Repeated Traumatic Brain Injury is Associated with Neurotoxic Plasma Autoantibodies Directed against the Serotonin 2A and Alpha 1 Adrenergic Receptors.

Objectives: Traumatic brain injury (TBI) was associated with increased plasma agonist autoantibodies targeting the serotonin 2A receptor. Repeated TBI exposure is associated with high risk for neurodegenerative and neuropsychiatric complications. Here we tested a hypothesis that repeated TBI is associated with plasma agonist autoantibodies targeting more than one kind of catecholamine G-protein coupled receptor. Methods: Protein-A affinity chromatography was used to isolate the IgG fraction of plasma in forty-two middle-aged and older adults who had experienced one or more TBI exposures. The Ig (1/40th dilution=7.5 ug/mL) were tested for neurotoxicity in mouse neuroblastoma cells using an acute neurite retraction assay indicative of Gq11/IP3/Ca2+ and RhoA/Rho kinase signaling pathways' activation. Three different linear synthetic peptides corresponding to the second extracellular loop of the alpha 1A, alpha 2A or serotonin 2A receptors were used as target antigen in different enzyme-linked immunoassays. The second extracellular loop receptor peptides themselves (alpha 1A, alpha 2A) or a fragment (serotonin 2A) were tested for ability to prevent Ig-induced neurite retraction. Results: Patients who had experienced either repeated TBI (N=10) or a single TBI with a co-morbid autoimmune disease (N=5) were significantly more likely to harbor neurotoxic plasma autoantibodies targeting both alpha 1 adrenergic and serotonin 2A receptors vs. patients having only a single TBI. Ig-induced neurotoxicity was significantly prevented by co-incubation with either 850 nM prazosin (alpha 1 adrenergic receptor) and/or 500 nM M100907 (serotonin 2A receptor) antagonists. Alpha 1 adrenergic receptor and serotonin 2A receptor Ig immunoreactive level and titer were significantly increased in repeated TBI and single TBI/autoimmune patients (N=7-8) compared to age-matched TBI patients without neurotoxic plasma Ig (N=4). SN.8, a linear synthetic peptide corresponding to a conserved region of the second extracellular loop (ECL) of the serotonin 2A receptor completely prevented neurite retraction induced by repeated TBI plasma Ig. A repeated TBI patient harboring alpha adrenergic receptor AAB alone experienced prospective steep decline in cognitive function over two years. Conclusions: Repeated TBI and TBI with associated autoimmunity harbored more than one kind of neurotoxic catecholaminergic agonist GPCR autoantibody each associated with high risk for steep rate of cognitive decline. Specific immunoassays using the second extracellular receptor loop as target antigen are needed to detect each specific different GPCR autoantibody. A fragment of the second ECL of the serotonin 2A receptor (SN.8) neutralized Ig-induced neurotoxicity in repeated TBI or TBI with associated systemic autoimmunity.

Neuroprotective Effects of a Serotonin Receptor Peptide Following Sham vs. Mild Traumatic Brain Injury in the Zucker Rat.

Aims: Accelerated cognitive decline frequently complicates traumatic brain injury. Obesity and type 2 diabetes mellitus drive peripheral inflammation which may accelerate traumatic brain injury-associated neurodegeneration. The Zucker rat harbors G-protein coupled receptor agonist IgG autoantibodies and in vitro neurotoxicity caused by these autoantibodies was prevented by a novel synthetic fragment of the serotonin 2A receptor. The aim of the present study was to test whether genetic obesity manifested in Zucker diabetic fatty rat is associated with greater spatial memory impairment before and after mild traumatic brain injury compared to Zucker lean rats. Furthermore, we investigated whether these neurodegenerative complications can be lessened by administration of a novel putative neuroprotective peptide comprised of a fragment of the second extracellular loop of the serotonin 2A receptor. Methods: Age-matched lean and fatty diabetic Zucker rats were tested in the Morris water maze (spatial memory) prior to receiving a sham-injury or lateral fluid percussion (LFP) mild traumatic brain injury. Behavioral testing was repeated at 1-week, 1-month, and 3-month intervals following injury. A synthetic peptide consisting of a portion of the 5-hydroxytryptamine (serotonin) 2A receptor (2 mg/kg) (vehicle, or an inactive scrambled version of the peptide (2 mg/kg)) was administered via intraperitoneal route every other day for 7 days after sham or LFP injury to lean rats or 7 days before and after sham or LFP injury to fatty rats. Results: Mild traumatic brain injury impaired recall of spatial memory in fatty and lean rats. Zucker fatty rats subjected to sham-injury or mild TBI experienced a significantly greater longitudinal decline in recall of spatial memory compared to lean Zucker rats. A synthetic peptide fragment of the 5-hydroxytryptamine 2A receptor significantly enhanced acquisition of spatial learning and it appeared to strengthen recall of spatial learning (one-week) after sham injury in Zucker rats. Conclusions: These data suggest that the Zucker diabetic fatty rat is a suitable animal model to investigate the role of metabolic factor(s) in accelerated cognitive decline. A novel synthetic peptide comprised of a fragment of the second extracellular loop of the human serotonin 2A receptor appeared to have neuroprotective effects on both acquisition and recall of spatial memory in subsets of Zucker rats, with relatively greater benefit in sham-injured, lean Zucker rats.

