Serial Serum Specimens Research Articles

A Swine Model of Neural Circuit Electromagnetic Fields: Effects of Immediate Electromagnetic Field Stimulation on Cortical Injury.

Background Neurologic diseases have profound disability, mortality, and socioeconomic effects worldwide. Treatment of these disorders varies but is largely limited to unique factors associated with neural physiology. Early studies have evaluated alterations in electromagnetic fields (EMF) due to neural disorders with subsequent modulation of EMF as a potential treatment modality. Swine models have begun to be evaluated as translational models in this effect. Methods EMF measurements of a Yucatan miniswine were recorded using proprietary non-contact, non-invasive induction sensors with a dual layer Mu-metal and interlaced copper mesh helmet. The swine then underwent controlled cortical impact (CCI) to simulate traumatic brain injury (TBI). Twenty minutes post-injuryafter surgical wound closure, the swine underwent targeted EMF signal modulation using a signal generator to stimulate the swine's injured cortical circuit using a sinusoidal wave individualized at 2.5 Hz with a 500mV positive offset at 1V. After 10 days of stimulation, settings were modified to another individualized frequency of 5.5 Hz, 500mV positive offset and 1V for stimulation. Behavioral patterns in swine were evaluated, and EMF measurements were recorded daily prior to, during, and after stimulation. Artificial intelligence (AI) models evaluated patterns in EMF signals.Histology of the stimulated swine cortex was evaluated using hematoxylin and eosin staining and pentachrome staining and compared to a control swine without stimulation and a swine that had received stimulation two days post-injury in a delayed fashion. Serial serum specimens and tissue at the time of euthanasia were obtained for assessment of neuron-specific enolase (NSE) concentration. Results Pre-operative and post-stimulation measurements demonstrated differences in patterns and activity early on. There was an identified peak at 1.6Hz, not frequently seen pre-operatively. There were convergent frequencies in both data setsat 10.5 Hz and 3.9 Hz. Plateaus and decreased variability of changes in slope were identified early in the post-injury phase. AI modeling identified early similarities in pre-operative and post-stimulation measurements through the patterns of peaks with similarities on postoperative day 10 and similarities in the valleys on postoperative day 17.Histologic specimens identified increased degrees of apoptosis and cellular death in the non-stimulated control compared to the stimulated swine. Similarly, the immediately stimulated swine had less apoptosis and increased histologic viability at the site of injury compared to the two-day delayed stimulation swine. There were increased levels of NSE noted in the stimulated swine at the site of injury compared to non-injured sites and the control swine. Conclusions Cortical function was appropriately measured through induction sensors and shielding in the form of a helmet and electromagnetic field channels. Early stimulation resulted in the early and durable recovery of neuronal circuit-driven electromagnetic field patterns. Histology identified increased viability of neurons with fewer apoptotic neurons and glial cells in stimulated swine with early stimulation identifying the best effect compared to a non-stimulated subject. This recovery identifies change and recovery at the circuit, cellular, and subcellular levels that potentiatethe need for further study of EMF modulation as a treatment modality in neurological disorders.

Trending Anti-E7 Serology Predicts Mortality and Recurrence of HPV-Associated Cancers of the Oropharynx.

High-risk human papillomavirus (HPV) is among the most common causes of head and neck cancer (HNC) with increasing incidence. HPV-associated HNC patients' clinical response to treatment varies drastically, which has made treatment de-escalation clinical trials challenging. To address the need for noninvasive biomarkers that differentiate patient outcomes, serum antibodies to E7 oncoprotein levels were evaluated in serial serum specimens from HPV-positive HNC patients (n = 48). We have found that increasing antibodies to E7 throughout treatment correlates with increased cancer recurrence or progression to mortality (p = .004) with 100% specificity as a predictive test.

