Publications

Resolution of leishmanial infections requires the expansion of specific type 1 T helper cells that secrete or express on their membrane lymphokines capable of activating macrophages that contain these parasites to a microbicidal state. Specific CD8+ T cells, which are triggered during infection, also appear to play a role in protective immunity, possibly through their ability to secrete interferon-gamma. In the mouse model of infection with Leishmania major, the expansion of specific type 2 T helper cells exacerbates disease, an effect that appears to result from the properties of type 2 T helper derived lymphokines to deactivate macrophages and inhibit release of activating cytokines by type 1 T helper cells. In the mouse, destruction of intracellular Leishmania by activated macrophages depends upon the L-arginine-dependent production of nitrogen oxides. Molecules from the parasite that can induce, and are the target of, the protective T-cell response are being characterized.

The v-rel oncogene product from the avian reticuloendotheliosis virus strain T corresponds to a member of the Rel-related family of enhancer-binding proteins that includes both the mammalian 50- and 65-kDa subunits of the NF-kappa B transcription factor complex. However, in contrast to NF-kappa B, v-Rel has been shown to function as a dominant-negative repressor of kappa B-dependent transcription in many mature cell types. We now demonstrate that a highly conserved motif within the Rel homology domain of v-Rel containing a consensus protein kinase A phosphorylation site is required for DNA binding, transcriptional repression, and cellular transformation mediated by this oncoprotein. However, replacement of the serine phosphate acceptor within the protein kinase A site with an alanine did not alter any of these functions of v-Rel, suggesting that phosphorylation at this site is not central to the regulation of this oncogene product. Rather, the inactive mutations appear to identify a functional domain within v-Rel required for these various biological activities. It is notable that these same mutations do not impair the ability of v-Rel to heterodimerize with the 50-kDa subunit of NF-kappa B, suggesting that v-Rel-mediated transcriptional repression likely involves direct nuclear blockade of the kappa B enhancer rather than indirect alterations in the composition of preformed cytoplasmic NF-kappa B complexes. Paradoxically, when introduced into undifferentiated F9 cells, v-Rel functions as a kappa B-specific transcriptional activator rather than as a dominant-negative repressor. These stimulatory effects of v-Rel require both the conserved protein kinase A phosphorylation site and additional unique C-terminal sequences not needed for v-Rel-mediated repression in mature cells. Retinoic acid-induced differentiation of these F9 cells restores the repressor function of v-Rel. These opposing biological actions of v-Rel occurring in cells at distinct stages of differentiation may have important implications for the mechanism of v-Rel-mediated transformation occurring in avian splenocytes.

A polymerase chain reaction (PCR)-based homology cloning strategy was used to define the spectrum of stromelysin-like matrix metalloproteinases (MMPs) synthesized by cultured glomerular mesangial cells (MC). Using this technique, cDNAs encoding an unusual, truncated member of the MMP family, punctuated (putative) metalloproteinase (PUMP-1), were exclusively isolated. Incubation with the cytokines interleukin 1 and tumour necrosis factor increased the abundance of PUMP-1 mRNA in mesangial cells. The mesangial PUMP-1 mRNA is processed in a tissue-specific manner, yielding a transcript containing repeated 3'-untranslated region ATTTA motifs commonly found in cytokines with limited mRNA stability. Polyclonal antibodies prepared against the C-terminal region of the PUMP-1 protein documented release of this enzyme by cultures of cytokine-stimulated MC and permitted identification of PUMP-1-expressing mesangial cells within clinical biopsy specimens of acute glomerulonephritis. These findings represent new molecular and clinical evidence that non-malignant cells process and secrete this unusual member of the MMP family in a cytokine-mediated, tissue-specific manner. Mesangial synthesis of PUMP-1 may contribute to the progression of injury during glomerular inflammatory states.

Interleukin-1 (IL-1) is synthesized as a 31 kDa precursor protein, whose multiple extracellular activities are attributed to receptor binding of a processed, carboxy-terminal 17 kDa peptide. Unlike other secreted proteins, the IL-1 precursor lacks a hydrophobic leader sequence and is not found in organelles composing the classical secretory pathway. In order to further clarify the intracellular processing of IL-1, we studied its site of synthesis in human monocytes. Secreted and integral membrane proteins are translated on membrane-bound polyribosomes, while intracellular proteins are translated on free polyribosomes. Free and membrane-bound polysomes were isolated from Lipid A-stimulated monocyte lysates and immunoblotted using antibodies specific to the N-terminal regions of the IL-1 alpha and beta precursors. Free polysome fractions showed multiple small bands consistent with nascent peptide chains; membrane-bound polysomes yielded no detectable IL-1. Polysome fractions were then analyzed by immunoelectron microscopy; nascent IL-1 alpha and beta peptide chains were readily seen emerging from cytoskeletal-associated free polyribosomes, but not membrane-bound polyribosomes. Electron microscopic in situ hybridization revealed IL-1 mRNA chains attached to cytoskeletal-associated free, but not membrane-bound polyribosomes. The intracellular distribution of the fully synthesized IL-1 beta precursor was studied in human mesangial cells (HMC), whose cytoskeletal organization is more readily evaluated than that of monocytes. Dual immunofluorescence microscopy of these cells revealed a complex intracellular distribution of the fully synthesized 31 kDa IL-1 precursors. IL-1 was asymmetrically distributed between cytosolic, microtubule, and nuclear compartments, without association with actin or intermediate filaments. This demonstration of the sites of IL-1 synthesis and patterns of intracellular distribution provide further evidence for an extracellular release mechanism which is clearly distinct from the classical secretory pathway.

In order to indicate priorities for possible occupational health care planning activities, we evaluated occupational health risks, health services, and occupational research and training in Israel from the perspective of occupational medicine in the United States. We used available public information as well as data collected in a previous regional assessment of occupational health in the Negev. We estimated that each year 35% of the workforce in Israel may be exposed to high levels of noise, 4-11% to workplace toxins, and 7% to work injuries, all hazards warranting attention by health planners. Reviewing occupational health services we found that programmatic deficiencies limit the effective use of existing resources. We also evaluated the potential benefits of strengthened expert review in setting funding priorities for research and training in occupational safety and health in Israel.