Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3.

Neuromedins are a family of peptides best known for their contractile activity on smooth muscle preparations. The biological mechanism of action of neuromedin U remains unknown, despite the fact that the peptide was first isolated in 1985. Here we show that neuromedin U potently activates the orphan G protein-coupled receptor FM3, with subnanomolar potency, when FM3 is transiently expressed in human HEK-293 cells. Neuromedins B, C, K, and N are all inactive at this receptor. Quantitative reverse transcriptase-polymerase chain reaction analysis of neuromedin U expression in a range of human tissues showed that the peptide is highly expressed in the intestine, pituitary, and bone marrow, with lower levels of expression seen in stomach, adipose tissue, lymphocytes, spleen, and the cortex. Similar analysis of FM3 expression showed that the receptor is widely expressed in human tissue with highest levels seen in adipose tissue, intestine, spleen, and lymphocytes, suggesting that neuromedin U may have a wide range of presently undetermined physiological effects. The discovery that neuromedin U is an endogenous agonist for FM3 will significantly aid the study of the full physiological role of this peptide.

Quantitation of human tissue and immune cell type II 14 kDa phospholipase A(2) by enzyme immunoassay.

The metabolism of arachidonic acid into inflammatory mediators (e.g. prostaglandin, leukotrienes) is dependent upon the rate-limiting enzyme phospholipase A(2). Localization and quantification of type II 14 kDa phospholipase A(2) (PLA(2)) in cells or tissue preparations has historically been accomplished through activity measurements, a process that can provide variable results due to interference by exogenous substances with hydrolysis assessment. Others have reported on the use of sandwich enzyme immunoassays (EIA) to measure 14 kDa PLA(2) by mass in serum and exudate fluids, e.g. synovial fluid. Herein, we report the utilization of a human recombinant type II 14 kDa PLA(2) sandwich EIA to directly measure cell or tissue-residing 14 kDa PLA(2). It is known that type II 14 kDa PLA(2) resists acid treatment, and this technique was applied to cell fractions which liberated the enzyme from cellular membrane components prior to quantitation by EIA. Two human immune cell populations were assessed and shown to contain measurable levels of 14 kDa PLA(2). Neutrophil or monocyte cytosolic fractions contained no measurable levels whereas the respective 100 000g particulate fractions contained 2.6+/-0.8 pg (neutrophil) and 2.1+/-0.6 pg (monocyte) 14 kDa PLA(2)/mug protein. Human placenta cytosolic fractions contained no measurable levels while 100 000g particulate contained approximately 25 ng 14 kDa PLA(2)/mg protein. This EIA, in conjunction with acid extraction, provides an easy and reproducible assay to identify and quantify this enzyme in cells and whole tissues, expanding our ability to study the relationship of this enzyme to inflammatory processes.

Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library.

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rIL-5. Spleens from two mice were used to generate hybridomas. Spleens from an additional three mice were used to construct a combinatorial library. In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rIL-5. These Abs were tested for the ability to block binding of iodinated rIL-5 to the alpha-chain of the human IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-dependent cells. By hybridoma technology, 16 mAbs were obtained, 11 of which blocked binding to IL-5R alpha, including three that inhibited proliferation. Quantitative binding assays and sequence analysis revealed that these latter three mAbs were closely related. Combinatorial cloning and selection by phage display was used to isolate 24 bacterial colonies secreting Fabs that bound to 125I-rIL-5 and to rIL-5-coated plates. Sequencing of 10 of the Fabs indicated that four unique Abs were obtained, comprising one predominant VH paired with one of two different VL. The sequence of the Fabs was distinct from the sequences of the neutralizing mAbs. In contrast to the mAbs, none of the Fabs blocked binding of 125I-IL-5 to IL-5R alpha or neutralized the biologic activity of IL-5. The inability to identify neutralizing Fabs was shown not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kappa light chains, retained neutralizing activity. By chain shuffling, pairing of the Fd fragment of the heavy chain of one of the neutralizing mAbs (2B6), with the light chain library derived from the IL-5-immunized mice, neutralizing Fabs were obtained. These Fabs contained light chain sequences closely related to the original light chain of 2B6. Hence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection. The results reveal differences in the Abs obtained from a combinatorial library vs hybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity.

Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations.

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.

Immune complexes in sarcoidosis: a correlation with activity and duration of disease.

Samples of serum from patients with sarcoidosis were examined for the presence of immune complexes, using the Raji cell line. An indirect immunofluorescent technique was used to detect the binding of immune complexes to the complement receptors of Raji cells. Preliminary studies indicated that the method was sensitive and specific. We studied 44 patients with sarcoidosis who were separable into the following three distinct clinical groups: (1) acute disease (one year or less); (2) chronic active disease (five years or more); and (3) resolved disease (five years or more). In the 26 patients with acute disease, 12 had circulating immune complexes; immune complexes were present in two of the ten patients with chronic active disease. In contrast, immune complexes were absent in patients with resolved disease and normal control subjects. In patients with active disease, there was no apparent correlation between the presence of immune complexes and the stage of disease; however, immune complexes were present in five of the 11 patients with extrapulmonary disease, in seven of 12 patients with elevated concentrations of gamma-globulin, and in four of five of those patients with autoantibodies to lymphocytes.