Immunization. Update: search for an AIDS vaccine.

Identifying a successful HIV preventive vaccine is among the highest research priorities of the US NIH. While therapies for HIV have brought hope to those who are already HIV-infected, stopping the worldwide epidemic will require safe, effective HIV vaccines. Here, we describe scientific and other obstacles to attaining that goal and provide a brief synopsis of clinical trial results, recent preclinical results, and future priorities.

Protective effect of radio-detoxified endotoxin (Tolerin) on the ultrastructure of pancreas in experimental endotoxin shock of rats.

The ultrastructural changes of pancreas exocrine cells were studied after the intravenous administration of endotoxin (LPS) or radio-detoxified endotoxin (150 kGy 60Co-gamma irradiated: RD-LPS or Tolerin). The LPS (1 mg/rat) induces an autolytic destruction in the membranes of the mitochondria of the pancreas exocrine cells. The RD-LPS given in similar dose does not produce any autolytic change. However, a small dose (100/micrograms/rat) of RD-LPS (Tolerin) as a pretreatment can protect the autolytic destruction of the mitochondria induced by LPS. This may be attributed to the membrane stabilizing effect of RD-LPS.

Elimination of endotoxin from the blood by extracorporeal activated charcoal hemoperfusion in experimental canine endotoxin shock.

Circulating endotoxin is an important factor in the pathogenesis and clinical symptoms of endotoxin shock. The effect of extracorporeal activated charcoal hemoperfusion was investigated in experimental endotoxin shock of dogs produced by i.v. injection of Escherichia coli 089 endotoxin (1 mg/kg body weight). The endotoxin was labeled with 99mTc. The aorta and vein cava caudalis of anesthetized dogs were cannulated through the arteria and vein femorales. The cannulae were contacted to the hemoperfusion charcoal cartridge. The efficiency of hemoperfusion was tested from the blood samples, and the endotoxin content of blood was measured biologically (in lead acetate-treated rats) and isotopically (99mTc radioactivity) at 15, 30, 60, 90, and 120 min after injection. It was demonstrated that extracorporeal activated charcoal hemoperfusion can eliminate the majority of circulating endotoxin from the blood within 30 min.

The DNA binding properties of the MutL protein isolated from Escherichia coli.

The mutL gene of Escherichia coli, which is involved in the repair of mispaired and unpaired nucleotides in DNA, has been independently cloned and the gene product purified. In addition to restoring methyl-directed DNA repair in extracts prepared from mutL strains, the purified MutL protein binds to both double and single stranded DNA. The affinity constant of MutL for unmethylated single stranded DNA was twice that of its affinity constant for methylated single stranded DNA and methylated or unmethylated double stranded DNA. The binding of MutL to double stranded DNA was not affected by the pattern of DNA methylation or the presence of a MutHLS-repairable lesion.

Intraoperative ultrasonography for common bile duct exploration during laparoscopic cholecystectomy.

The "Endomedix Laparoscan Diagnostic Ultrasound" from the Medilas Ltd. and the "Leopard" and "Panther" type intraoperative ultrasound instruments from the Green Sound Ltd. have been used with success in sixty cases during laparoscopic cholecystectomy (LC) by the authors. Of these patients in four cases common bile duct (CBD) stones and in one case only sludge have been found. Of those with CBD stones, in one case four little stones have been observed despite of negative intraoperative cholangiography (IC) and in one case calculus in the retropancreatic CBD part was detected. On the basis of preoperative findings the CBD stones have not been expected. According to the authors the intraoperative ultrasound (IOUS) can be used with no exception in all cases eligible for LC, and also it can be used for the examination of neighboring organs (liver, pancreas, hepatoduodenal ligament). The method is extremely useful, performance quick, simple and informative as well as it can replace IC during LC.

Transcriptional activity of the human immunodeficiency virus-1 LTR promoter in fission yeast Schizosaccharomyces pombe.

We have analyzed the transcriptional activity of the human immunodeficiency virus type I (HIV-1) LTR promoter in the fission yeast Schizosaccharomyces pombe (S.pombe). The ability of a series of 5'-deleted forms of the HIV-1 LTR promoter to direct transcription of the chloramphenicol acetyltransferase reporter gene was studied. We found that the HIV-1 promoter is functional in S.pombe and that deletion of sequences upstream of the NF-kB binding site previously identified to contain the negative regulatory element (NRE) in mammalian cells, resulted in about thirty-fold increase in transcriptional activity. Sequences in the HIV-1 promoter that bind NF-kB were found to be essential for transcriptional activation in S.pombe. In mammalian cells, transactivation of the HIV-1 LTR requires TAR sequences and the viral Tat protein. In fission yeast, Tat failed to transactivate the HIV-1 LTR, suggesting that S.pombe may lack a cellular factor(s) required for the Tat transactivation process.