LTBK-03. Targeted mass spectrometry of serial CSF and serum specimens from children with diffuse intrinsic pontine glioma treated with intracranial B7-H3 CAR T cells

Considering the high expression of B7-H3 (CD276) on diffuse intrinsic pontine glioma (DIPG) and preliminary evidence that repeated locoregional delivery of CAR T cells to patients with CNS tumors was feasible and tolerable, we open the phase 1 trial BrainChild-03 (NCT04185038). Children with DIPG in Dose Regimen 1 received 40 intraventricular B7H3CAR doses, with the two patients enrolling post-progression surviving >400 days from initial CAR T infusion. To evaluate for immune and tumor responses that may correlate with clinical and radiographic evaluations, we performed targeted proteomic analysis on serial CSF and serum biospecimens.MRM-MS is a targeted mass spectrometry that provides sensitive measurement of proteins in cancer tissues and fluids. In 2 patients with longitudinal biospecimens, we identified 50 CSF and 59 serum proteins above level of detection. In general, there were fewer serum fluctuations compared to the CSF, supporting that intracranial delivery provides local immune activation. Sharp fluctuations of several immunoregulatory peptides were measured in the CSF at pre and post infusion timepoints, including BCL10, CXCL13, TIM-3, ICOSLG, and PD-L2. Notably, several analytes tracked consistently in the CSF of both patients, including markers of macrophage maturation and immune cell recruitment, including CD14, CD163, CD44, CSF-1, CXCL13 and VCAM-1. B7-H3 was detected in the CSF and serum of both patients. S005, who progressed on protocol therapy, had a sharp increase in B7-H3 in the CSF during Course 2, while S008, who had clinical improvement on protocol therapy, had consistently lower B7-H3 present. Notably, serum B7-H3 steadily declined in both patients over time, with the exception of a transient increase in S005 between Courses 4 and 5. Future work for all enrolled patients with DIPG will explore the correlation of these targeted proteomic measurements with clinical end points for potential use as markers of efficacy of therapy or adverse events.

Clinical usefulness of a novel high-sensitivity hepatitis B core-related antigen assay to determine the initiation of treatment for HBV reactivation.

A fully automated, novel, high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developing. The purpose of this study is to evaluate the efficacy of measuring HBcrAg, using that assay, to diagnose HBV reactivation in a multi-center setting, compared with ultra-high-sensitivity HBsAg (iTACT-HBsAg) and HBV DNA assays. Forty-four patients with HBV reactivation from 2008 to 2020 were enrolled in four hospitals. Serial serum specimens from the patients were assessed retrospectively for their HBcrAg levels by iTACT-HBcrAg (lower limit of detection; 2.0 log U/mL) and HBsAg levels by iTACT-HBsAg (lower limit of detection; 0.0005IU/mL); these were compared to the HBV DNA levels. HBV reactivation was defined as detection of serum HBV DNA, including unquantifiable detection. At HBV reactivation and/or thereafter, HBV DNA levels were quantified (≥ 1.3 log IU/mL) in the sera of 27 patients, and were below the level of quantification (< 1.3 log IU/mL) in the sera of 17 patients. Of the 27 patients with HBV reactivation and whose serum HBV DNA was quantified, the sera of 26 and 24 patients (96.3% and 88.9%) were positive by iTACT-HBcrAg and iTACT-HBsAg, respectively. HBcrAg was detectable by iTACT-HBcrAg before HBV DNA was quantifiable in 15 of the 27 patients. Of the 11 patients with HBV reactivation and undetectable HBcrAg by iTACT-HBcrAg at HBV reactivation and/or thereafter, 10 had unquantifiable HBV DNA and none developed HBV reactivation-related hepatitis. The iTACT-HBcrAg assay is useful for monitoring HBV reactivation to determine the initiation of treatment with nucleos(t)ide analogues.

