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Background and ObjectiveMultiple studies highlighting the diagnostic utility of neurofascin-155 (NF155)–immunoglobulin G4 (IgG4) in chronic demyelinating inflammatory polyradiculoneuropathy (CIDP) have been published. However, few studies comprehensively address the long-term outcomes or clinical utility of NF155–immunoglobulin M (IgM) or NF155–immunoglobulin G (IgG) in the absence of NF155-IgG4. We evaluated phenotypic and histopathologic specificity and differences in outcomes between these NF155 antibody isotypes or IgG subclasses. We also compare NF155-IgG4-seropositive cases to other seropositive demyelinating neuropathies.MethodsNeuropathy patient sera at Mayo Clinic were tested for NF155-IgG4, NF155-IgG, and NF155-IgM autoantibodies. Demographic and clinical data of all seropositive cases were reviewed.ResultsWe identified 32 NF155 cases (25 NF155-IgG-positive [20 NF155-IgG4-positive], 7 NF155-IgM-seropositive). NF155-IgG4-seropositive patients clinically presented with distal more than proximal muscle weakness, positive sensory symptoms (prickling, asymmetric paresthesia, neuropathic pain), and gait ataxia. Cranial nerve involvement (11/20 [55%]) and papilledema (4/12 [33%]) occurred in many. Electrodiagnostic testing (EDX) demonstrated demyelinating polyradiculoneuropathy (19/20 [95%]). Autonomic involvement occurred in 45% (n = 9, median composite autonomic scoring scale score 3.5, range 1–7). Nerve biopsies from the NF155-IgG4 patients (n = 11) demonstrated grouped segmental demyelination (50%), myelin reduplication (45%), and paranodal swellings (50%). Most patients needed second- and third-line immunosuppression but had favorable long-term outcomes (n = 18). Among 14 patients with serial EDX over 2 years, all except one demonstrated improvement after treatment. NF155-IgG-positive, NF155-IgG4-negative (NF155-IgG-positive) and NF155-IgM-positive patients were phenotypically different from NF155-IgG4-seropositive patients. Sensory ataxia, neuropathic pain, cerebellar dysfunction, and root/plexus MRI abnormalities were significantly more common in NF155-IgG4-positive compared to myelin-associated glycoprotein (MAG)–IgM neuropathy. Chronic immune sensory polyradiculopathy (CISP)/CISP-plus phenotype was more common among contactin-1 neuropathies compared to NF155-IgG4-positive cases. NF155-IgG4-positive cases responded favorably to immunotherapy compared to MAG-IgM-seropositive cases with distal acquired demyelinating symmetric neuropathy (p < 0.001) and had better long-term clinical outcomes compared to contactin-1 IgG (p = 0.04).DiscussionWe report long-term follow-up and clinical outcome of NF155-IgG4 cases. NF155-IgG4 but not IgM or IgG cases have unique clinical–electrodiagnostic signature. We demonstrate NF155-IgG4-positive patients, unlike classical CIDP with neuropathic pain and dysautonomia common at presentation. Long-term outcomes were favorable.Classification of EvidenceThis study provides Class III evidence that NF155-IgG4-seropositive patients, compared to patients with typical CIDP, present with distal more than proximal muscle weakness, positive sensory symptoms, and gait ataxia.

