to light (5.75% vs 8.07% [P = 0.002]). All pupils dilated to ≥6.0 mm, with equivalent proportions achieving ≥7.0 mm for TCP and CP (P = 0.18). CONCLUSIONS: TCP and CP elicited equivalent cycloplegic effects. Mydriatic differences between the regimens, although statistically significant in dark irides, were of limited clinical magnitude, and all pupils achieved sufficient dilation for funduscopy.

Pupillary manifestations of Marfan syndrome: from the Marfan eye consortium of Chicago.

BACKGROUND: Marfan syndrome (MFS) is a genetic disorder that affects multiple organ systems, including the eye. The most common ocular manifestations include ectopia lentis and retinal detachment. The current literature qualitatively cites that MFS patients have miotic or "poorly dilating" pupils. This study was the first to quantitatively assess pupillary function in MFS patients. MATERIALS AND METHODS: 57 eyes from 29 MFS patients, 36 eyes from 18 pediatric age- and gender-matched controls, and 44 eyes from 22 adult age-matched controls were measured in a clinic-based cross sectional study. Pupillometry data were measured in scotopic conditions using the handheld NeurOptics PLR-200™ Pupillometer (NeurOptics, Irvine, CA, USA). Data obtained with the pupillometer were maximum and minimum diameter, constriction percentage, latency, average and maximum constriction velocities, average dilation velocity, and 75% recovery time (T75). RESULTS: Pediatric patients with MFS had significantly slower average constriction velocity measurements (ß = 0.65, p = 0.0003), maximum constriction velocity measurements (ß = 0.51, p = 0.0150) and average dilation velocity measurements (ß = -0.19, p = 0.0029) compared to control patients. In the adult cohort, results indicated significantly slower average dilation velocity measurements (ß = -0.13, p = 0.0077) compared to controls. CONCLUSIONS: Our data highlight pupillary parameters within a population of MFS patients under scotopic conditions. Constriction and dilation velocities were slower in the pediatric MFS patients compared to age- and gender-matched controls, and dilation velocities were slower in the adult MFS patients compared to age-matched controls. These findings, for the first time, quantitatively demonstrated differences in pupillary function in patients with MFS.

Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein.

Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004-0.174, 13), day 2 (0.020, 0.002-0.109, 10), and day 3 (0.018, 0.003-0.058, 23) compared with febrile (0.705, 0.187-1.778, 22) and healthy (0.7, 0.4-1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2-54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3-20.6, 11). MMP-9/TIMP-1 ratios were significantly lower, while A-FaBP and mrProANP were higher in septic patients compared to the control groups. Each biomarker was associated with hospital morbidity and length of stay. These results suggest that these biomarkers merit further prospective study for the early identification of children with sepsis.

Thrombin decreases expression of the glutamate transporter GLAST and inhibits glutamate uptake in primary cortical astrocytes via the Rho kinase pathway.

Astrocyte glutamate transporters GLAST and GLT1 play a key role in regulating neuronal excitation and their levels are altered in patients with epilepsy, and after traumatic brain injury. The mechanisms which regulate their expression are not well understood. We tested the hypothesis that exposure of astrocytes to high levels of thrombin, as may occur after a compromise of the blood-brain barrier, would reduce astrocyte glutamate transporter levels. In isolated rat cortical astrocytes we examined the effects of thrombin on the expression and function of glutamate transporters, and the signaling pathways involved in these responses by using Western blotting and selective inhibitors. Thrombin induced a selective decrease in the expression of GLAST but not GLT1, with a corresponding decrease in the capacity of astrocytes to take up glutamate. Activation of the thrombin receptor PAR-1 with an activating peptide induced a similar decrease in the expression of GLAST and compromise of glutamate uptake. The downregulation of GLAST induced by thrombin was mediated by the mitogen activated protein kinases p38 MAPK, ERK and JNK, but inhibition of these kinases did not prevent the decrease in glutamate uptake induced by thrombin. In contrast, inhibition of the Rho kinase pathway using the specific inhibitor, Y27632, suppressed both the decrease in the expression of GLAST and the decrease in glutamate uptake induced by thrombin. In hippocampal astrocyte cultures, thrombin caused a decrease in both GLAST and GLT1. In tissue resected from brains of children with intractable epilepsy, we found a decrease in the integrity of the blood-brain barrier along with a reduction in immunoreactivity for both transporters which was associated with an increase in cleaved thrombin and reactive astrogliosis. The in vitro results suggest a specific mechanism by which thrombin may lead to a compromise of astrocyte function and enhanced synaptic excitability after the blood-brain barrier is compromised. The human in vivo results provide indirect support evidence linking the compromise of the blood-brain barrier to thrombin-induced reduction in glutamate transporter expression and an increase in neuronal excitation.

Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways.

BACKGROUND: Astrocytes are an integral component of the blood-brain barrier (BBB) which may be compromised by ischemic or traumatic brain injury. In response to trauma, astrocytes increase expression of the endopeptidase matrix metalloproteinase (MMP)-9. Compromise of the BBB leads to the infiltration of fluid and blood-derived proteins including albumin into the brain parenchyma. Albumin has been previously shown to activate astrocytes and induce the production of inflammatory mediators. The effect of albumin on MMP-9 activation in astrocytes is not known. We investigated the molecular mechanisms underlying the production of MMP-9 by albumin in astrocytes. METHODS: Primary enriched astrocyte cultures were used to investigate the effects of exposure to albumin on the release of MMP-9. MMP-9 expression was analyzed by zymography. The involvement of mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS) and the TGF-ß receptor-dependent pathways were investigated using pharmacological inhibitors. The production of ROS was observed by dichlorodihydrofluorescein diacetate fluorescence. The level of the MMP-9 inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 produced by astrocytes was measured by ELISA. RESULTS: We found that albumin induces a time-dependent release of MMP-9 via the activation of p38 MAPK and extracellular signal regulated kinase, but not Jun kinase. Albumin-induced MMP-9 production also involves ROS production upstream of the MAPK pathways. However, albumin-induced increase in MMP-9 is independent of the TGF-ß receptor, previously described as a receptor for albumin. Albumin also induces an increase in TIMP-1 via an undetermined mechanism. CONCLUSIONS: These results link albumin (acting through ROS and the p38 MAPK) to the activation of MMP-9 in astrocytes. Numerous studies identify a role for MMP-9 in the mechanisms of compromise of the BBB, epileptogenesis, or synaptic remodeling after ischemia or traumatic brain injury. The increase in MMP-9 produced by albumin further implicates both astrocytes and albumin in the acute and long-term complications of acute CNS insults, including cerebral edema and epilepsy.

Evaluation of asymmetric dimethylarginine, arginine, and carnitine metabolism in pediatric sepsis.