The human anti-ganglioside GM1 autoantibody response following traumatic and surgical central nervous system insults

Predictors of the central nervous system (CNS) directed autoantibody response after acute CNS injury are poorly understood. We analyzed titers of IgG and IgM autoantibodies to ganglioside GM1 in serial serum specimens collected from human patients following acute spinal cord injury (SCI), traumatic brain injury (TBI) and brain tumor resection. We also assessed putative predictors of the autoantibody titers. We enrolled 19 patients with acute SCI, 14 patients with acute severe TBI, and 19 patients undergoing brain tumor resection. We also enrolled 25 control subjects. Some SCI, TBI and tumor patients exhibited elevated IgG titers as compared with control values; some SCI and TBI patients exhibited an acute peak in IgG titers, most commonly 14 days after insult. Some clinical and radiographic measures of injury severity correlated with IgG titer elevation in SCI and TBI patients but not tumor patients. Our study demonstrates that diverse CNS insults are followed by increased IgG autoimmune antibody titers to the CNS antigen ganglioside GM1, however the response inherent to each insult type is unique. IgG autoimmune antibody titers to GM1 merit further study as a biomarker of traumatic injury severity that can be measured in delayed fashion after CNS insult. These human data help to inform which patients with CNS insults are at risk for CNS-directed autoimmunity as well as the time course of the response.

Developing a model of placental lesions prediction in varicose veins patients

The objective of the study was to identify predictive biomarkers and generate the model to predict placental lesions in women with varicose veins. We collected serial serum specimens from 128 women with varicose veins between 22 and 24 weeks’ gestation. The investigation includes ultrasound findings, blood analysis of endothelin-1, vascular endothelial growth factor (VEGF), CRP, coagulation factors as well as BMI. We used machine learning algorithm and multivariable logistic regression with Lasso method to predict placental lesions among the pregnant patients with varicose veins.A total of 47 (36.7 %) women with varicose veins subsequently developed placental insufficiency. Mean serum VEGF were higher in women who developed placental insufficiency – 29 (27–31) pg/ml, as compared with women without varicose disease – 24 (22–25) pg/ml, p &lt; 0.001. The performance of the model trained with all the most valuable tests (VEGF, endothelin, CRP, D-dimers, fibrinogen, CEAP class) is admissible (AUC 0.94; CI 0.842–0.956; p &lt; 0.001).We identified novel combination of clinical and laboratory predictive markers that provide pathophysiological insights and could help future improvements of diagnosis and treatment of placental lesions in women with varicose veins.

Performance, Correlation and Kinetic Profile of Circulating Serum Fungal Biomarkers of Invasive Aspergillosis in High-Risk Patients with Hematologic Malignancies.

As conventional microbiological documentation of invasive aspergillosis (IA) is difficult to obtain, serum fungal biomarkers are important adjunctive diagnostic tools. Positivity rates and the kinetic profiles of galactomannan (GM), 1,3-β-D-glucan (BDG) and Aspergillus DNA (PCR) were studied in high-risk patients with hematologic malignancies. GM, BDG and PCR data from serial serum specimens (n = 240) from 93 adult hematology patients with probable (n = 8), possible (n = 25) and no (n = 60) IA were retrospectively analyzed. Positivity rates and sensitivity/specificity/positive/negative predictive values (NPV) of each fungal biomarker alone and in combination were estimated. The three markers were compared head-to-head and correlated with various biochemical, demographic and patient characteristics. The positivity rates for patients with probable/possible/no IA were 88%/8%/0% for GM (X2 = 55, p < 0.001), 62%/46%/35% for BDG (X2 = 2.5, p = 0.29), 62%/33%/27% for PCR (X2 = 3.9, p = 0.15), 50%/4%/0% for GM + BDG and GM + PCR (X2 = 31, p < 0.001), 50%/8%/22% for BDG + PCR (X2 = 6.5, p = 0.038) and 38%/4%/0% for GM + BDG + PCR (X2 = 21, p < 0.001). Higher agreement (76%) and negative correlation (rs = −0.47, p = 0.0017) was found between GM index and PCR Ct values. The sensitivity and NPV was 45–55% and 90–92% when biomarkers assessed alone and increased to 75–90% and 93–97%, respectively when combined. Weak significant correlations were found between GM, PCR and BDG results with renal/liver function markers (r = 0.11–0.57) with most GM+ and PCR+ samples found in the first and second week of clinical assessment, respectively and BDG later on. Different positivity rates, time profiles and performances were found for the three biomarkers advocating the combination of GM with PCR for the early diagnosis of IA, whereas the high NPV of combined biomarkerscould help excluding IA.

