Successful pharmacotherapy for the treatment of severe feeding aversion with mechanistic insights from cross-species neuronal remodeling.

Pediatric feeding disorders affect up to 5% of children, causing severe food intake problems that can result in serious medical and developmental outcomes. Behavioral intervention (BI) is effective in extinguishing feeding aversions, and also expert-dependent, time/labor-intensive and not well understood at a neurobiological level. Here we first conducted a double-blind, placebo-controlled trial comparing BI with BI plus d-cycloserine (DCS). DCS is a partial N-methyl-d-aspartate (NMDA) receptor agonist shown to augment extinction therapies in multiple anxiety disorders. We examined whether DCS enhanced extinction of feeding aversion in 15 children with avoidant/restrictive food intake disorder (ages 20-58 months). After five treatment days, BI improved feeding by 37%. By contrast, BI+DCS improved feeding by 76%. To gain insight into possible mechanisms of successful intervention, we next tested the neurobiological consequences of DCS in a murine model of feeding aversion and avoidance. In mice with conditioned food aversion, DCS enhanced avoidance extinction across a broad dose range. Confocal fluorescence microscopy and three-dimensional neuronal reconstruction indicated that DCS enlarged dendritic spine heads-the primary sites of excitatory plasticity in the brain-within the orbitofrontal prefrontal cortex, a sensory-cognition integration hub. DCS also increased phosphorylation of the plasticity-associated extracellular signal-regulated kinase 1/2. In summary, DCS successfully augments the extinction of food aversion in children and mice, an effect that may involve plasticity in the orbitofrontal cortex. These results warrant a larger-scale efficacy study of DCS for the treatment of pediatric feeding disorders and further investigations of neural mechanisms.

Consolidation and reconsolidation of contextual fear memory requires mammalian target of rapamycin-dependent translation in the dorsal hippocampus.

The mammalian target of rapamycin (mTOR) pathway is important for regulating protein translation. The present study characterized the role of mTOR-dependent translation in the dorsal hippocampus (DH) during the consolidation and reconsolidation of contextual fear memory. We first showed that fear conditioning resulted in increased phosphorylation of p70s6 kinase (p70s6K) in the DH and that infusion of the mTOR inhibitor rapamycin (RAP) into the DH immediately after training disrupted formation of long-term contextual fear memory. Additionally we showed that p70s6K was activated after retrieval of a previously stored fear memory, and inhibition of mTOR by DH infusion of RAP blocked the reconsolidation of contextual fear memory. Together these results demonstrate that within the DH translational control through the mTOR pathway is important for consolidation as well as the stability of fear memory after retrieval.

The formation of auditory fear memory requires the synthesis of protein and mRNA in the auditory thalamus.

The medial geniculate nucleus of the thalamus responds to auditory information and is a critical part of the neural circuitry underlying aversive conditioning with auditory signals for shock. Prior work has shown that lesions of this brain area selectively disrupt conditioning with auditory stimuli and that neurons in the medial geniculate demonstrate plastic changes during fear conditioning. However, recent evidence is less clear as to whether or not this area plays a role in the storage of auditory fear memories. In the current set of experiments rats were given infusions of protein or messenger RNA (mRNA) synthesis inhibitors into the medial geniculate nucleus of the thalamus 30 min prior to auditory fear conditioning. The next day animals were tested to the auditory cue and conditioning context. Results showed that rats infused with either inhibitor demonstrated less freezing to the auditory cue 24 h after training, while freezing to the context was normal. Autoradiography confirmed that the doses used were effective in disrupting synthesis. Taken together with prior work, these data suggest that the formation of fear memory requires the synthesis of new protein and mRNA at multiple brain sites across the neural circuit that supports fear conditioning.

Multianalyte assay system developed for drugs of abuse.

A simple, 10-min immunoassay system has been developed that simultaneously screens for five different classes of drugs of abuse in a urine sample. This system tests for amphetamines, cannabinoids, cocaine metabolites, opiates, and phencyclidine, and each assay has a specific preset cutoff concentration. Accuracy is > 99% for reporting positive or negative results for samples with 200% or 50%, respectively, of the cutoff concentrations of the drugs. Tests of a panel of 96 compounds yielded only three cases of nonspecific reactivity (at a drug concentration of 100 mg/L). Another panel of 12 compounds that could normally be found in urine samples was also evaluated and no interferences were observed. Concordance was > 95% between this system and the comparable automated immunoassays for detecting drugs of abuse. Greater than 98% of GC/MS-confirmed positive samples gave positive results with this assay system.

