In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood mononuclear cells of patients with myasthenia gravis.

We studied the in vitro synthesis of antibodies to acetylcholine receptor (anti-AChR) by peripheral blood mononuclear cells (PBM) of patients with myasthenia gravis (MG) and normal subjects (NS). PBM from three of eight patients with generalized MG (MG-G) synthesized anti-AChR in vitro in the absence of pokeweed mitogen (PWM), and seven of eight did so in the presence of PWM. In individual subjects with MG-G, the levels of anti-AChR secreted in vitro by PBM correlated with serum anti-AChR antibody levels (r = 0.77) but not with the amount of IgG secreted in vitro (r = 0.44). No anti-AChR secretion was seen in culture of PBM from a patient with ocular MG, a patient with thymoma without MG, or six NS.

In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood cells: role of suppressor T cells in normal subjects.

Peripheral blood mononuclear cells of 15 of 20 patients with generalized myasthenia gravis synthesized antibodies to acetylcholine receptor (AChR) when the cells were stimulated in vitro with pokeweed mitogen. In contrast, mononuclear cells of 1 of 16 normal subjects synthesized detectable AChR antibodies. Peripheral blood mononuclear cells of five normal subjects were studied before and after putative suppressor T cells (OKT8+) were removed by a fluorescent activated cell sorter. Depletion of OKT8+ cells did not result in production of AChR antibodies, but pokeweed mitogen-induced polyclonal IgG synthesis and activation of B cells to form immunoglobulin-secreting cells (reverse hemolytic plaque assay) were increased. Therefore, failure of blood mononuclear cells of normal subjects to synthesize detectable anti-AChR in response to pokeweed mitogen is not due to suppression by OKT8+ cells.

Analysis of B-cell activation of cerebrospinal fluid lymphocytes in multiple sclerosis.

We have developed a microculture system to study pokeweed mitogen (PWM)-induced B-cell differentiation responses of CSF lymphocytes from patients with multiple sclerosis (MS) and other neurologic diseases (OND). B-cell differentiation was assessed by (1) enumeration of immunoglobulin-secreting cells (IgSC) by a protein A reverse hemolytic plaque assay; and (2) quantitation of supernatant IgG by ELISA. Cultures of MS CSF cells and OND CSF cells responded to PWM with a similar frequency, with responses in CSF cell cultures exceeding responses in corresponding blood cell cultures in several instances in both groups of patients. Numbers of IgSC in unstimulated cultures of MS CSF cells exceeded numbers in cultures of autologous peripheral blood mononuclear cells (PBM). Results suggest that CSF cells may be a particularly reactive population compared with PBM.

Expression of neurotrophin and its tropomyosin-related kinase receptors (Trks) during axonal regeneration following spinal cord injury in larval lamprey.

Exogenous neurotrophins reduce neuronal atrophy and promote regeneration following spinal cord injury but little is known about the endogenous expression of neurotrophins and their tropomyosin-related kinase (Trk) receptors in the injured spinal cord. For this purpose, we used the larval lamprey because it recovers from complete spinal transection and axons regenerate selectively in their correct paths. We cloned lamprey neurotrophin (NT) and its two Trk receptors and assessed their mRNA expression by in situ hybridization and QRT-PCR in control animals and after spinal cord transection. Control lampreys showed a longitudinal array of NT-expressing neurons along length of the spinal cord. At 2 weeks post-transection, NT expression was downregulated in neurons close to the transection, but was little affected remote from the lesion. By 4 weeks, NT expression returned to control levels in spinal cord neurons rostral and caudal to the lesion, although it was upregulated in reactive microglia at 14 and 30 days post-transection. Double-label in situ hybridization for Trk1 and Trk2 showed that Trk transcripts were expressed in several giant reticulospinal neurons, including the Mauthner neurons. After spinal cord transection, Trk1 mRNA expression was downregulated, but Trk2 mRNA expression was not changed or was increased. Thus, our data suggest that spinal cord injury in larval lampreys modulate expression of endogenous neurotrophin and induces proliferation of macrophage/microglial cells that express neurotrophin.

The sea lamprey tyrosine hydroxylase: cDNA cloning and in situ hybridization study in the brain.