Objectives To describe CSF‐defined neuronal intermediate filament (NIF) autoimmunity. Methods NIF‐IgG CSF‐positive patients (41, 0.03% of 118599 tested, 1996–2019) were included (serum was neither sensitive nor specific). Criteria‐based patient NIF‐IgG staining of brain and myenteric NIFs was detected by indirect immunofluorescence assay (IFA); NIF‐specificity was confirmed by cell‐based assays (CBAs, alpha internexin, neurofilament light [NF‐L]), heavy‐[NF‐H] chain). Results Sixty‐one percent of 41 patients were men, median age, 61 years (range, 21–88). Syndromes were encephalopathy predominant (23), cerebellar ataxia predominant (11), or myeloradiculoneuropathies (7). MRI abnormalities (T2 hyperintensities of brain, spinal cord white matter tracts. and peripheral nerve axons) and neurophysiologic testing (EEG, EMG, evoked potentials) co‐localized with clinical neurological phenotypes (multifocal in 29%). Thirty patients (73%) had ≥ 1 immunological perturbation: cancer (paraneoplastic), 22; systemic infection (parainfectious [including ehrlichosis, 3] or HIV), 7; checkpoint‐inhibitor cancer immunotherapy, 4; other, 5. Cancers were as follows: neuroendocrine‐lineage carcinomas, 12 (small cell, 6; Merkel cell, 5; pancreatic, 1 [11/12 had NF‐L‐IgG detected, versus 8/29 others, P = 0.0005]) and other, 11. Onset was predominantly subacute (92%) and accompanied by inflammatory CSF (75%), and immunotherapy response (77%). In contrast, CSF controls (15684 total) demonstrated NIF‐IgG negativity (100% of test validation controls), and low frequencies of autoimmune diagnoses (20% of consecutively referred clinical specimens) and neuroendocrine‐lineage carcinoma diagnosis (3.1% vs. 30% of NIF cases), P

ObjectiveTo describe paraneoplastic neuronal intermediate filament (NIF) autoimmunity.MethodsArchived patient and control serum and CSF specimens were evaluated by tissue-based indirect immunofluorescence assay (IFA). Autoantigens were identified by Western blot and mass spectrometry. NIF specificity was confirmed by dual tissue section staining and 5 recombinant NIF-specific HEK293 cell-based assays (CBAs, for α-internexin, neurofilament light [NfL], neurofilament medium, or neurofilament heavy chain, and peripherin). NIF–immunoglobulin Gs (IgGs) were correlated with neurologic syndromes and cancers.ResultsAmong 65 patients, NIF-IgG-positive by IFA and CBAs, 33 were female (51%). Median symptom onset age was 62 years (range 18–88). Patients fell into 2 groups, defined by the presence of NfL-IgG (21 patients, who mostly had ≥4 NIF-IgGs detected) or its absence (44 patients, who mostly had ≤2 NIF-IgGs detected). Among NfL-IgG-positive patients, 19/21 had ≥1 subacute onset CNS disorders: cerebellar ataxia (11), encephalopathy (11), or myelopathy (2). Cancers were detected in 16 of 21 patients (77%): carcinomas of neuroendocrine lineage (10) being most common (small cell [5], Merkel cell [3], other neuroendocrine [2]). Two of 257 controls (0.8%, both with small cell carcinoma) were positive by both IFA and CBA. Five of 7 patients with immunotherapy data improved. By comparison, the 44 NfL-IgG-negative patients had findings of unclear significance: diverse nervous system disorders (p= 0.006), as well as limited (p= 0.003) and more diverse (p< 0.0001) cancer accompaniments.ConclusionsNIF-IgG detection by IFA, with confirmatory CBA testing that yields a profile including NfL-IgG, defines a paraneoplastic CNS disorder (usually ataxia or encephalopathy) accompanying neuroendocrine lineage neoplasia.

Objective A novel autoimmune central nervous system (CNS) disorder with glial fibrillary acidic protein (GFAP)-IgG as biomarker was recently characterized. Here, 102 patients with GFAP-IgG positivity are described. Methods The 102 included patients had: (1) serum, cerebrospinal fluid (CSF), or both that yielded a characteristic astrocytic pattern of mouse tissue immunostaining; (2) confirmation of IgG reactive with specific GFAP isoforms (α, ɛ, or κ) by cell-based assays; and (3) clinical data available. Control specimens (n = 865) were evaluated by tissue (n = 542) and cell-based (n = 323) assays. Results Median symptom onset age was 44 years (range = 8–103), and 54% were women. The predominant phenotype (83 patients; 81%) was inflammation of meninges, brain, spinal cord, or all 3 (meningoencephalomyelitis). Among patients, highest specificity for those phenotypes was observed for CSF testing (94%), and highest sensitivity was for the GFAPα isoform (100%). Rare GFAP-IgG positivity was encountered in serum controls by tissue-based assay (0.5%) or cell-based assay (1.5%), and in CSF controls by cell-based assay (0.9%). Among patients, striking perivascular radial enhancement was found on brain magnetic resonance imaging in 53%. Although cases frequently mimicked vasculitis, angiography was uniformly negative, and spinal imaging frequently demonstrated longitudinally extensive myelitic lesions. Diverse neoplasms encountered were found prospectively in 22%. Ovarian teratoma was most common and was predicted best when both N-methyl-D-aspartate receptor–IgG and aquaporin-4–IgG coexisted (71%). Six patients with prolonged follow-up had brisk corticosteroid response, but required additional immunosuppression to overcome steroid dependency. Interpretation GFAPα-IgG, when detected in CSF, is highly specific for an immunotherapy-responsive autoimmune CNS disorder, sometimes with paraneoplastic cause. Ann Neurol 2017;81:298–309