Immunoglobulin secretion by cell lines derived from African and American undifferentiated lymphomas of Burkitt's and non-Burkitt's type.

As part of an investigation of the cellular origins of undifferentiated lymphomas of the Burkitt's and non-Burkitt's types, we have examined immunoglobulin secretion by cell lines and biopsy samples from these tumors and have compared it with other lymphoblastoid cell lines. The majority of American lymphoma cell lines and a smaller fraction of African lymphoma cell lines secreted monoclonal IgM. No other Ig classes were secreted. Ig secretion by the tumor samples was also found to be exclusively IgM. The secreted IgM was polymeric and associated with J chain. Secretion of free light chain by most of the tumor cell lines was observed. Quantitation by immunoassay demonstrated that the American lymphoma cell lines secreted significantly more IgM than the African lymphoma cell lines. We have proposed that the lymphoma cells represent an early stage of B cell differentiation, during which antigen-independent secretion occurs.

A stable, sensitive assay for human fibronectin.

A solid-phase radioimmunoassay system to quantitate human fibronectin has been developed. This assay utilizes 125I-labeled affinity purified IgG directed against human plasma fibronectin. The sensitivity of this system is comparable to conventional (labeled antigen) radioimmunoassays having a detection limit of approximately 0.5 ng. This assay is relatively rapid (less than 24 h), the reagents are stable at 4 degrees C (less than 2 months), and the reproducibility is excellent. Both human plasma and cellular fibronectin react equivalently in this assay.

Serum levels of fibronectin and a fibronectin-like DNA-binding protein in patients with various diseases.

The levels of fibronectin and the fibronectin-like, malignant disease-associated DNA-binding protein (MAD-2) were determined in sera from patients with various diseases. The levels of both proteins were elevated in most serum samples from patients with various types of cancer and in some patients with nonmalignant diseases. The highest levels of these proteins were found in patients with carcinomas of the breast and lung. A subset of the lung cancer patients with small cell carcinoma had either normal or depressed marker levels. The levels of fibronectin and MAD-2 varied concordantly in the different patient populations studied, which suggests that the serum levels of the fragment are directly proportional to the total amount of fibronectin in the serum.

Isolation and identification of a human serum fibronectin-like protein elevated during malignant disease.

A human DNA-binding protein, designated MAD-2, has recently been found to be elevated in the serum from patients with malignant diseases. MAD-2 has been purified approximately 500-fold from peritoneal and pleural fluids collected from cancer patients. Immunodiffusion studies have indicated that MAD-2 is immunochemically identical to human plasma fibronectin. The purified material has been resolved by sodium dodecyl sulfate gel electrophoresis into two major protein chains with molecular weights in the range of 200,000 to 210,000 in either the presence or absence of disulfide bond-reducing agents. These results suggest that MAD-2 is a fibronectin fragment which has been generated through proteolysis. A quantitative assay system capable of detecting ng quantities of MAD-2 has been developed and used to verify the presence of elevated MAD-2 levels in DNA-binding protein fractions isolated from the serum of individuals with malignant diseases.

Detection of a human serum DNA-binding protein associated with malignant disease.

Human serum DNA-binding proteins were fractionated by DNA-cellulose affinity chromatography. Electrophoretic resolution of these protein fractions allowed the direct comparison of DNA-binding proteins from pools of serum from cancer patients, fetuses, or normal humans. A protein band detected in the fraction eluted by 0.6 M NaCl was elevated in pooled cancer serum, compared to normal or fetal serum. This malignant disease-associated DNA-binding protein (MAD-2) did not react with a variety of antisera directed against specific human serum proteins, blood components, or tumor markers. A chromatography-electrophoresis assay system for MAD-2 that allows the simultaneous determination of 20-30 serum samples was developed. This assay system was used for a preliminary clinical study, which revealed elevated MAD-2 levels in sera from pregnant women and individuals with various carcinomas, compared to levels in sera obtained from normal individuals, patients with nonmalignant diseases, or fetal cord samples.