Lampreys belong to the oldest group of extant vertebrates, the agnathans or cyclostomes. Thus, they occupy a key phylogenetic position near the root of the vertebrate tree, which makes them important to the study of nervous system evolution. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis and is considered a marker of catecholaminergic neurons. In the present study, we report partial cloning of the sea lamprey tyrosine hydroxylase (TH) cDNA and the pattern of TH transcript expression in the adult brain by means of in situ hybridization. Sea lamprey TH mRNA is characterized by the presence of a large untranslated sequence in the 3' end that contains a typical polyadenylation signal (ATTAAA). The deduced partial TH protein sequence presents a conserved domain with two His residues coordinating Fe(2+) binding and a conserved cofactor binding site. Neurons expressing the TH transcript were observed in the preoptic, postoptic commissure, dorsal hypothalamic, ventral hypothalamic, mammillary and paratubercular nuclei of the prosencephalon. In situ hybridization experiments also confirmed the existence of a catecholaminergic (dopaminergic) striatal population in the brain of the adult sea lamprey. A few granule-like cells in the olfactory bulbs also showed weak TH transcript expression. No cells showing TH transcript expression were observed in the rostral rhombencephalon, which suggests the absence of a locus coeruleus in the sea lamprey. Comparison of the pattern of TH mRNA expression in the prosencephalon between lampreys and teleost fishes revealed both similarities and differences. Our results suggest that the duplication of the TH gene might have occurred before the separation of agnathans and gnathostomes.

Virulence variation of white spot syndrome virus in Pacific white shrimp Litopenaeus vannamei.

The virulence of seven geographic isolates of white spot syndrome virus (WSSV; genus Whispovirus; China [strain CH1995], Nicaragua [strain N2000], Honduras [strain H2000], Ecuador [strains E-L1999 and E-LT2002], and Mexico [strains M-M2001 and M-LP2001]) was compared using a series of challenge experiments, each lasting 10 d. For each isolate, four quantified dilutions (10(-6), 10(7), 10(-8), and 10(-9)) of a viral inoculum were prepared from WSSV-infected shrimp tissue. Each viral inoculum was injected into 10 specific pathogen-free juvenile Pacific white shrimp Litopenaeus vannamei (0.25-1.50 g); controls received injections of marine crustacean physiological saline (3.2%). The minimum dose of viral inoculum that killed 50% of injected shrimp (LD50) was calculated for dilution, tissue concentration, and viral DNA amount. The CH1995 and M-M2001 isolates were the least virulent, with LD50 values of 10(-6) to 10(-7) of viral inoculum. The isolates could be grouped into three virulence clusters (CH1995 and M-M2001; N2000 and E-LT2002; and H2000, E-L1999, and M-LP2001). Virulence clusters were not altered by LD50 values based on viral DNA concentration, although a slight shifting of order in regards to virulence was seen among the three most virulent isolates (E-L1999, H2000, and M-LP2001). Overall, results indicate that there is a measurable virulence difference among WSSV isolates, which may correspond to geographical region.

Fluorescent-antibody technique in detection of salmonellae in animal feed and feed ingredients.

Comparative studies of fluorescent antibody procedure and a cultural method for the detection of Salmonella were made on 1,013 feed and feed-ingredient samples. The agreement between the two methods was 92.1%. There were more false positives (5.7%) than false negatives (2.2%). Of the 22 false negatives, 15 (68%) were obtained on meat meal. Of the total number of samples, 37% were meat meal. An additional study of 73 samples of meat meal indicated that correlation between methods was better than correlation between samples.

Suppressor T cells in myasthenia gravis and antibodies to acetylcholine receptor.

We cultured blood mononuclear cells of patients with myasthenia gravis both with and without removal of T8 suppressor cells. The levels of synthesized antibodies to acetylcholine receptor and IgG as well as the frequency of immunoglobulin-secreting cells were all higher in pokeweed mitogen-stimulated cultures depleted of T8+ cells. Thus, autologous suppressor cells within the T8+ population exert some regulatory control over antiacetylcholine receptor-producing cells in patients with myasthenia gravis.

Identification of the sterol- and actin-binding domains of plasma vitamin D binding protein (Gc-globulin).

The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of the sterol binding site with the radioactive electrophilic ligand, 3 beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.