Plasma Serotonin 2A Receptor Autoantibodies Predict Rapid, Substantial Decline in Neurocognitive Performance in Older Adult Veterans with TBI.

Aim: Traumatic brain injury (TBI) was associated with increased plasma serotonin 2A receptor (5-HT2AR) autoantibodies in adults who experienced neurodegenerative complications. We tested whether the baseline presence of plasma serotonin 2A receptor (5-HT2AR) autoantibodies was a significant predictor of the two-year rate of cognitive decline in middle-aged and older adult TBI. Methods: Plasma from thirty-five middle-aged and older adult veterans (mean 65 years old) who had suffered traumatic brain injury was subjected to protein-A affinity chromatography. One-fortieth dilution of the resulting immunoglobulin (Ig) G fraction was tested for binding (in ELISA) to a linear synthetic peptide corresponding to the second extracellular loop region of the human 5-HT2A receptor. All available patients completed baseline and two-year follow-up neurocognitive tests of memory (St Louis University Mental Status), processing speed (Digit Symbol Substitution Test) and executive function (Trails-making Test, Part B). Change in cognitive performance was computed as (two-year - baseline) raw test score. Results: Eighteen patients completed both baseline and two-year follow up neurocognitive tests. TBI patients harboring plasma 5-HT2AR autoantibodies at the baseline examination (n=13) did not differ significantly in their baseline clinical characteristics (age, education level) compared to TBI patients lacking baseline plasma autoantibodies (n=5). Plasma serotonin 2AR antibody-positive patients experienced a significantly greater post-baseline decline in performance on the St Louis University Mental Status test (P=0.0118) and in the Digit Symbol Substitution Test (P=0.011), but not in Trails-making Part B (P=0.129) compared to serotonin 2AR antibody-negative patients. In multivariable linear regression analyses that adjusted for age, baseline presence of plasma 5-HT2AR autoantibody was a significant predictor of the two-year rate of decline in memory, and processing speed. Binding of plasma autoantibody to the serotonin 2A receptor peptide in the enzyme linked immunosorbent assay was also significantly higher (at 1/160th titer of the protein-A eluate= 1 µg/mL IgG) in TBI patients harboring vs. those not harboring baseline plasma 5-HT2AR autoantibodies. Conclusion: These data suggest that plasma 5-hydroxytryptamine 2A receptor autoantibodies which were increased in approximately two-thirds of middle-aged and older adults following traumatic brain injury predicts rapid and substantial declines in cognitive function (memory and processing speed), independent of age.

Myristolated Serotonin 2A Receptor Peptide Promotes Long-Lasting Blood Pressure-Lowering and Reno protection in Hypertensive Rat Species.

Aim: The aim of the present study was to test whether conjugation of a synthetic peptide corresponding to a fragment of the second extracellular domain of the human serotonin 2A receptor substantially alters the in vivo pharmacodynamic blood pressure-lowering profile of the peptide in different hypertensive rat strains. Methods: Sertuercept (SCLLADDN) was synthesized and modified using pegylation or myristolation. The two different peptide conjugates were tested in male Zucker diabetic fatty rats for acute and long-lasting blood pressure-lowering effects following single intraperitoneal administration. The myristolated Sertuercept was administered intraperitoneally to female Zucker fatty and male spontaneously hypertensive rats (SHR) and blood pressure was monitored either using tail cuff measurement (female Zucker) or by telemetry (SHR) rats. Plasma immunoglobulin G obtained by Protein G affinity chromatography in 25-week-old female Zucker or male spontaneously hypertensive rats was tested for binding to a linear synthetic peptide corresponding to the second extracellular loop of the serotonin 2A receptor. A cohort of male Zucker diabetic fatty rats was randomized to seven weeks of once-weekly myristolated Sertuercept or scrambled peptide (injections) and the kidneys were examined histologically for differences in total kidney lesions or fibrosis. Results: Pegylated Sertuercept promoted substantial blood pressure-lowering lasting approximately 30-48 hours in male Zucker diabetic fatty rats. Blood pressure-lowering following a single injection of Myristolated Sertuercept was much longer-lasting (6-11 days) and it was effective in male Zucker diabetic fatty rats, male spontaneously hypertensive rats and in a subset of hypertensive female Zucker fatty rats. Seven weeks' treatment with once-weekly Myristolated Sertuercept (2mg/kg) was associated with significantly fewer kidney lesions and less interstitial fibrosis compared to scrambled peptide in 25-week-old male Zucker diabetic fatty rats. Male spontaneously hypertensive rats (4 of 4 tested) harbored plasma IgG which bound significantly to serotonin 2A receptor peptide, and a subset of female Zucker fatty rats harboring IgG were responsive to blood pressure-lowering from the myristolated Sertuercept peptide. Summary: Myristolated-Sertuercept, an epitope-specific peptide comprised of a portion of second extracellular loop of the human serotonin 2A receptor was safe, well-tolerated and effectively lowered blood pressure for one week or longer in two different strains of male hypertensive rats. These data provide proof-of-concept that once-weekly systemic drug administration is feasible to achieve not only long-lasting hypertension control, but also substantial renoprotection.

Gene Expression Changes in a Model Neuron Cell Line Exposed to Autoantibodies from Patients with Traumatic Brain Injury and/or Type 2 Diabetes.