A Prospective Multicenter Cohort Surveillance Study of Invasive Aspergillosis in Patients with Hematologic Malignancies in Greece: Impact of the Revised EORTC/MSGERC 2020 Criteria.

Data concerning the incidence of invasive aspergillosis (IA) in high-risk patients in Greece are scarce, while the impact of the revised 2020 EORTC/MSGERC consensus criteria definitions on the reported incidence rate of IA remains unknown. A total of 93 adult hematology patients were screened for IA for six months in four tertiary care Greek hospitals. Serial serum specimens (n = 240) the sample was considered negative by PCR were collected twice-weekly and tested for galactomannan (GM) and Aspergillus DNA (PCR) detection. IA was defined according to both the 2008 EORTC/MSG and the 2020 EORTC/MSGERC consensus criteria. Based on the 2008 EORTC/MSG criteria, the incidence rates of probable and possible IA was 9/93 (10%) and 24/93 (26%), respectively, while no proven IA was documented. Acute myeloid leukemia was the most (67%) common underlying disease with most (82%) patients being on antifungal prophylaxis/treatment. Based on the new 2020 EORTC/MSGERC criteria, 2/9 (22%) of probable and 1/24 (4%) of possible cases should be reclassified as possible and probable, respectively. The episodes of probable IA were reduced by 33% when GM alone and 11% when GM + PCR were used as mycological criterion. The incidence rate of IA in hematology patients was 10%. Application of the 2020 EORTC/MSGERC updated criteria results in a reduction in the classification of probable IA particularly when PCR is not available.

Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.

Clinical utility of AQP4-IgG titers and measures of complement-mediated cell killing in NMOSD.

ObjectiveTo investigate whether aquaporin-4–immunoglobulin G (AQP4-IgG) titers and measures of complement-mediated cell killing are clinically useful to predict the occurrence of relapse, relapse severity, and/or disability in neuromyelitis optica spectrum disorder (NMOSD).MethodsWe studied 336 serial serum specimens from 82 AQP4-lgG–seropositive patients. NMOSD activity at blood draw was defined as preattack (24 [7.1%], drawn within 30 days preceding an attack), attack (108 [32.1%], drawn on attack onset or within 30 days after), or remission (199 [59.2%], drawn >90 days after attack onset and >30 days preceding a relapse). For each specimen, we documented the attack type and severity and immunotherapy status. Complement-mediated cell killing was quantitated by flow cytometry using an M23-AQP4 cell-based assay.ResultsThe estimated logarithmic means of AQP4-IgG titers in preattack, attack, and remission samples were 3.302, 3.657, and 3.458, respectively, p = 0.21. Analyses of 81 attack/remission pairs in 42 patients showed no significant titer differences (3.736 vs 3.472, p = 0.15). Analyses of 13 preattack/attack pairs in 9 patients showed no significant titer differences (3.994 vs 3.889, p = 0.67). Of 5 patients who converted to seronegative status, 2 continued to have attacks. Titers for major and minor attacks (n = 70) were not significantly different (3.905 vs 3.676, p = 0.47). Similarly, measures (titers) of complement-mediated cell killing were not significantly associated with disease course, attack severity, or disability at 5 years.Conclusions and relevanceAQP4-IgG titer and complement-mediated cell killing lack significant prognostic or predictive utility in NMOSD. Although titers may drop in the setting of immunotherapy, seroconversion to negative status does not preclude ongoing clinical attacks.Classification of evidenceThis study provides Class II evidence that in patients with NMOSD, AQP4-IgG titers and measures of complement-mediated cell killing activity do not predict relapses, relapse severity, or disability.

Temporal profile of HEV RNA concentration in blood and stool from patients with acute uncomplicated hepatitis E in a region with genotype 1 predominance.