Serum ribonuclease activity in cancer patients.

A study was made of the levels of ribonuclease (RNase) in human serum, using 2 independently collected banks of samples from Scripps Clinic and Research Foundation and the Mayo Clinic, each bank representing more than 100 individuals. These serum samples originated from a cross-section of normal individuals, smokers, patients with benign tumours, and patients with a variety of neoplasms. Elevated levels of serum RNase occurred in 68% of the samples from individuals with malignant disease. Elevated levels also occurred in 24% of the samples from individuals with benign tumours and in 38% of the smoker controls from the Mayo Clinic serum bank. Using ion-exchange chromatography, pooled sera from normal individuals and cancer patients were fractionated by differential salt elution. Each pool showed 2 distinct peaks of RNase activity, and both peaks were elevated to the same degree in the cancer serum pools. Similar results were obtained after thin-layer-gel isoelectric focusing of both normal and cancer sera; no new species of RNase could be detected in the sera of patients with malignant diseases. The results suggested a generalized nonspecific increase in serum RNase in these patients.

A correlation of serum C3DP levels with chemotherapeutic management of cancer patients.

Serial serum C3DP levels of 33 patients at our institution have been followed for up to 10 months. Individuals experiencing periods free of symptoms and signs of proliferating or expanding malignant disease, had C3DP levels which remained below 150 microgram/ml. Patients with active or recurrent disease, while on chemotherapy, had elevated C3DP values (greater than 150 microgram/ml). Serum C3DP values declined abruptly following treatment which resulted in major reduction of tumor mass. Decreases in C3DP levels from values above 150 microgram/ml to values within the normal range (50-150 microgram/ml) were observed during 89% (25/28) of the favorable clinical responses which have been followed with C3DP assays. Increases in C3DP levels from values within the normal range to values above 150 microgram/ml were observed either prior to or coordinate with clinical symptoms of disease recurrence 83% of the time (10/12 cases). These studies suggest that serial C3DP determinations offer an excellent prognostic aid for evaluating the response of malignant tumors during chemotherapeutic management.

A clinical evaluation of serum C3DP levels in individuals with malignant diseases.

A human serum DNA-binding protein (C3DP) derived from complement component C3 has been found in elevated concentrations in the sera of individuals with malignant diseases. An assay system has been devised which reveals quantitative values of serum C3DP levels. Results obtained using this system indicate that normal human sera have an average C3DP level of 242 mug/ml (range, 40 to 146), whereas sera from individuals with active carcinomas have an average C3DP level of 242 mug/ml (range, 146 to 400). Sera from individuals with active leukemias, lymphomas, and melanomas all had elevated levels of C3DP, whereas sera from individuals with polycythemia vera or other nonmalignant diseases had normal or only slightly elevated C3DP concentrations. No tissue specificity seems to be required for malignant growths to result in elevated C3DP serum concentrations. The levels of C3DP in 79% of the individuals who experienced disease remission were found to decline to normal values, concurrent with the disease regression. Patients who did not respond to therapy regimens retained high C3DP levels.

Purification and identification of a human-serum DNA-binding protein associated with malignant diseases.

A malignancy-associated human serum DNA-binding protein (C3DP protein), which was previously detected using dodecylsulfate gel electrophoresis, has been purified and characterized. This protein was isolated from human fetal cord serum by DNA-cellulose affinity chromatography, ammonium sulfate fractionations, DEAE-cellulose chromatography, and Sephadex gel filtration. The molecular weight of purified C3DP protein has been shown to be 135000 by ultracentrifugation, gel filtration, and dodecylsulfate gel electrophoresis. Dodecylsulfate gel electrophoresis following disulfide bond reduction has revealed that this protein is composed of three subunits having molecular weights of 74000, 40000 and 22000. Carbohydrate has been demonstrated to be attached to the 74000 and 22000 molecular weight components. Immunochemical studies have revealed that the C3DP protein is a fragment of human complement component C3, which closely resembles C3c.