Widespread neuronal expression of the presenilin-1 early-onset Alzheimer's disease gene in the murine brain.

Mutations in the presenilin-1 (S182) gene have been genetically linked to early-onset Alzheimer's disease. To clarify the underlying molecular mechanism through which presenilin-1 is involved in the pathogenesis of this neurodegenerative disorder, the regional and cellular transcription profile of this gene was characterized in primary cells isolated from the murine brain by Northern blot hybridization using digoxigenin-labeled riboprobes. Our results indicate that presenilin-1 mRNA transcripts are widely distributed throughout the adult mouse brain. Furthermore, immunohistochemical labeling of hybridized sections indicates that expression was predominantly localized to neuronal cells. Neurons in the hippocampus and cerebral cortex, which are severely compromised in Alzheimer's disease, showed prominent expression of presenilin-1. In contrast, white matter areas and endothelial cells do not appear to express presenilin-1 to detectable levels. presenilin-1 transcripts, however, are also present less frequently in certain nonneuronal cell populations such as ependymal cells in the choroid plexus. Analysis of primary cells isolated from murine brain supported the results obtained by in situ hybridization and showed that cultured primary neurons and astrocytes express presenilin-1. Overall, it appears that the pattern of presenilin-1 gene expression parallels that previously described for the amyloid precursor protein.

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

Effect of cellular differentiation on cytokine-induced expression of human immunodeficiency virus in chronically infected promonocytic cells: dissociation of cellular differentiation and viral expression.

Cellular differentiation is thought to play an important role in the susceptibility of monocytic lineage cells to human immunodeficiency virus (HIV) infection as well as in their ability to support virus replication. In addition, virus replication in monocytes/macrophages has been demonstrated in vitro to be strongly modulated by several cytokines such as tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The purpose of the present study was to investigate the interaction between cellular differentiation and cytokines in the regulation of HIV expression from chronically infected monocytic lineage cells. U1, a persistently HIV-infected promonocytic cell line, is characterized by low levels of virus expression which can be modulated by several cytokines. 1 alpha,-25-Dihydroxyvitamin D3 (Vit.D3), a well-known differentiating agent for myelomonocytic cells which has been previously reported to modulate HIV replication in other in vitro systems, induced maturation of U1 cells toward a macrophage-like phenotype, as demonstrated by the induction of the differentiation-associated cell surface markers CD14 and CD11b. Vit.D3-induced differentiation did not result in induction of HIV expression; however, when U1 cells were stimulated with tumor necrosis factor alpha in the presence of Vit.D3, a synergistic induction of cell differentiation and viral expression was demonstrated. In contrast, Vit.D3 suppressed the induction of HIV expression in U1 cells stimulated with gamma interferon, interleukin-6, and granulocyte-macrophage colony-stimulating factor, although synergy between Vit.D3 and these cytokines was observed in terms of cellular differentiation. These data suggest that differentiation of monocytic cells does not necessarily correlate with increased HIV expression.

CXCR4 and CCR5 genetic polymorphisms in long-term nonprogressive human immunodeficiency virus infection: lack of association with mutations other than CCR5-Delta32.

Polymorphisms in the coding sequences of CCR5 and CXCR4 were studied in a group of human immunodeficiency virus (HIV)-infected long-term nonprogressors. Two different point mutations were found in the CXCR4 coding sequence. One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. The well-described 32-bp deletion within the CCR5 coding sequence (CCR5-Delta32) was found in 4 of 13 nonprogressors, and 12 different point mutations were found scattered over the CCR5 coding sequence from 8 nonprogressors. Most of the mutations created either silent or conservative changes in the predicted amino acid sequence: only one of these mutations was found in more than a single nonprogressor. All nonsilent mutations were tested in an HIV envelope-dependent fusion assay, and all functioned comparably to wild-type controls. Polymorphisms in the CXCR4 and CCR5 coding sequences other than CCR5-Delta32 do not appear to play a dominant mechanistic role in nonprogression among HIV-infected individuals.