Traumatic brain injury and adult type 2 diabetes mellitus are each associated with the late occurrence of accelerated cognitive decline and Parkinson's disease through unknown mechanisms. Previously, we reported increased circulating agonist autoantibodies targeting the 5-hydroxytryptamine 2A receptor in plasma from subsets of Parkinson's disease, dementia, and diabetic patients suffering with microvascular complications. Here, we use a model neuron, mouse neuroblastoma (N2A) cell line, to test messenger RNA expression changes following brief exposure to traumatic brain injury and/or type 2 diabetes mellitus plasma harboring agonist 5-hydroxytryptamine 2A receptor autoantibodies. We now report involvement of the mitochondrial dysfunction pathway and Parkinson's disease pathways in autoantibody-induced gene expression changes occurring in neuroblastoma cells. Functional gene categories upregulated significantly included cell death, cytoskeleton-microtubule function, actin polymerization or depolymerization, regulation of cell oxidative stress, mitochondrial function, immune function, protein metabolism, and vesicle function. Gene categories significantly downregulated included microtubule function, cell adhesion, neurotransmitter release, dopamine metabolism synaptic plasticity, maintenance of neuronal differentiation, mitochondrial function, and cell signaling. Taken together, these results suggest that agonist 5-hydroxytryptamine receptor autoantibodies (which increase in Parkinson's disease and other forms of neurodegeneration) mediate a coordinating program of gene expression changes in a model neuron which predispose to neuro-apoptosis and are linked to human neurodegenerative diseases pathways.

Severe COVID-19 Pneumonia is Associated with Increased Plasma Immunoglobulin G Agonist Autoantibodies Targeting the 5-Hydroxytryptamine 2A Receptor.

AIMS: To test whether plasma autoantibodies targeting the 5-hydroxytryptamine 2A receptor increase in COVID-19 infection; and to characterize the pharmacologic specificity, and signaling pathway activation occurring downstream of receptor binding in mouse neuroblastoma N2A cells and cell toxicity of the autoantibodies. METHODS: Plasma obtained from nineteen, older COVID-19 patients having mild or severe infection was subjected to protein-A affinity chromatography to obtain immunoglobulin G fraction. One-fortieth dilution of the protein-A eluate was tested for binding to a linear synthetic peptide QN.18 corresponding to the second extracellular loop of the human 5-hydroxytryptamine 2A receptor. Mouse neuroblastoma N2A cells were incubated with COVID-19 IgG autoantibodies in the presence or absence of selective inhibitors of G-protein coupled receptors, signaling pathway antagonists, or a novel decoy receptor peptide. RESULTS: 5-hydroxytryptamine 2A receptor autoantibody binding occurred in 17 of 19 (89%) patients with acute COVID-19 infection and increased level was significantly correlated with increased severity of COVID-19 infection. The agonist autoantibodies mediated acute neurite retraction in mouse neuroblastoma cells by a mechanism involving Gq11/PLC/IP3R/Ca2+ activation and RhoA/Rho kinase pathway signaling occurring downstream of receptor binding which had pharmacologic specificity consistent with binding to the 5-HT2A receptor. A novel synthetic peptide 5-HT2AR fragment, SN..8, dose-dependently blocked autoantibody-induced neurotoxicity. The COVID-19 autoantibodies displayed acute toxicity in bovine pulmonary artery endothelial cells (stress fiber formation, contraction) and modulated proliferation in a manner consistent with known 'biased agonism' on the 5-HT2A receptor. CONCLUSION: These data suggest that 5-HT2AR targeting autoantibodies are highly prevalent may contribute to pathophysiology in acute, severe COVID-19 infection.

A Serotonin 2A-Receptor Decoy Peptide Potently Lowers Blood Pressure in Male Zucker Diabetic, Fatty, Hypertensive Rats.

AIMS: To test whether a novel 5-hydroxytryptamine 2A decoy receptor peptide, SN..8 (Sertuercept), administered via intraperitoneal injection, acutely lowers arterial blood pressure in obese, hypertensive male Zucker diabetic rats (ZDF). To examine the safety, tolerability and possible reno-protective effects following chronic alternate daily administration of Sertuercept (for 10 weeks) in the male ZDF rat. METHODS: Systolic and diastolic blood pressure were determined at baseline and regular intervals for up to 48 hours after a single IP administration of either Sertuercept (2 mg/kg), vehicle (saline) or an identical concentration of a scrambled sequence of the decoy receptor peptide, LN…8, in male ZDF and Zucker lean rats using tail cuff plethysmography. Plasma autoantibodies were obtained in thirteen male ZDF rats for determination of 5-hydroxytryptamine 2A receptor-mediated neurotoxicity using an acute neurite retraction assay in mouse neuroblastoma cells. Rats were sacrificed at 25-weeks of age, the kidneys were perfused, fixed and sections were stained using Masson's trichrome for semi-quantitative determination of glomerular and interstitial fibrosis. RESULTS: Sertuercept (2 mg/kg IP) potently lowered systolic and diastolic blood pressure in both 11-week-old and 25-week-old male ZDF rats and in a subset of hypertensive Zucker lean rats. There was no significant blood pressure-lowering effect of vehicle (saline) or scrambled peptide sequence (LN.8). Blood pressure-lowering was rapid in onset (15-30 minutes following IP injection) and sustained for at least 24 hours. Alternate daily IP administration of 2 mg/kg dose of Sertuercept vs. scrambled peptide (for 10 weeks) was safe, well-tolerated and associated with a significant decrease in glomerulosclerosis in 25-week-old male ZDF rats. Plasma autoantibody-induced neurotoxicity correlated significantly with the global index of renal fibrosis severity in 25-week-old male ZDF rats. CONCLUSIONS: These data indicate potent arterial blood pressure-lowering efficacy from a decoy receptor peptide comprised of a second extracellular loop region of the human 5-hydroxytryptamine receptor. Chronic administration of the decoy receptor peptide (10 weeks) was safe, well-tolerated and protected against renal glomerulosclerosis in the male ZDF rat.