OBJECTIVE: Increased plasma concentrations of the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine, decreased arginine bioavailability, and mitochondrial dysfunction have been reported in adult sepsis. We studied whether asymmetric dimethylarginine, arginine, and carnitine metabolism (a measure of mitochondrial dysfunction) are altered in pediatric sepsis and whether these are clinically useful biomarkers. DESIGN: : Prospective, observational study. SETTING: Pediatric intensive care unit at an academic medical center. PATIENTS: : Ninety patients ≤ 18 yrs old, 30 with severe sepsis or septic shock, compared with 30 age-matched febrile and 30 age-matched healthy control subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma asymmetric dimethylarginine and whole blood arginine, citrulline, ornithine, and acylcarnitine:free carnitine ratio were measured daily for septic patients and once for control subjects using tandem mass spectrometry. Plasma asymmetric dimethylarginine concentration (median; interquartile range µmol/L) on day 1 was lower in severe sepsis and septic shock (0.38; 0.30-0.56) compared with febrile (0.45; 0.40-0.59) and healthy (0.60; 0.54-0.67) control subjects (p < .001), although decreased asymmetric dimethylarginine was predominantly found in neutropenic patients. Day 1 arginine was lower in septic (10; interquartile range, 7-20 µmol/L) compared with healthy patients (32; interquartile range, 23-40; p < .001), and the arginine:ornithine ratio was decreased in sepsis, indicating increased arginase activity (an alternative pathway for arginine metabolism). The arginine:asymmetric dimethylarginine and acylcarnitine:free carnitine ratios did not differ between septic and control patients. Asymmetric dimethylarginine was inversely correlated with organ dysfunction by Pediatric Logistic Organ Dysfunction score (r = -0.50, p = .009), interleukin-6 (r = -0.55, p = .01), and interleukin-8 (r = -0.52, p = .03) on admission. Arginine, arginine:asymmetric dimethylarginine, and acylcarnitine:free carnitine were not associated with organ dysfunction or outcomes. CONCLUSIONS: Asymmetric dimethylarginine was decreased in pediatric sepsis and was inversely associated with inflammation and organ dysfunction. This suggests that inhibition of nitric oxide synthase by asymmetric dimethylarginine accumulation is unlikely to impact sepsis pathophysiology in septic children despite decreased arginine bioavailability. We did not find an association of asymmetric dimethylarginine with altered carnitine metabolism nor were asymmetric dimethylarginine, arginine, and acylcarnitine:free carnitine useful as clinical biomarkers.

Mild stretch-induced injury increases susceptibility to interleukin-1ß-induced release of matrix metalloproteinase-9 from astrocytes.

Traumatic brain injury (TBI) results in the activation of glia and the release of proinflammatory cytokines, including interleukin (IL)-1ß. The response of astrocytes to mild TBI has not been well studied. We used an in vitro model of cell stretch to investigate the effects of mild mechanical insult on astrocyte injury (lactate dehydrogenase and propidium iodide), and on mediators of inflammation including IL-1ß, the chemokine CX3CL1, and nitrite. Here, we tested the hypothesis that a mild mechanical insult would increase susceptibility of astrocytes to delayed exposure to IL-1ß, including enhanced release of the matrix metalloproteinease-9 (MMP-9). We investigated the role of the mitogen protein-activated kinase (MAPK) pathway in these responses. Cells subjected to a mild stretch show an increase in activation of the ERK1/2 and JNK pathways, and an increase in lactate dehydrogenase (LDH), but no change in the levels of inflammatory mediators. An early increase in LDH was dependent on ERK activation. Exposure to IL-1ß, or to stretch alone, did not increase MMP-9. In contrast, the combination of mild stretch followed by IL-1ß resulted in greater activation of the ERK pathway compared to either stimulus alone, and also resulted in an increase in the production of MMP-9 by astrocytes. Inhibition of the ERK pathway suppressed the increase in MMP-9 induced by the combination of stretch and IL-1ß treatment. These results suggest that a primary mild mechanical injury renders astrocytes more susceptible to a secondary exposure to a proinflammatory cytokine such as IL-1ß via the activation of the ERK pathway, and suggest a mechanism by which a mild head injury may confer increased susceptibility to neurologic injury caused by a subsequent insult.

Albumin causes increased myosin light chain kinase expression in astrocytes via p38 mitogen-activated protein kinase.

Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton, leading to disruption of vascular barrier integrity in multiple organs, including the blood-brain barrier (BBB), after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia, and has been linked to TGF-ß receptor signaling. We investigated the role of albumin in the increase of MLCK in astrocytes and the signaling pathways involved in this increase. After midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MLCK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-ß receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK, or rho kinase, also prevented this increase. These results are further evidence of a role of MLCK in the mechanisms of BBB compromise following TBI and identify astrocytes as a cell type, in addition to endothelium in the BBB, that expresses MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB.

Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. Mutations in copper/zinc superoxide dismutase 1 (SOD1) have been implicated in the pathophysiology of this disease. Using a high-throughput screening assay expressing mutant G93A SOD1, two bioactive chemical hit compounds (1 and 2), identified as arylsulfanyl pyrazolones, were identified. The structural optimization of this scaffold led to the generation of a more potent analogue (19) with an EC(50) of 170nM. To determine the suitability of this class of compounds for further optimization, 1 was subjected to a battery of pharmacokinetic assays; most of the properties of 1 were good for a screening hit, except it had a relatively rapid clearance and short microsomal half-life stability. Compound 2 was found to be blood-brain barrier penetrating with a brain/plasma ratio=0.19. The optimization of this class of compounds could produce novel therapeutic candidates for ALS patients.

Albumin activates the canonical TGF receptor-smad signaling pathway but this is not required for activation of astrocytes.

The use of albumin as a resuscitation fluid is considered safe for most critically ill patients. However, clinical data suggest albumin may increase mortality in neurotrauma, but improve outcome after stroke. Albumin has been shown to activate glia, and to play a role in the mechanisms of epileptogenesis via the TGFß-receptor (TGFßR). We have previously shown that albumin induces the production of inflammatory mediators including IL-1ß via activation of MAPK pathways in primary astrocytes and microglia. The extracellular signaling mechanisms leading to the activation of glial cells in response to albumin are not well understood. Here, we investigated the role of the TGFßR and the canonical TGFß receptor-smad signaling pathway in astrocyte activation by albumin. In primary astrocyte cultures, albumin activated the smad pathway downstream of the TGFßR by increasing the phosphorylation of smad2, and in the level of smad3 and smad4 translocated to the nucleus. Albumin produced an increase in IL-1ß which was not dependent on smad activation, but was prevented by blockade of the TGFßR. Increase in the chemokine CX3CL1, and the decrease in S100B produced by albumin were independent of the TGFßR and smad activation. Albumin induced an increase in LDH release that was inhibited by blockade of the TGFßR and by inhibition of smad activation. These findings show that albumin activates the canonical TGF receptor-smad signaling pathway. The albumin-induced increase in the pro-epileptogenic cytokine IL-1ß involves the TGFßR, but is independent of smad activation. Taken together, the effects of albumin on both IL-1ß and activation of the TGFßR pathway are further evidence for a role for albumin in neurotrauma-related epileptogenesis.

Albumin activates astrocytes and microglia through mitogen-activated protein kinase pathways.

Following acute brain injury, albumin may gain access to the brain parenchyma. Clinical studies indicate a protective role for albumin in stroke but an increase in mortality associated with albumin administration following traumatic brain injury. We investigated the effects of albumin on astrocyte and microglial activation, and the role of mitogen-activated protein kinases (MAPK) in these responses. Albumin activated ERK1/2, p38 MAPK and JNK signaling pathways in astrocytes, and induced the production of interleukin (IL)-1beta, inducible nitric oxide (NO) synthase, the NO metabolite nitrite, and the chemokine CX3CL1 while reducing the level of S100B. The release of inflammatory markers by astrocytes was partially dependent on p38 MAPK and ERK1/2 pathways, but not JNK. In microglia, albumin exposure activated all three MAPK pathways and produced an increase in IL-1beta and nitrite. Inhibition of p38 MAPK in microglia leads to an increased level of IL1beta, while inhibition of all three MAPKs suppressed the release of nitrite. These results suggest that albumin activates astrocytes and microglia, inducing inflammatory responses involved both in the mechanisms of cellular injury and repair via activation of MAPK pathways, and thereby implicate glial activation in the clinical responses to administration of albumin.

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood-brain barrier penetration.

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood-brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.

Analogues of 2-aminopyridine-based selective inhibitors of neuronal nitric oxide synthase with increased bioavailability.

Overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) has been linked to several neurodegenerative diseases. We have recently designed potent and isoform selective inhibitors of nNOS, but the lead compound contains several basic functional groups. A large number of charges and hydrogen bond donors can impede the ability of molecules to cross the blood brain barrier and thereby limit the effectiveness of potential neurological therapeutics. Replacement of secondary amines in our lead compound with neutral ether and amide groups was made to increase bioavailability and to determine if the potency and selectivity of the inhibitor would be impacted. An ether analogue has been identified that retains a similar potency and selectivity to that of the lead compound, and shows increased ability to penetrate the blood brain barrier.

A novel p38 alpha MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model.

BACKGROUND: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 alpha MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A beta) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 alpha MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes. METHODS: A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model. RESULTS: A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38 alpha MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits. CONCLUSION: The p38 alpha MAPK pathway is quantitatively important in the A beta-induced production of proinflammatory cytokines in hippocampus, and brain p38 alpha MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Glia proinflammatory cytokine upregulation as a therapeutic target for neurodegenerative diseases: function-based and target-based discovery approaches.

Inflammation is the body's defense mechanism against threats such as bacterial infection, undesirable substances, injury, or illness. The process is complex and involves a variety of specialized cells that mobilize to neutralize and dispose of the injurious material so that the body can heal. In the brain, a similar inflammation process occurs when glia, especially astrocytes and microglia, undergo activation in response to stimuli such as injury, illness, or infection. Like peripheral immune cells, glia in the central nervous system also increase production of inflammatory cytokines and neutralize the threat to the brain. This brain inflammation, or neuroinflammation, is generally beneficial and allows the brain to respond to changes in its environment and dispose of damaged tissue or undesirable substances. Unfortunately, this beneficial process sometimes gets out of balance and the neuroinflammatory process persists, even when the inflammation-provoking stimulus is eliminated. Uncontrolled chronic neuroinflammation is now known to play a key role in the progression of damage in a number of neurodegenerative diseases. Thus, overproduction of proinflammatory cytokines offers a pathophysiology progression mechanism that can be targeted in new therapeutic development for multiple neurodegenerative diseases. We summarize in this chapter the evidence supporting proinflammatory cytokine upregulation as a therapeutic target for neurodegenerative disorders, with a focus on Alzheimer's disease. In addition, we discuss the drug discovery process and two approaches, function-driven and target-based, that show promise for development of neuroinflammation-targeted, disease-modifying therapeutics for multiple neurodegenerative disorders.

Protection against endotoxic shock as a consequence of reduced nitrosative stress in MLCK210-null mice.

This study investigated the consequences of deletion of the long isoform of myosin light chain kinase (MLCK210) on the cardiovascular changes induced by the bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation puncture using MLCK210-/- mice. Here, we provide evidence that deletion of MLCK210 enhanced survival after intraperitoneal injection of LPS or cecal ligation puncture. LPS-induced vascular hyporeactivity to vasoconstrictor agents was completely prevented in aorta from MLCK210-/- mice. This was associated with a decreased up-regulation of nuclear facor-kappaB expression and activity, inducible nitric-oxide synthase, and level of oxidative stress in the vascular media. Furthermore, LPS-induced increase of nitric oxide production in the circulation and tissues (including heart, liver, and lung) that was correlated with an increased expression of inducible nitric-oxide synthase was also reduced in MLCK210-/- mice. These data demonstrate a role for MLCK210 in endotoxin shock injury associated with oxidative and nitrosative stresses and vascular hyporeactivity.

Development of a novel therapeutic suppressor of brain proinflammatory cytokine up-regulation that attenuates synaptic dysfunction and behavioral deficits.

We report the development of a novel, aqueous-soluble, safe, small molecule, experimental therapeutic that suppresses injury-induced, proinflammatory cytokine increases in the brain, with resultant attenuation of synaptic protein biomarker loss and improvement in hippocampus-dependent behavioral deficits. A GMP production scheme for the active pharmaceutical ingredient, compound 17, is presented. The development and large-scale availability of this novel compound allow exploration of new, potentially disease-modifying, therapeutic approaches to CNS disorders.