In this prospective evaluation of serum and CSF samples, all but two CSF GFAPα-IgG positive patients had autoimmune meningoencephalomyelitis while serum GFAPα-IgG positivity alone was less specific. Phenotypes were diverse among patients that were serum positive only. Adult and pediatric clinical presentations were similar. Most patients were immunotherapy responsive. Co-existing NMDA-R-IgG and cancer were associated with lack of response to first-line immunotherapy. Among patients with follow-up information, 18% had relapses. This study demonstrates CSF GFAPα-IgG is a specific autoimmune meningoencephalomyelitis biomarker, with favorable corticosteroid response. Lack of response should prompt evaluation for co-existing NMDA-R-IgG or malignancy.

We determined the effect of hyperandrogenemia as observed in polycystic ovary syndrome (PCOS) on fasting and glucose-stimulated proatherogenic inflammation markers in lean healthy reproductive-age women. Sixteen lean healthy ovulatory reproductive-age women were treated with 130 mg of DHEA or placebo (n=8 each) for 5 days. Interleukin-6 (IL-6) mRNA and IL-6 release from mononuclear cells (MNC), plasma IL-6 and C-reactive protein (CRP), and MNC-derived (matrix metalloproteinase-2) MMP-2 protein were quantified in the fasting state and 2 h after glucose ingestion, before and after treatment. Before treatment, subjects receiving dehydroepinadrosterone (DHEA) or placebo exhibited no differences in androgens, or any proatherogenic inflammation markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-sulfate (DHEA-S), and increased the percent change from baseline in fasting IL-6 mRNA, IL-6 release, plasma IL-6, and CRP and MMP-2 protein. However, there were no differences in any of the proatherogenic inflammation markers following glucose ingestion after DHEA administration. We conclude that in lean reproductive-age women, proatherogenic inflammation in the fasting state increases after raising circulating androgens to levels observed in PCOS. However, this hyperandrogenemia-induced MNC activation does not provoke a similar response to subsequent glucose ingestion.

A novel autoimmune central nervous system (CNS) disorder with glial fibrillary acidic protein (GFAP)-IgG as biomarker was recently characterized. Here, 102 patients with GFAP-IgG positivity are described.The 102 included patients had: (1) serum, cerebrospinal fluid (CSF), or both that yielded a characteristic astrocytic pattern of mouse tissue immunostaining; (2) confirmation of IgG reactive with specific GFAP isoforms (α, ɛ, or κ) by cell-based assays; and (3) clinical data available. Control specimens (n = 865) were evaluated by tissue (n = 542) and cell-based (n = 323) assays.Median symptom onset age was 44 years (range = 8-103), and 54% were women. The predominant phenotype (83 patients; 81%) was inflammation of meninges, brain, spinal cord, or all 3 (meningoencephalomyelitis). Among patients, highest specificity for those phenotypes was observed for CSF testing (94%), and highest sensitivity was for the GFAPα isoform (100%). Rare GFAP-IgG positivity was encountered in serum controls by tissue-based assay (0.5%) or cell-based assay (1.5%), and in CSF controls by cell-based assay (0.9%). Among patients, striking perivascular radial enhancement was found on brain magnetic resonance imaging in 53%. Although cases frequently mimicked vasculitis, angiography was uniformly negative, and spinal imaging frequently demonstrated longitudinally extensive myelitic lesions. Diverse neoplasms encountered were found prospectively in 22%. Ovarian teratoma was most common and was predicted best when both N-methyl-D-aspartate receptor-IgG and aquaporin-4-IgG coexisted (71%). Six patients with prolonged follow-up had brisk corticosteroid response, but required additional immunosuppression to overcome steroid dependency.GFAPα-IgG, when detected in CSF, is highly specific for an immunotherapy-responsive autoimmune CNS disorder, sometimes with paraneoplastic cause. Ann Neurol 2017;81:298-309.