Inverse Association between Serotonin 2A Receptor Antagonist Medication Use and Mortality in Severe COVID-19 Infection.

Advanced age and medical co-morbidity are strong predictors of mortality in COVID-19 infection. Yet few studies (to date) have specifically addressed risk factors associated with COVID-19 mortality in a high-risk subgroup of older US adults having one or more chronic diseases. Our hypothesis is that medications having 'off-target' anti-inflammatory effects may play a role in modulating the immune response in COVID-19 infection. We analyzed baseline risk factors associated with respiratory failure or death in 55 older adult US military veterans hospitalized for COVID-19 infection during (March-June 2020) the peak of the pandemic in New Jersey. Fifty-three percent (29/55) of patients experienced respiratory failure and thirty-one percent (17/55) died. In adjusted logistic regression analysis, baseline neutrophil to lymphocyte ratio (NLR) (P=0.0035) and body mass index (P=0.03) were significant predictors of the risk for respiratory failure. Age (P=0.05) and non-use (vs. use) of psychotropic medications having serotonin 2A receptor antagonist properties (odds ratio 5.06; 95% confidence intervals 1.18-21.7; P= 0.029) was each a significant predictor of an increased risk of death. There was a significant interaction effect of age and non-use (vs.. use) of psychotropic serotonin 2A receptor antagonist medications on the odds ratio (OR) for death (P=0.011). In selected, ventilator-dependent COVID-19 pneumonia patients treated with psychotropic serotonin 2A receptor antagonist medications to control agitation and ICU delirium, there was an apparent positive association between medication use and significant rise in the absolute lymphocyte count and decrease in the neutrophil: lymphocyte ratio. Taken together, these data are the first to suggest that certain psychotropic medications used in the treatment of chronic psychiatric illness and/or for acute delirium are inversely associated with mortality in severe COVID-19 infection by unknown mechanism which may involve (in part) immunomodulatory effects.

Serotonin 2A Receptor Autoantibodies Increase in Adult Traumatic Brain Injury In Association with Neurodegeneration.

OBJECTIVE: Traumatic brain injury (TBI) is associated with an increased risk of late neurodegenerative complications via unknown mechanisms. Circulating neurotoxic 5-hydroxytryptamine 2A receptor (5-HT2AR) autoantibodies were reported to increase in subsets of obese type 2 diabetes having microvascular complications. We tested whether 5-HT2AR autoantibodies increase in adults following traumatic brain injury in association with neurodegenerative complications. METHODS: Plasma from thirty-five middle-aged and older adult veterans (mean 65 years old) who had suffered traumatic brain injury was subjected to protein-A affinity chromatography. The resulting immunoglobulin (Ig) G fraction was tested for neurotoxicity (acute neurite retraction, and accelerated cell death) in mouse N2A neuroblastoma cells or for binding to a linear synthetic peptide corresponding to the second extracellular loop region of the human 5-HT2A receptor. RESULTS: Nearly two-thirds of traumatic brain injured-patients harbored 5-HT2AR autoantibodies in their circulation. Active TBI autoantibodies caused neurite retraction in mouse N2A neuroblastoma cells and accelerated N2A cell loss which was substantially prevented by co-incubation with a two hundred and fifty nanomolar concentration of M100907, a highly selective 5-HT2AR antagonist. Antagonists of RhoA/Rho kinase and Gq11/phospholipase C/inositol triphosphate receptor signaling pathways blocked TBI autoantibody-induced neurite retraction. Following traumatic brain injury, autoantibody binding to a 5-HT2A receptor peptide was significantly increased in patients having co-morbid Parkinson's disease (n=3), dementia (n=5), and painful neuropathy (n=8) compared to TBI subsets without neurologic or microvascular complication (n=20). Autoantibody titer was significantly elevated in TBI subsets experiencing multiple neurotraumatic exposures vs. single TBI. Plasma white blood cell, a marker of systemic inflammation, correlated significantly (correlation coefficient r =0.52; P < 0.01) with, 5-HT2A receptor peptide binding of the TBI-autoantibody. CONCLUSION: These data suggest that circulating neurotoxic 5-hydroxytryptamine 2A receptor agonist autoantibodies increase in adults following traumatic brain injury in association with late neurodegenerative complications.

Type 2 Diabetes Predicts Increased Risk of Neurodegenerative Complications in Veterans Suffering Traumatic Brain Injury.