Acute hepatitis E virus (HEV) is associated with viremia and faecalexcretion of the virus. The information on duration and temporal pattern of viremia and faecal shedding in HEV infection is important, but is not available. Serial serum and stool specimens were collected from patients with acute hepatitis E (typical clinical picture, serum alanine aminotransferase levels>5-folds the upper limit of normal and presence of IgM anti-HEV), beginning from within 7days of the onset of symptoms. HEV RNA concentrations were measured in sera and 10% stool suspensions, using a real-time Taqman-based nucleic acidamplification assay. Seventeen patients (median age 25 [range 19-61] years; all men) were enrolled within a median of 5 (range 3-8) days of the onset of the first symptom and provided 113 serum specimens and 71 stool specimens. The median (range) highest levels of serum bilirubin, alanine aminotransferase and aspartate aminotransferase in the patients were 10.3 (5.9-43.4) mg/dL, 1817 (442-4642) IU/L and 1016 (88-4561) IU/L, respectively. All the 17 patients had demonstrable viremia, and 12 of the13 patientswho were tested had faecal excretion at one or more time points. The HEV RNA titres were the highest in the early phase of disease and declined rapidly with time, becoming nondetectable in the serum by day 20 and in thestool by day 21. In most of the patients with acute uncomplicated acute hepatitis E, the degree of viremia and faecal shedding decline quickly after the onset of clinical illness and rapidly disappear in parallel with each other.

Correlation of Cytomegalovirus (CMV) Disease Severity and Mortality With CMV Viral Burden in CMV-Seropositive Donor and CMV-Seronegative Solid Organ Transplant Recipients.

BackgroundThe rate of cytomegalovirus (CMV) viral load increase and peak viral loads are associated with CMV disease in kidney and liver transplant recipients, but relationships to disease severity or mortality have not been shown.MethodsUsing stored serial serum specimens from renal (n = 59) and liver (n = 35) transplant recipients (D+R-; CMV-seropositive donors, CMV-seronegative recipients) from 2 prospective, randomized, controlled, interventional prophylaxis trials of CMV immune globulin (CMVIG), CMV viral load was measured using the COBAS quantitative polymerase chain reaction assay and the World Health Organization CMV standard. Patients with severe CMV-associated disease were classified according to trial definitions. Pairwise comparisons of mean viral load among deceased, surviving diseased, and nondiseased patients were analyzed by 2-way analysis of variance. To determine if viral load could predict mortality, receiver operating characteristic (ROC) curves were constructed using area under the curve (AUC) of the viral load and peak viral concentration (Vmax).ResultsViral load (mean log10 [AUC], peak viral load [Vmax]) for patients with severe CMV disease was significantly higher compared with nondiseased patients (P < .001). Similarly, higher viral burden was significantly associated with mortality (P < .001). Viral load AUC and Vmax AUROCs for predicting mortality were 0.796 and 0.824, respectively, for renal patients, and 0.769 and 0.807, respectively, for liver patients.ConclusionsUsing specimens from studies preceding the antiviral prophylaxis era, CMV viral load was associated with severe CMV disease and death, supporting CMV viral load quantification as a proxy for CMV disease severity and disease-associated mortality end points in solid organ transplantation.

Temporal characterization of serum metabolite signatures in lung cancer patients undergoing treatment.

Lung cancer causes more deaths in men and women than any other cancer related disease. Currently, few effective strategies exist to predict how patients will respond to treatment. We evaluated the serum metabolomic profiles of 25 lung cancer patients undergoing chemotherapy ± radiation to evaluate the feasibility of metabolites as temporal biomarkers of clinical outcomes. Serial serum specimens collected prospectively from lung cancer patients were analyzed using both nuclear magnetic resonance (1H-NMR) spectroscopy and gas chromatography mass spectrometry (GC–MS). Multivariate statistical analysis consisted of unsupervised principal component analysis or orthogonal partial least squares discriminant analysis with significance assessed using a cross-validated ANOVA. The metabolite profiles were reflective of the temporal distinction between patient samples before during and after receiving therapy (1H-NMR, p < 0.001: and GC–MS p < 0.01). Disease progression and survival were strongly correlative with the GC–MS metabolite data whereas stage and cancer type were associated with 1H-NMR data. Metabolites such as hydroxylamine, tridecan-1-ol, octadecan-1-ol, were indicative of survival (GC–MS p < 0.05) and metabolites such as tagatose, hydroxylamine, glucopyranose, and threonine that were reflective of progression (GC–MS p < 0.05). Metabolite profiles have the potential to act as prognostic markers of clinical outcomes for lung cancer patients. Serial 1H-NMR measurements appear to detect metabolites diagnostic of tumor pathology, while GC–MS provided data better related to prognostic clinical outcomes, possibility due to physiochemical bias related to specific biochemical pathways. These results warrant further study in a larger cohort and with various treatment options.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-016-0961-5) contains supplementary material, which is available to authorized users.