Context:Hyperandrogenism and oxidative stress are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate oxidative stress.Objective:The purpose of this study was to determine the effect of oral androgen administration on fasting and glucose-stimulated leukocytic reactive oxygen species (ROS) generation, reduced nicotinamide adenine dinucleotide phosphate oxidase p47phox subunit gene expression, and plasma thiobarbituric acid-reactive substances (TBARS) in lean healthy reproductive-age women.Participants, Design, and Setting:Sixteen lean healthy ovulatory reproductive-age women were treated with 130 mg dehydroepiandrosterone (DHEA) or placebo (n = 8 each) for 5 d in this randomized, controlled, double-blind study that was performed at an an academic medical center.Main Outcome Measures:Leukocytic ROS generation, p47phox gene expression, and plasma TBARS were quantified in the fasting state and 2 h after glucose ingestion, before and after treatment.Results:Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any prooxidant markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-sulfate, increased the percent change in glucose-challenged p47phox RNA content, and increased the percent change in fasting and glucose-challenged ROS generation from mononuclear cells and polymorphonuclear cells, p47phox protein content, and plasma TBARS.Conclusion:Elevation of circulating androgens comparable to what is present in PCOS increases leukocytic ROS generation, p47phox gene expression, and plasma TBARS to promote oxidative stress in lean healthy reproductive-age women. Thus, hyperandrogenemia activates and sensitizes leukocytes to glucose in this population.

The MIS pathway is a potential therapeutic target in epithelial ovarian cancer (EOC): signaling requires both type II (T2R) and type I receptors (T1R), and results in growth inhibition. MISR2 is expressed in EOC, but the prevalence and relative contributions of candidate T1R remain unknown. We sought to: a) determine expression of T1R in EOC; b) assess impact of T1R expression with clinical outcomes; c) verify MIS-dependent Smad signaling and growth inhibition in primary EOC cell cultures. Tissue microarrays (TMA) were developed for analysis of T1Rs (ALK2/3/6) and MISR2 expression. Primary cell cultures were initiated from ascites harvested at surgery which were used to characterize response to MIS. TMA's from 311 primary cancers demonstrated the most common receptor combinations were: MISR2+/ALK2+3+6+ (36%); MISR2+/ALK2+3+6- (34%); MISR2-/ALK2+3+6- (18%); and MISR2-/ALK2+3+6+ (6.8%). No differences in overall survival (OS) were noted between combinations. The ALK6 receptor was least often expressed T1R and was associated with lower OS in early stage disease only (p =0.03). Most primary cell cultures expressed MISR2 (14/22 (63.6%)): 95% of these express ALK 2 and ALK3, whereas 54.5% expressed ALK6. MIS-dependent Smad phosphorylation was seen in the majority of cultures (75%). Treatment with MIS led to reduced cell viability at an average of 71% (range: 57-87%) in primary cultures. MIS signaling is dependent upon the presence of both MISR2 and specific T1R. In the majority of EOC, the T1R required for MIS-dependent signaling are present and such cells demonstrate appropriate response to MIS.