AIMS: Obese type 2 diabetes and traumatic brain injury are associated with persistent peripheral and neuro-inflammation, respectively. We tested whether adult type 2 diabetes increased the hazard rate for neurodegeneration complications following traumatic brain injury. METHODS: Retrospective chart review of patients treated at the Veterans Affairs New Jersey Healthcare System between 2016-2019 and having a diagnosis of prior traumatic brain injury was performed in adult veterans, age 50 years or older. Cox proportional hazards regression analysis was used to identify risk factors predictive of an increased risk of neurodegeneration, i.e. worsening major depression, dementia or Parkinson's disease following traumatic brain injury. RESULTS: Type 2 diabetes predicted a nearly three-fold increased hazard ratio (HR = 2.95, 95% CI 1.15-7.56, P =0.02) for the occurrence of worsening major depression, dementia or Parkinson's disease in eighty adults age 50 years or older who had experienced prior traumatic brain injury. After adjusting for other covariates, hypertension (HR= 4.15, 95% CI 1.21-14.29, P =0.02) was significant and body mass index (HR=1.14, 95% CI 0.99-1.30; P=0.06) modestly significant predictors of the risk for the time to first occurrence of the composite neurodegenerative outcome. CONCLUSION: Type 2 diabetes, hypertension and higher body mass index increase the hazard for the occurrence of worsening depression, Parkinson's disease and dementia following traumatic brain injury in middle-aged and older adults.

Schizophrenia Plasma Autoantibodies Promote 'Biased Agonism' at the 5-Hydroxytryptamine 2A Receptor: Neurotoxicity is Positively Modulated by Metabotropic Glutamate 2/3 Receptor Agonism.

AIMS: To test whether neurite-inhibitory plasma autoantibodies in chronic schizophrenia activate Gq/11- and Gi- coupled signaling pathways downstream of 5-hydroxytryptamine 2A receptor activation; and for modulation of serotonergic signaling by the metabotropic 2/3 receptor agonist LY379268. METHODS: Plasma from five older adults with chronic schizophrenia and eight age-matched patients having another neuropsychiatric, immune or metabolic disorder was subjected to Protein-A affinity chromatography to obtain IgG autoantibodies. Mean neurite retraction (5 minutes) or cell survival (24 hours) was determined in mouse N2A neuroblastoma cells incubated with autoantibodies in the presence or absence of specific antagonists of the Gq/11/PLC/IP3R signaling pathway, Gi-coupled, beta-arrestin2-directed pathways, or LY379268. RESULTS: Chronic schizophrenia plasma autoantibodies- mediated dose- and time-dependent acute N2A neurite retraction was completely prevented by M100907, a selective 5-hydroxytryptamine 2A receptor antagonist. LY379268 promoted autoantibody-induced neurite retraction causing a shift-to-the-left in the dose-response curve. Antagonists of the RhoA/Rho kinase and Gq/11/PLC/IP3R signaling pathways blocked autoantibody-mediated neurite retraction. Chronic schizophrenia plasma autoantibodies mediated increased N2A cell survival which was blocked by LY379268, pertussis toxin, and antagonists of PI3-kinase- mediated survival signaling. CONCLUSION: Schizophrenia plasma autoantibodies activate the 5-hydroxytryptamine 2A receptor positively coupled to Gq/11/PLC/IP3R pathway and RhoA/Rho kinase signaling activation in promoting acute N2A cell neurite retraction. Autoantibodies in a subset of patients experiencing hallucinations promoted increased N2A cell survival mediated (in part) via a pertussis-toxin sensitive, Gi-coupled, PI3-kinase-dependent mechanism. Positive modulation of 5-HT2AR-mediated neurite retraction by LY379268 suggests the autoantibodies may target (in part) the 5-HT2AR/mGlu2R heteromer.

Endothelial Cell Growth Promoting Activity in Graves' Disease Sera is Neutralized by Anti-Basic Fibroblast Growth Factor Antibodies in Patients with Fat Expansive but Not Infiltrative Orbitopathy.

PURPOSE: To report a case of orbital fat expansion leading to globe prolapse in a Graves' disease patient undergoing high-dose glucocorticoid therapy. To evaluate the growth factor receptor specificities of plasma autoantibodies in Graves' disease patients who exhibited contrasting subtypes of thyroid-associated ophthalmopathy, i.e. orbital fat expansion-type vs. infiltrative. METHODS: Sera from Graves' orbitopathy and control patients with or without Graves' disease were subjected to protein-A affinity chromatography to obtain immunoglobulin G. A (1/50th to 1/1600th) range in dilutions of the protein-A eluate fraction was incubated for four days at 37 degrees C with bovine pulmonary artery endothelial cells to test for endothelial cell inhibition or stimulation. Growth stimulatory autoantibodies were co-incubated with specific neutralizing anti-insulin like growth factor 1 receptor antibodies or anti-basic fibroblast growth factor antibodies to assess autoantibody specificity in contrasting Graves' orbitopathy subtypes. RESULTS: We observed increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from eighteen patients with active Graves' disease (117 ± 28%, n = 18) compared to mean endothelial cell activity (89 ± 10%, n = 13, P = 0.003) in thirteen adults without Graves' disease. The protein-A eluate fraction in acute infiltrative-type Graves' orbitopathy contained a high titer (> 1:1000) of endothelial cell stimulatory activity which was significantly neutralized by specific monoclonal anti-human insulin-like growth factor 1 receptor antibodies. The protein-A eluate fraction in fat expansion-type Graves' orbitopathy contained endothelial cell inhibitory activity (at low titers) and stimulatory activity (at high titers), and the latter stimulatory activity was completely neutralized by specific anti-basic fibroblast growth factor antibodies. CONCLUSION: Graves' disease suffering globe prolapse secondary to marked orbital fat-expansion had coexisting plasma fibroblast growth factor-inhibitory and -stimulatory autoantibodies. The latter was completely neutralized by anti-basic fibroblast growth factor antibodies.