Effects of host response and antifungal therapy on serum and BAL levels of galactomannan and (1→3)-β-D-glucan in experimental invasive pulmonary aspergillosis.

Galactomannan and (1→3)-β-D-glucan are useful biomarkers of invasive pulmonary aspergillosis (IPA). However, the effects of immunosuppression on levels of galactomannan or (1→3)-β-D-glucan in IPA are not well understood or quantified. We therefore studied the simultaneous levels of galactomannan and (1→3)-β-D-glucan in two rabbit models of experimental IPA: (1) AraC-induced neutropenia in untreated (UC-AraC) and liposomal amphotericin B-treated (LAMB-AraC) rabbits; and (2) nonneutropenic cyclosporine-methylprednisolone immunosuppression in untreated (UC-CsA+M) and LAMB-treated (LAMB-CsA+M) rabbits. Simultaneous levels of galactomannan and (1→3)-β-D-glucan were determined in bronchoalveolar lavage (BAL) fluid and serial serum specimens and correlated with pulmonary host response. Serum galactomannan index (GMI) and (1→3)-β-D-glucan concentration-time-curves were higher in UC-AraC vs. UC-CsA+M (Mann-Whitney U-test, P < .05). Serum galactomannan and (1→3)-β-D-glucan in treatment groups demonstrated therapeutic responses with similarly lower levels in comparison to UC (P < .01) in both models. Host differences did not affect BAL fluid GMI or (1→3)-β-D-glucan but did affect galactomannan and (1→3)-β-D-glucan levels in serum. The higher serum GMI and (1→3)-β-D-glucan concentration-time-curves in UC-AraC correlated with extensive pulmonary infiltration by angioinvasive hyphae and minimal inflammation, while the lower concentration-time-curves in UC-CsA+M were associated with shorter and fewer hyphae in lung tissue and an intensive neutrophil response to Aspergillus hyphae. Thus, serum levels of galactomannan and (1→3)-β-D-glucan in IPA depended upon immunosuppression, which also affected severity of infection and hyphal morphology, while BAL fluid galactomannan and (1→3)-β-D-glucan were sensitive biomarkers not affected by host response.

Aquaporin 4 IgG Serostatus and Outcome in Recurrent Longitudinally Extensive Transverse Myelitis

