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Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of the Limulus antilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF 31-52 peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-␥) and IFN-␣. Analysis of the effect of LALF 31-52 on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF 31-52 protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-␣ levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.Lipopolysaccharide (LPS) is believed to be the initiator of the systemic inflammatory cascade that culminates in the organ damage and multiorgan failure found in septic patients (17,47). In recent years, attention has been focused on the release by macrophages of cytokines, particularly interleukin 1␤ (IL-1␤), IL-6, and tumor necrosis factor alpha (TNF-␣), that are important mediators of septic shock pathology (9, 39). Recent data have substantially advanced our knowledge on the complex timing and interactions among events like LPS exposure, cytokine release (32), and the development of septic shock. These data strongly involve LPS and macrophage cytokine release as primary effectors in the etiology of septic shock. They also suggest that LPS blockade, cytokine blockade, or specific cytokine modulation may be of therapeutic benefit in septic patients.Therapeutic approaches based on molecules that bind and neutralize LPS are particularly attractive, because they directly block the primary stimulus for the proinflammatory cytokine cascade. Recent studies have mainly involved rBPI (recombinant bactericidal permeability-increasing protein) (1) and peptides derived from it (13, 36). The Limulus anti-LPS factor (LALF) is a small, basic protein found in hemocytes from both Tachypleus tridentatus and Limulus polyphemus, which inhibits the endotoxin-mediated activation of the coagulation cascade (30), apparently by binding to LPS. This prot...

Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.

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Dialysable Leucocyte Extract (DLE) is a low molecular weight dialysable material of disrupted peripheral human leucocytes with widespread effects on the immune system. We described the in vitro anti-HIV activity of DLE as well as its three chromatographic fractions (Fa, Fb and Fc). To determine the levels of inhibition on HIV replication by DLE we infected MT-4 cell cultures, using the Bru viral isolate at 0.05, 0.1, 0.5 and 1 m.o.i. Previously, MT-4 cells cultures were treated with DLE or fractions at non-toxic concentrations. Reverse transcriptase (RT) activity and p24 antigen were evaluated in culture supernatants at seven days postinfection. No effect was observed when MT-4 cells were incubated with DLE for 3 h. Whereas inhibition of HIV production was observed when MT-4 cells were pre-treated for a longer period of time. DLE inhibited p24 production and RT activity more than 50% at 0.1 m.o.i. More than 80% of inhibition was observed for all doses of DLE tested at 0.05 m.o.i. Higher viral doses (m.o.i. 0.5 and 1) were used to assess the antiviral activity of DLE fractions. Fraction Fb inhibits viral production more than 80%. Otherwise, fractions Fa and Fc did not show inhibitory effect for any viral dose used. These results indicate that DLE is able to modulate cell susceptibility to viral infection in vitro.