Thyroid Hormone Resistance in Identical Twin Sisters with Atrial Fibrillation: Case Report and Review of the Literature.

AIM: To report identical twin sisters harboring the A317T mutation in the thyroid hormone beta receptor gene (TR ß) who developed atrial fibrillation and refractory congestive heart failure in the sixth decade of life. To critically assess whether the A317T mutation may be responsible for increased cardiotoxicity compared to other thyroid hormone beta receptor gene mutations. METHODS: A 59-year-old woman referred for evaluation of abnormal thyroid function tests had been experiencing frequent spells of tachycardia associated with dyspnea, and dizziness necessitating multiple hospitalizations. Elevation in free thyroxine (T4), total triiodothyronine (T3) and inappropriately normal thyroid stimulating hormone (TSH) was consistent with a clinical diagnosis of thyroid hormone resistance. Magnetic resonance imaging of the brain was negative for a TSH-secreting pituitary adenoma. A blood sample was sent for thyroid hormone receptor gene mutational analysis, but it would require eight weeks to complete processing. RESULTS: A modified L-T3 suppression test was used to assess thyroid-pituitary axis feedback. After three weeks' of cytomel (L-T3) (25 micrograms daily) TSH decreased by 50%, and free T4 level decreased by 22% compared to baseline levels. Genetic testing revealed a heterozygous A317T mutation in the thyroid hormone beta receptor gene. Serial two-dimensional echocardiography demonstrated evolution to left atrial enlargement over a three-year period. Prior published literature suggests a less than 10% prevalence of atrial fibrillation in adults with thyroid hormone resistance harboring various TR-ß gene mutations. Yet all five of five (100%) adults having the A317T mutation were reported to experience atrial fibrillation by age 50. CONCLUSIONS: A new kindred with resistance to thyroid hormone harboring the A317T disease-causative mutation is described in which identical twin sisters had a mid-life onset of atrial fibrillation and refractory congestive heart failure.

Circulating Neurotoxic 5-HT2A Receptor Agonist Autoantibodies in Adult Type 2 Diabetes with Parkinson's Disease.

AIMS: To test whether circulating neurotoxic autoantibodies increase in adult type 2 diabetes mellitus with Parkinson's disease (PD) or dementia. To identify the G-protein coupled receptor on neuroblastoma cells mediating neural inhibitory effects in diabetic Parkinson's disease plasma autoantibodies. To determine the mechanism of accelerated neuroblastoma cell death and acute neurite retraction induced by diabetic Parkinson's disease and dementia autoantibodies. METHODS: Protein-A eluates from plasma of twelve older adult male diabetic patients having Parkinson's disease (n=10) or dementia (n=2), and eight age-matched control diabetic patients were tested for ability to cause accelerated N2A neuroblastoma cell death and acute neurite retraction. Specific antagonists of G protein coupled receptors belonging to the G alpha q subfamily of heterotrimetric G-proteins, the phospholipase C/inositol triphosphate/Ca2+ pathway, or the RhoA/Rho kinase pathway were tested for ability to block diabetic Parkinson's disease/dementia autoantibody-induced neurite retraction or N2A accelerated cell loss. Sequential Liposorber LA-15 dextran sulfate cellulose/protein-A affinity chromatography was used to obtain highly-purified fractions of diabetic Parkinson's disease autoantibodies. RESULTS: Mean accelerated neuroblastoma cell loss induced by diabetic Parkinson's disease or dementia autoantibodies significantly exceeded (P = 0.001) the level of N2A cell loss induced by an identical concentration of the diabetic autoantibodies in control patients without these two co-morbid neurodegenerative disorders. Co-incubation of diabetic Parkinson's disease and dementia autoantibodies with two-hundred nanomolar concentrations of M100907, a highly selective 5-HT2AR antagonist, completely prevented autoantibody-induced accelerated N2A cell loss and neurite retraction. A higher concentration (500 nM-10µM) of alpha-1 adrenergic, angiotensin II type 1, or endothelin A receptor antagonists did not substantially inhibit autoantibody-induced neuroblastoma cell death or prevent neurite retraction. Antagonists of the inositol triphosphate receptor (2-APB, 50µM), the intracellular calcium chelator (BAPTA-AM, 30 µM) and Y27632 (10 µM), a selective RhoA/Rho kinase inhibitor, each completely blocked acute neurite retraction induced by sixty nanomolar concentrations of diabetic Parkinson's disease autoantibodies. Co-incubation with 2-APB (1-2 µM) for 8 hours' prevented autoantibody-induced N2A cell loss. The highly-purified fraction obtained after Liposorber LA/protein-A affinity chromatography in hypertriglyceridemic diabetic dementia and Parkinson's disease plasmas had apparent MWs > 30 kD, and displayed enhanced N2A toxicity requiring substantially higher concentrations of 5-HT2AR antagonists (M100907, ketanserin, spiperone) to effectively neutralize. CONCLUSION: These data suggest increased autoantibodies in older adult diabetes with Parkinson's disease or dementia cause accelerated neuron loss via the 5-hydroxytryptamine 2 receptor coupled to inositol triphosphate receptor-mediated cytosolic Ca2+ release.