Studies focused on recurrent longitudinally extensive transverse myelitis (rLETM) are lacking. To determine the aquaporin 4 (AQP4) IgG detection rate using recombinant human APQ4-based assays in sequential serum specimens collected from patients with rLETM categorized as negative by first-generation tissue-based indirect immunofluorescence (IIF) assay and to define the clinical characteristics and motor disability outcomes in AQP4-IgG-positive rLETM. A search of the Mayo Clinic computerized central diagnostic index (October 1, 2005, through November 30, 2011), cross-linked with the Neuroimmunology Laboratory database, identified 48 patients with rLETM, of whom 36 (75%) were positive and 12 (25%) negative for neuromyelitis optica (NMO) IgG (per IIF of serial serum specimens). Stored serum specimens from "seronegative" patients were retested with recombinant human AQP4-based assays, including enzyme-linked immunosorbent, transfected cell-based, and fluorescence-activated cell-sorting assays. Control patients included 140 AQP4-IgG-positive patients with NMO, of whom a subgroup of 20 initially presented with 2 attacks of transverse myelitis (rLETM-onset NMO). AQP4-IgG serostatus, clinical characteristics, and Expanded Disability Status Scale score. Six patients with negative IIF results were reclassified as AQP4-IgG positive, yielding an overall AQP4-IgG seropositivity rate of 89%. Fluorescence-activated cell-sorting, cell-based, and enzyme-linked immunosorbent assays improved the detection rate to 89%, 85%, and 81%, respectively. The female to male ratio was 2:3 for AQP4-IgG-negative rLETM and 5:1 for AQP4-IgG-positive patients. The AQP4-IgG-positive patients with rLETM or rLETM-onset NMO were similar in age at onset, sex ratio, attack severity, relapse rate, and motor disability. From Kaplan-Meier analyses, 36% of AQP4-IgG-positive patients with rLETM are anticipated to need a cane to walk within 5 years after onset. For patients with rLETM-onset NMO, the median time from onset to first optic neuritis attack (54 months) was similar to the median disease duration for AQP4-IgG-positive patients with rLETM (59 months). The median number of attacks was 3 for AQP4-IgG-positive patients with rLETM (range, 2-22), and the first optic neuritis attack for those with rLETM-onset NMO followed a median of 3 myelitis attacks (range, 2-19). Immunosuppressant therapy reduced the relapse rate in both AQP4-IgG-positive and AQP4-IgG-negative patients with rLETM. Recombinant antigen-based assays significantly increase AQP4-IgG detection in patients with rLETM, and AQP4-IgG-negative adults with rLETM are rare. Evolution to NMO can be anticipated in AQP4-IgG-positive patients. Early initiation of immunotherapy may result in a more favorable motor outcome.

Usefulness of Highly Sensitive AFP-L3 and DCP in Surveillance for Hepatocellular Carcinoma in Patients with a Normal Alpha-Fetoprotein

Background and aims: Early detection of Hepatocellular Carcinoma (HCC) is crucial for effective management. Incidence of HCC has increased in the United States largely attributed to hepatitis B and C virus. Lens culinaris agglutinin-reactive Alpha-Fetoprotein (AFP-L3) and Des-Gamma-Carboxy Prothrombin (DCP) are being recognized specific biomarkers for HCC. Methods: We measured AFP-L3 and DCP in serial serum specimens of a cohort of chronic hepatitis patients on HCC surveillance and compared these markers to abdominal imaging. Among fifty patients who developed HCC during surveillance, 30 were included in the study with available sera 1-2 years before, at diagnosis and post ablation of HCC. For controls, three consecutive annual sera were examined from 106 chronic hepatitis patients without HCC during surveillance for 5-10 years. The μTASWako i30 auto analyzer was used for the assay that utilizes the microfluidics chip based assay platform. It can fractionate AFP-L3 glycoform and calculates AFP-L3% if AFP level is ≥ 0.6 ng/mL. Results: Combination of AFP, AFP-L3 and DCP showed high sensitivity of 83% in all patients and 75% in patients with AFP<20 ng/mL. AFP-L3 and DCP assays were useful in patients with low levels of AFP (<20 ng/mL) and could detect significant AFP-L3% elevation in some patients more than one year before the diagnosis of HCC. Furthermore, AFP-L3 predicted recurrence of HCC. Conclusions: This is the first study in the U.S. patients using the μTASWako i30 analyzer to test these HCC biomarkers. Our results suggest that combinations of these biomarkers are highly useful for early detection of HCC.

&amp;lt;i&amp;gt;Blastomyces dermatitidis&amp;lt;/i&amp;gt; Antibody Responses in Serial Serum Specimens from Dogs with Blastomycosis: Comparison of Different Yeast Lysate Antigens

The systemic fungal organism, Blastomyces dermatitidis causes blastomycosis in animals and hu-mans. This study was designed to evaluate antibody detection in 55 serial serum specimens from 9 dogs with blastomycosis using B. dermatitidis yeast lysate antigens produced from two human isolates (B5896; B5931) and two dog isolates (ERC-2; T-58) with the indirect enzyme linked im-munosorbent assay (ELISA; peroxidase system) to determine an optimal lysate antigen(s) for use in the ELISA to detect antibody in the dog serum specimens. The mean absorbance values when the lysate antigens were compared with respect to their ability to detect antibody in the day 0 sera from the 9 dogs were 1.024 (ERC-2), 1.351 (B5896), 1.700 (B5931) and 2.084 (T-58) respectively. All of the reagents exhibited a high level of sensitivity and in all instances the amount of antibody declined as the time interval post-treatment increased, but the T-58 lysate prepared from the dog isolate from Tennessee was the optimal reagent. We continue to evaluate antigens for B. derma-titidis antibody detection in different immunodiagnostic assays.