BackgroundGlycine receptor alpha-1 subunit (GlyRα1)-immunoglobulin G (IgG) is diagnostic of stiff-person syndrome (SPS) spectrum but has been reported detectable in other neurologic diseases for which significance is less certain.MethodsTo assess GlyRα1-IgGs as biomarkers of SPS spectrum among patients and controls, specimens were tested using cell-based assays (binding [4°C] and modulating [antigen endocytosing, 37°C]). Medical records of seropositive patients were reviewed.ResultsGlyRα1-IgG (binding antibody) was detected in 21 of 247 patients with suspected SPS spectrum (8.5%) and in 8 of 190 healthy subject sera (4%) but not CSF. Among 21 seropositive patients, 20 had confirmed SPS spectrum clinically, but 1 was later determined to have a functional neurologic disorder. Sera from 9 patients with SPS spectrum , but not 7 controls, nor the functional patient, caused GlyRα1 modulation (100% specificity). SPS spectrum phenotypes included progressive encephalomyelitis with rigidity and myoclonus (PERM) (8), classic SPS (5), stiff limb (5), stiff trunk (1), and isolated exaggerated startle (hyperekplexia, 1). Neuropsychiatric symptoms present in 12 patients (60%) were anxiety (11), depression (6), and delirium (3). Anxiety was particularly severe in 3 patients with PERM. Objective improvements in SPS neurologic symptoms were recorded in 16 of 18 patients who received first-line immunotherapy (89%, 9/10 treated with corticosteroids, 8/10 treated with IVIg, 3/4 treated with plasma exchange, and 1 treated with rituximab). Treatment-sparing maintenance strategies were successful in 4 of 7 patients (rituximab [2/3], azathioprine [1/1], and mycophenolate [1/3]).ConclusionsGlyRα1-modulating antibody improves diagnostic specificity for immunologically treatable SPS spectrum disorders.Classification of evidenceThis study provides Class IV evidence that GlyRα1-modulating antibody accurately identifies patients with treatable SPS spectrum disorders.

Among all of the genetic factors associated with multiple sclerosis (MS) susceptibility, MHC class II molecules have the strongest association. Although a direct role of DR alleles in MS have been confirmed, it has been difficult to understand the role of DQ alleles in disease pathogenesis due to strong linkage disequilibrium with certain DR alleles. Population studies have indicated that DQ alleles may play a modulatory role in progression of MS. Using HLA class II transgenic (Tg) mice, we investigated gene complementation between DR and DQ genes in the disease process. Previously, using single Tg mice (expressing HLA-DR or DQ gene), we showed that PLP91–110 peptide induced experimental autoimmune encephalomyelitis (EAE) only in DR3.Aβ° mice, suggesting that DR3 (DRB1*0301) is a disease susceptibility gene in the context of PLP. We also showed that DQ6 protects development of EAE in DQ6/DR3 double Tg mice by production of anti-inflammatory IFN-γ. In this study, we investigated the ability of DQ8 to modulate disease in DR3/DQ8 double Tg mice. Introduction of DQ8 onto DR3 Tg mice led to higher disease incidence and increased disease severity on immunization with PLP91–110, indicating that DQ8 had an exacerbating effect on the development of EAE. Increased susceptibility in DR3/DQ8 Tg mice was due to increased production of proinflammatory cytokine IL-17 by DQ8-restricted T cells. HLA-DR3/DQ8 mice with EAE also demonstrated increased inflammation and demyelination in CNS as compared with single DR3 Tg mice. Thus double Tg mouse provides a novel model to study epistatic interactions between HLA class II molecules in inflammatory and demyelinating disease.

In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of “shared epitope” and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE°. DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4+ T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II+ CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.

The human MHC class II genes are associated with genetic susceptibility to multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS of presumed autoimmune origin. These genes encode for proteins responsible for shaping immune response. The exact role of HLA-DQ and -DR genes in disease pathogenesis is not well-understood due to the high polymorphism, linkage disequilibrium, and heterogeneity of human populations. The advent of HLA class II-transgenic (Tg) mice has helped in answering some of these questions. Previously, using single-Tg mice (expressing the HLA-DR or -DQ gene), we showed that proteolipid protein (PLP)91–110 peptide induced classical experimental autoimmune encephalomyelitis only in DR3.Aβ° mice, suggesting that DR3 (DRB1*0301) is a disease susceptible gene in the context of PLP. Human population studies have suggested that HLA-DQ6 (DQB1*0601) may be a protective gene in MS. To test this disease protection in an experimental model, we generated double-Tg mice expressing both HLA-DR3 and -DQ6. Introduction of DQ6 onto DR3-Tg mice led to a decrease in disease incidence on immunization with PLP91–110 peptide indicating a dominant protective role of DQ6. This protective effect is due to high levels of IFN-γ produced by DQ6-restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis. Our study indicates that DQ6 modifies the PLP91–110-specific T cell response in DR3 through anti-inflammatory effects of IFN-γ, which is protective for experimental autoimmune encephalomyelitis. Thus, our double-Tg mouse provides a novel model in which to study epistatic interactions between HLA class II molecules in MS.

