Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production.

Using a recently described method for efficiently deriving homozygous targeted alleles in embryonic stem cells, we produced chimeric mice whose tissues were derived partially from embryonic stem cells bearing homozygous deletion of the mouse immunoglobulin heavy-chain joining (JH) region. Characterization of these chimeric mice indicated that homozygous JH deletion leads to arrest of B-cell development at an early stage, resulting in a total lack of peripheral B cells and serum IgM. These results were confirmed in mice containing the homozygous JH deletion in their germ line. This novel B-cell-deficient mouse strain provides a tool for studying the recombination and expression of exogenous immunoglobulin genes introduced into the mouse germ line.

Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis.

Human placental tissue contains regulatory molecules that may prevent allo-sensitization. Recently, a 14 kDa beta-galactoside binding protein with demonstrated immunoregulatory properties has been cloned using cDNA from human placenta and expressed in Escherichia coli. The present study assesses the ability of this recombinant immunomodulatory lectin (rIML-1), to prevent experimental autoimmune encephalomyelitis (EAE), a paralytic T cell-mediated disease directed against myelin basic protein (BP). Injection of rIML-1 into Lewis rats inhibited the induction of both clinical and histological signs of EAE, apparently by blocking sensitization of encephalitogenic BP-specific T cells and inducing BP-dependent suppressor cells. Because it is neither immunogenic nor toxic, rIML-1 may have application in humans, and would have distinct advantages over unselective cytotoxic immunosuppressive agents used currently in the treatment of autoimmune diseases and transplantation.

Enzyme immunoconjugates utilizing glucose oxidase and myeloperoxidase are cytotoxic to Candida tropicalis.

A dual-enzyme immunoconjugate system was evaluated for its cytotoxic effect on Candida tropicalis. Glucose oxidase, which generates hydrogen peroxide in the presence of glucose and oxygen and myeloperoxidase, which catalyzes the oxidation of halides in the presence of hydrogen peroxide, were each conjugated to a C. tropicalis-specific monoclonal antibody. Neither the glucose oxidase nor the myeloperoxidase conjugates exhibited any significant cytotoxic effect by themselves. A combination of glucose oxidase conjugate (3.2 ng/ml) and myeloperoxidase conjugate (12.8 ng/ml) in the presence of 5 mg of glucose per ml, 150 mM chloride, and 50 microM iodide was cytotoxic to C. tropicalis, killing 99.9% of the treated sample. Flow cytometry was used to characterize the binding of the conjugates to yeast cells and demonstrated that the binding of both conjugates to the yeast cell surface is required for cytotoxicity. In addition, the concentrations of conjugates required for a cytotoxic effect were below the concentrations required to saturate all of the yeast cell surface antibody-binding sites.

Molecular cloning, characterization, and expression of a human 14-kDa lectin.

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.