Angiogenic biomarkers for prediction of early preeclampsia onset in high-risk women

Objective: Chronic hypertension, pregestational diabetes mellitus, history of prior preeclampsia and obese nulliparity are maternal conditions associated with increased preeclampsia risk. Whether altered maternal angiogenic factor levels allow for prediction of pending disease is unclear. Our objective was to evaluate angiogenic factors for early preeclampsia prediction in high-risk women.Methods: Serial serum specimens were collected from 157 women at high preeclampsia risk and 50 low-risk controls between 23 and 36 weeks gestation in 3 windows (23–27.6, 28–31.6, and 32–35.6 weeks) in a two-center observational cohort. Soluble fms-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF) and soluble endoglin (sEng) were measured by ELISA.Results: Multivariate parsimonious logistic regression analyses using backward elimination for prediction of early-preeclampsia (diagnosed < 34 weeks) found the best-fitting model included the predictors (1) sFlt1 measured in the second window (28–31.6 weeks) with AUC 0.85, sensitivity 67% and specificity 96% and (2) sFlt1 measured in the first window (23–27.6 weeks) and sEng change between first and second window with AUC 0.91, sensitivity 86% and specificity 96%.Conclusions: Two-stage sampling screening protocol utilizing sFlt1 and sEng is promising for prediction of preeclampsia diagnosed before 34 weeks. Larger studies are needed to confirm these findings.

Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer

Objective Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Methods Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. Results The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum–tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. Conclusions This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer.

Early Tumor Progression Associated with Enhanced EGFR Signaling with Bortezomib, Cetuximab, and Radiotherapy for Head and Neck Cancer

A phase I clinical trial and molecular correlative studies were conducted to evaluate preclinical evidence for combinatorial activity of the proteasome inhibitor bortezomib, the epidermal growth factor receptor (EGFR) inhibitor cetuximab, and radiation therapy. Patients with radiotherapy-naive stage IV or recurrent squamous cell carcinoma of the head and neck (SCCHN) were studied. Escalating doses of bortezomib (0.7, 1.0, and 1.3 mg/m²) were given intravenously twice weekly on days 1, 4, 8, and 11, every 21 days, with weekly cetuximab beginning 1 week prior and concurrently with intensity-modulated radiotherapy, delivered in 2 Gy fractions to 70 to 74 Gy. Molecular effects were examined in serial serum and SCCHN tumor specimens and the cell line UMSCC-1. Seven patients were accrued before the study was terminated when five of six previously untreated patients with favorable prognosis oropharyngeal SCCHN progressed within 1 year (progression-free survival = 4.8 months; 95% CI, 2.6-6.9). Three patients each received bortezomib 0.7 or 1.0 mg/m², without dose-limiting toxicities; one patient treated at 1.3 mg/m² was taken off study due to recurring cetuximab infusion reaction and progressive disease (PD). Expected grade 3 toxicities included radiation mucositis (n = 4), dermatitis (n = 4), and rash (n = 1). SCCHN-related cytokines increased in serial serum specimens of patients developing PD (P = 0.029). Bortezomib antagonized cetuximab- and radiation-induced cytotoxicity, degradation of EGFR, and enhanced prosurvival signal pathway activation in SCCHN tumor biopsies and UMSCC-1. Combining bortezomib with cetuximab and radiation therapy showed unexpected early progression, evidence for EGFR stabilization, increased prosurvival signaling, and SCCHN cytokine expression, warranting avoidance of this combination.