Background Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer. Methods/Principal Findings Using patient tissue microarray (TMA), in vitro and in vivo studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The in vitro effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H2S producing chemical Na2S. Silencing CBS in a cisplatin resistant orthotopic model in vivo by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics. Conclusion The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer.

Hyperandrogenism and chronic low-grade inflammation are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate inflammation. We determined the effect of oral androgen administration on fasting and glucose-stimulated nuclear factor-κB (NF-κB) activation and expression and related markers of inflammation in mononuclear cells (MNC) of lean reproductive-age women. Sixteen lean, ovulatory reproductive-age women were treated with 130 mg of DHEA or placebo ( n = 8 each) for 5 days in a randomized, controlled, double-blind fashion. Nuclear activation of NF-κB, p65 and p105 NF-κB subunit RNA, TNFα and IL-1β mRNA, and NF-κB p65 and inhibitory-κB (IκB) protein were quantified from MNC obtained while fasting and 2 h after glucose ingestion, before and after DHEA or placebo administration. Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any inflammatory markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-S, increased the percent change in fasting and glucose-challenged activated NF-κB, p65, p105, TNFα, and IL-1β RNA and p65 protein, and decreased the percent change in fasting and glucose-challenged IκB protein. We conclude that elevation of circulating androgens to the range observed in PCOS upregulates the NF-κB inflammation pathway in lean reproductive-age women. Thus, hyperandrogenemia activates and sensitizes MNC to glucose in this population.

Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the US. Müllerian inhibiting substance (MIS) is a glycoprotein hormone that causes Müllerian duct regression through a complex of TGF- β family homologous receptors: a type II ligand-binding receptor (T2R) and a tissue-specific type I receptor (T1R) that initiates downstream signaling. MIS has been proposed as potential therapeutic agent in OC. We have previously shown that specific combination of T2R/T1R receptors are expressed on OC cells, however data is lacking on the MIS-dependent pathways activated in OC cells in a T1R-specific manner. These limitations hinder further development of MIS as a therapy. We sought to determine the downstream signaling pathways activated by MIS in a T1R specific manner. We designed a chimeric OC cell model system in which GM-CSF can function as the ligand to specifically activate candidate T2R/T1R combinations (MISRII with either ALK2, ALK3, or ALK6). RNA-Seq technology was used to identify differentially expressed early and late response genes. Over 15,032 genes were analyzed, restricted to genes in which there were > 32 counts for duplicate samples. Pathway analysis was performed using Ingenuity's Integrative Pathway Analysis Tool (IPA) for genes which were expressed over 2 fold, with an False Discovery Rate (FDR)-0.001. We observed 56, 37, and 51 early response (1hr) genes which were up- or down-regulated after stimulation in OC cells engineered to express either ALK2, ALK3, and ALK6, respectively. We identified a subset of 18 of genes (e.g., ID1, SMAD7, FOSB) which were upregulated after treatment in all MISRII/T1R combinations, suggesting redundancy amongst the three T1R partner receptors. These observations will be confirmed by RT-qPCR and immunoblotting. A majority of these redundant genes are known to play roles in TGF-β signaling and cell proliferation pathways. Importantly, as we have previously shown that not all T1R candidates are expressed in OC, we report subsets of genes (e.g., RASSF6, C/EBPBβ, CSMD3) which were uniquely altered after stimulation only in the presence of a specific T1R. This indicates specific pathway activation is dependent upon T1R expression pattern in OC and likely determines response. In addition to previously known TGF- β signaling pathway, we found gene signatures (e.g. FOSB, RASSF6, LRP1B, ID1) suggesting pathways which affect cell proliferation, apoptosis, chemotherapy resistance, and DNA repair in response to MIS. This is the first study to begin to define the specific roles candidate T1R's play in signaling in response to MIS in an OC model. An understanding of these pathways is a necessary prerequisite to examining MIS as a therapy. These preliminary data suggest a role for MIS in apoptosis, cytotoxic drug resistance and DNA repair. This can be used to test combination therapy with currently available drugs and MIS in OC treatment. Citation Format: Qing Zhang, Eati Basal, Jaime I. Davila, Xueqian Yin, Edward B. Leof, William A. Cliby. Comparative RNA-Seq analysis of MIS signaling: Potential relevance as therapeutic strategy in ovarian cancer treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2014-3462

