Michael Paskind

Mice deficient in IL-1β-converting enzyme are defective in production of mature IL-1β and resistant to endotoxic shock

Abstract IL-1β-converting enzyme (ICE) cleaves pro-1L-1β to generate mature IL-1β. ICE is homologous to other proteins that have been implicated in apoptosis, including CED-3 and Nedd-2/Ich-1. We generated ICE -deficient mice and observed that they are overtly normal but have a major defect in the production of mature IL-1β after stimulation with lipopolysaccharide. IL-1α production is also impaired. ICE -deficient mice are resistant to endotoxic shock. Thymocytes and macrophages from the ICE -deficient animals undergo apoptosis normally. ICE therefore plays a dominant role in the generation of mature IL-1β, a previously unsuspected role in production of IL-1α, but has no autonomous function in apoptosis.

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a γ2a variable region

To examine germ line genes of the heavy chain variable region (VH) that might contribute to formation of antibodies of the NPb family, we have derived cDNA clones from two hybridomas making NPb antibodies. One, B1-8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the VH region, had very different D segments and had identical J segments (J2). A set of closely related germ line VH genes was then cloned from a partial Eco RI library of C57Bl/6 DNA. By comparing the germ line VH regions to the cDNA VH regions, we identified seven potential candidates for encoding the VH regions of NPb antibodies. The seven VH regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1-8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1-8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other VH genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their VH regions from the same germ line gene. Certain of the sequenced VH genes contain crippling mutations; the repertoire of germ line VH genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hydridization.

Dependence of Moloney murine leukemia virus production on cell growth

Abstract Production of Moloney murine leukemia virus (MuLV) ceases in cultures of infected mouse fibroblasts made stationary by growth in limiting medium. Stimulation of the cells to reenter the growth cycle synchronously was followed by two waves of virus release. The first wave, occurring within 4 hr after the cells were released from G 0 , was unaffected by 0.1 μg/ml of actinomycin D. The second wave, coinciding roughly with the mitotic period of the following cell cycle, was prevented by actinomycin D. Both waves of virus release were inhibited by 10 μg/ml of cycloheximide. The first wave of released virus appears to contain viral RNA already present in the resting cells, while the second wave of released virus particles carry newly formed viral RNA.

Biochemical and functional analysis of mice deficient in expression of the CD45-associated phosphoprotein LPAP.

The role of the CD45‐associated phosphoprotein (LPAP / CD45‐AP) during an immune response remains unclear. To understand better the function of LPAP we generated LPAP‐deficient mice by disrupting exon 2 of the LPAP gene. LPAP‐null mice were healthy and did not show gross abnormalities compared to their wild‐type littermates. However, immunofluorescence analysis of T and B lymphocytes revealed a reduced expression of CD45, which did not affect a particular subpopulation. In contrast to a recent report (Matsuda et al., J. Exp. Med. 1998. 187: 1863 – 1870) we neither observed significant alterations of the assembly of the CD45 / lck‐complex nor of polyclonal T‐cell responses. However, lymphnodes from LPAP‐null mice showed increased cellularity, which could indicate that expression of LPAP might be required to prevent expansion of lymphocytes in particular lymphatic organs rather than potentiating immune responses.