Increased Neuronal Depolarization Evoked by Autoantibodies in Diabetic Obstructive Sleep Apnea: Role for Inflammatory Protease(s) in Generation of Neurotoxic Immunoglobulin Fragment.

11 AIM: Obstructive sleep apnea increases in diabetes and morbid obesity. We tested a hypothesis that circulating autoantibodies in adult type 2 diabetes which increase in association with morbid obesity are capable of causing long-lasting neuronal depolarization and altered calcium release in mouse atrial cardiomyocytes. 12 METHODS: Protein-A eluates from plasma of 14 diabetic obstructive sleep apnea patients and 17 age-matched diabetic patients without sleep apnea were tested for effects on depolarization and neurite out growth in N2a mouse neuroblastoma cells. The mechanism of autoantibody-mediated neurite outgrowth inhibition was investigated in co-incubation experiments of diabetic obstructive sleep apnea autoantibodies with specific antagonists of G-protein coupled receptors or the RhoA/Rho kinase signaling pathway. Following long-term storage of the protein-A eluates (to allow spontaneous proteolysis and IgG subunit dissociation), plasma autoantibodies from diabetic obstructive sleep apnea, cancer or control patients were compared for enhancement of inhibitory effects on endothelial cell survival. Size exclusion chromatography performed (in the presence or absence of a specific membrane type 1-matrix metalloproteinase inhibitor) was used to characterize the IgG autoantibody subunit(s) or fragments associated with peak neurotoxicity in diabetic obstructive sleep apnea. 13 RESULTS: Diabetic obstructive sleep apnea (n = 14) autoantibodies caused a significant increase (P = 0.01) in membrane depolarization in N2a mouse neuroblastoma cells compared to control diabetic patients (n = 15) not suffering with obstructive sleep apnea. Process extension in N2A mouse neuroblastoma cells was significantly inhibited (P = 0.01) by diabetic obstructive sleep apnea (n = 9) autoantibodies compared to effects from identical 10 µg/mL concentrations of control diabetic autoantibodies in patients without obstructive sleep apnea. Ten micromolar concentrations of SCH-202676, a G-protein coupled receptor antagonist (n = 5) or ten micromolar concentration of Y27632, a selective Rho kinase inhibitor (n = 6), each significantly prevented (P < 0.001) neurite outgrowth inhibition by diabetic obstructive sleep apnea autoantibodies. Autoantibodies in representative patients with obstructive sleep apnea and either atrial fibrillation or left ventricular hypertrophy evoked acute large increases in intracellular Ca2+ in HL-1 mouse atrial cardiomyocytes. The magnitude of intracellular Ca2+ release was dose-dependently significantly correlated to the electrocardiographic Cornell voltage-duration product. Gel filtration of diabetic obstructive sleep apnea autoantibodies revealed peak neurotoxicity associated with MWs corresponding to IgG light chain dimer(s), monomers or half-light chains as well as a novel ∼ 5.5 kD putative light chain fragment. 14 CONCLUSIONS: These results suggest that diabetic obstructive sleep apnea autoantibodies may induce strong depolarization in neuronal cells and alter Ca2+ signaling in atrial cardiomyocytes consistent with a role in pathophysiology in subsets of diabetic obstructive sleep apnea having co-morbid atrial fibrillation or another clinically significant cardiac rhythm disturbance.

Diabetes Autoantibodies Mediate Neural- and Endothelial Cell- Inhibitory Effects Via 5-Hydroxytryptamine- 2 Receptor Coupled to Phospholipase C/Inositol Triphosphate/Ca2+ Pathway.

AIMS: To identify the G-protein coupled receptor(s) on neuroblastoma and endothelial cells which mediate neural- and endothelial cell-inhibitory effects in plasma autoantibodies from a subset of older type 2 diabetes with neurologic and vascular co-morbidity. To determine the mechanism(s) of neurite retraction induced by diabetic pathologies' auto antibodies. METHODS: Protein-A eluates from plasma of 11 diabetic patients having nephropathy, moderate-severe obesity and/or complications in which increased inflammation plays a role (depression, Parkinson's disease, atrial fibrillation, obstructive sleep apnea) were tested for neurite retraction and decreased survival in N2A neuroblastoma cells, and decreased survival in pulmonary artery endothelial cells. Specific antagonists of G protein coupled receptors belonging to the G alpha q subfamily of hetero trimetric G proteins or the phospholipase C/inositol triphosphate/Ca2+ pathway were tested for modulatory effects on diabetic pathologies' autoantibody-induced N2A neurite retraction, or cell survival. RESULTS: Co-incubation with specific antagonists of the 5-hydroxytryptamine- 2A receptor significantly prevented acute N2A neurite retraction induced by 50-100 nM concentrations of diabetic pathologies' autoantibodies. Protection against neurite retraction (M100907> spiperone> ketanserin) closely paralleled the antagonists' potency order at the 5-HT2-AR. Neuroblastoma or endothelial cell death (after 24 hours incubation) with 50-100 nM autoantibodies was completely or nearly completely (91%) prevented by co-incubation with 200 nM M100907, a highly selective 5-HT2-AR antagonist. Alpha-1 adrenergic, angiotensin II, metabotropic glutamate 5, or endothelin A (100 nM-10µM) receptor antagonists did not substantially inhibit autoantibody-induced cell death. The intracellular calcium chelator (BAPTA-AM, 50 µM) and inhibitors of the inositol triphosphate (IP3) receptor (2-APB, 50µM), and phospholipase C-gamma (U73144, 1µM) each significantly protected against autoantibody-induced acute N2A neurite retraction. CONCLUSION: These data suggest that neural- and endothelial- inhibitory effects in autoantibodies from older adult diabetes with nephropathy and obesity/inflammation-associated complications are mediated by agonist autoantibodies directed against the 5-hydroxytryptamine 2 receptor positively coupled to the phospholipase C/inositol triphosphate/ cytosolic Ca2+ release pathway.