Background: Ovarian cancer (OC) is commonly associated with ascites and predominantly remains in the peritoneal cavity. Soluble factors present in ascites such as growth factors and cytokines likely play a key role in local tumor behavior including stage, invasiveness and resectability and survival. Members of IL-12 family (IL-12p70, IL-23, IL-27 and IL-35) play a critical role in the regulation of inflammatory response. However, their role as local factors in ovarian cancer growth has not been well studied. We hypothesized that that the level of these important immunomodulatory and pro-inflammatory cytokines are associated with the clinical behavior of OC. Methods: Ascites fluid was collected and analyzed from women undergoing maximal debulking surgery for presumed OC, and relevant clinical endpoints were abstracted. Concentrations of IL-12 related cytokines (IL-23, IL-27, IL-35, and IL-12p70) were assessed by ELISA. Cytokines were also assessed on matching pre-surgery plasma samples. Cytokine concentrations between matched ascites and plasma samples were examined using scatter plots and Spearman correlations. Associations between ascites concentrations and clinical characteristics were assessed using Wilcoxon rank-sum tests. Associations between ascites levels with overall survival in newly diagnosed invasive OC patients were assessed by Cox proportional hazards models using a log2 transformation. Results: Ascites fluid was analyzed from 20 patients with primary epithelial OC including 18 serous histology, 19 patients with stage III or IV disease: 16 (80%) were optimally debulked. We observed that higher levels of IL-12p70 were associated with locally invasive disease including higher rates of sub-optimal debulking (p=0.0074) and stage IV disease (p=0.012). For other IL-12 family members we observed similar associations between higher cytokine levels and suboptimal disease and stage IV disease including IL-23 (p=0.73, p=0.17), IL-27 (p=0.041, p=0.018), and IL35 (p=0.12, p=0.11), respectively. In addition, increase in IL-23 was associated with worse overall survival (log2 HR = 1.85, p=0.031). Conclusions: Higher levels of IL-12 family of cytokines in ascitic fluid are associated with several surrogates of biological aggressiveness in OC including stage, resectability and survival. Given the importance of local tumor environment on clinical behavior, a detailed understanding of IL-12 pathway may help in developing novel therapeutic strategies to treat OC. Citation Format: Eati Basal, Mathew S. Block, Matthew J. Maurer, Krista Goergen, Courtney L. Erskine, Marshall Behrens, Kimberly R. Kalli, Keith Knutson, William Cliby. Levels of IL-12 family cytokines in ascitic fluid are associated with extent and resectability of ovarian cancer and correlate with overall survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2013-1434

BACKGROUND:Despite low incidence, ovarian cancer is the fifth leading cause of cancer deaths and it has the highest mortality rate of all gynecologic malignancies among US women. The mortality rate would be reduced with an early detection marker. The folate receptor alpha (FRalpha) is one logical choice for a biomarker because of its prevalent overexpression in ovarian cancer and its exclusive expression in only a few normal tissues. In prior work, it was observed that patients with ovarian cancer had elevated serum levels of a protein that bound to a FRalpha-specific monoclonal antibody relative to healthy individuals. However, it was not shown that the protein detected was intact functional FRalpha. In the current study, the goal was to determine whether ovarian cancer patients (n = 30) had elevated serum levels of a fully functional intact FRalpha compared to matched healthy controls (n = 30). METHODOLOGY/PRINCIPAL FINDINGS:FRalpha levels in serum were analyzed by two methods, immunoblotting analysis and a radiolabeled folic acid-based microfiltration binding assay. Using the immunoassay, we observed that levels of FRalpha were higher in serum of ovarian cancer patients as compared to controls. Similar results were also observed using the microfiltration binding assay, which showed that the circulating FRalpha is functional. Importantly, we also found that the levels of FRalpha were comparable between early and advanced stage patients. CONCLUSIONS:Our results demonstrate that ovarian cancer patients have elevated levels of functional intact FRalpha. These findings support the potential use of circulating FRalpha as a biomarker of early ovarian cancer.