Predictors of Cognitive Decline in Older Adult Type 2 Diabetes from the Veterans Affairs Diabetes Trial.

AIMS: Cognitive decline disproportionately affects older adult type 2 diabetes. We tested whether randomized intensive (INT) glucose-lowering reduces the rate(s) of cognitive decline in adults with advanced type 2 diabetes (mean: age, 60 years; diabetes duration, 11 years) from the Veterans Affairs Diabetes Trial. METHODS: A battery of neuropsychological tests [digit span, digit symbol substitution (DSym), and Trails-making Test-Part B (TMT-B)] was administered at baseline in ~1700 participants and repeated at year 5. Thirty-seven risk factors were evaluated as predictors of cognitive decline in multivariable regression analyses. RESULTS: The mean age-adjusted DSym or TMT-B declined significantly in all study participants (P < 0.001). Randomized INT glucose-lowering did not significantly alter the rate of cognitive decline. The final model of risk factors associated with 5-year decline in age-adjusted TMT-B included as significant predictors: longer baseline diabetes duration (beta = -0.028; P = 0.0057), lower baseline diastolic blood pressure (BP; beta = 0.028; P = 0.002), and baseline calcium channel blocker medication use (beta = -0.639; P < 0.001). Higher baseline pulse pressure was significantly associated with decline in age-adjusted TMT-B suggesting a role for both higher systolic and lower diastolic BPs. Baseline thiazide diuretic use (beta = -0.549; P = 0.015) was an additional significant predictor of 5-year decline in age-adjusted digit symbol score. Post-baseline systolic BP-lowering was significantly associated (P < 0.001) with decline in TMT-B performance. There was a significant inverse association between post-baseline plasma triglyceride-lowering (P = 0.045) and decline in digit symbol substitution task performance. CONCLUSION: A 5-year period of randomized INT glucose-lowering did not significantly reduce the rate of cognitive decline in older-aged adults with type 2 diabetes. Systolic and diastolic BPs as well as plasma triglycerides appeared as modifiable risk factors of the rate of cognitive decline in older adult type 2 diabetes.

Toxic Immunoglobulin Light Chain Autoantibodies are Associated with a Cluster of Severe Complications in Older Adult Type 2 Diabetes.

AIMS: To assess neuronal depolarization evoked by autoantibodies in diabetic depression compared to depolarization evoked by autoantibodies in control patients. To determine whether a subset of severe (late-onset) diabetic complications may be mediated in part by toxic immunoglobulin light chains that may increase in diabetic nephropathy. METHODS: Protein-A eluates from plasma of 21 diabetic depression patients and 37 age-matched controls were tested for depolarization in hippocampal or immature neurons. Subsets of depolarizing or non-depolarizing autoantibodies were tested for neurite outgrowth inhibition in N2A neuroblastoma cells or the ability to modulate Ca2+ release in HL-1 atrial cardiomyocytes or in endothelial cells. The stability of depolarizing autoantibodies was investigated by heat treatment (56°C × 30 minutes) or following prolonged exposure to the pro-protein convertase, furin. Gel filtration of active depolarizing autoantibodies was performed to determine the apparent molecular mass of peak neurotoxicity associated with the autoantibodies. RESULTS: Diabetic depression (n = 21) autoantibodies caused significantly greater mean depolarization in neuroblastoma cells (P < 0.01) compared to autoantibodies in diabetic (n = 15) or non-diabetic (n = 11) patients without depression. Depolarizing autoantibodies caused significantly more (P=0.011) inhibition of neurite outgrowth in neuroblastoma cells than non-depolarizing autoantibodies (n = 10) and they evoked sustained, global intracellular Ca2+ release in atrial cardiomyocytes or in endothelial cells. A subset of older diabetic patients suffering with a cluster of nephropathy, non-ischemic cardiomyopathy and/or depression demonstrated the presence of stable light chain dimers having apparent MW of 46 kD and associated with peak neurotoxicity in neuroblastoma cells. CONCLUSION: These data suggest that autoantibodies in older adult diabetic depression cause long-lasting depolarization in hippocampal neurons including adult dentate gyrus neural progenitor cells. The autoantibodies may impair adult dentate gyrus neurogenesis associated with treatment-refractory depression via several mechanisms including suppression of neurite outgrowth, and alteration of membrane excitability. Stable, toxic light chain autoantibody components may contribute to a cluster of severe (late-onset) complications characterized by dysfunction in highly vascularized tissues.