Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

IMPORTANCE: HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

Identification of Anti-gp41 Monoclonal Antibodies That Effectively Target Cytotoxic Immunoconjugates to Cells Infected with Human Immunodeficiency Virus, Type 1.

We are developing cytotoxic immunoconjugates (CICs) targeting the envelope protein (Env) of the Human Immunodeficiency Virus, type 1 (HIV) to purge the persistent reservoirs of viral infection. We have previously studied the ability of multiple monoclonal antibodies (mAbs) to deliver CICs to an HIV-infected cell. We have found that CICs targeted to the membrane-spanning gp41 domain of Env are most efficacious, in part because their killing is enhanced in the presence of soluble CD4. The ability of a mAb to deliver a CIC does not correlate with its ability to neutralize nor mediate Ab-dependent cellular cytotoxicity. In the current study, we seek to define the most effective anti-gp41 mAbs for delivering CICs to HIV-infected cells. To do this, we have evaluated a panel of human anti-gp41 mAbs for their ability to bind and kill two different Env-expressing cell lines: persistently infected H9/NL4-3 and constitutively transfected HEK293/92UG. We measured the binding and cytotoxicity of each mAb in the presence and absence of soluble CD4. We found that mAbs to the immunodominant helix-loop-helix region (ID-loop) of gp41 are most effective, whereas neutralizing mAbs to the fusion peptide, gp120/gp41 interface, and the membrane proximal external region (MPER) are relatively ineffective at delivering CICs. There was only a weak correlation between antigen exposure and killing activity. The results show that the ability to deliver an effective IC and neutralization are distinct functions of mAbs.

Distinct Metabolic States Are Observed in Hypoglycemia Induced in Mice by Ricin Toxin or by Fasting.

Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using 1H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.

Parenteral Exposure of Mice to Ricin Toxin Induces Fatal Hypoglycemia by Cytokine-Mediated Suppression of Hepatic Glucose-6-Phosphatase Expression.

Ricin toxin is an agent of biodefense concern and we have been developing countermeasures for ricin threats. In doing so, we sought biomarkers of ricin toxicosis and found that in mice parenteral injection of ricin toxin causes profound hypoglycemia, in the absence of other clinical laboratory abnormalities. We now seek to identify the mechanisms underlying this hypoglycemia. Within the first hours following injection, while still normoglycemic, lymphopenia and pro-inflammatory cytokine secretion were observed, particularly tumor necrosis factor (TNF)-α. The cytokine response evolved over the next day into a complex storm of both pro- and anti-inflammatory cytokines. Evaluation of pancreatic function and histology demonstrated marked islet hypertrophy involving predominantly ß-cells, but only mildly elevated levels of insulin secretion, and diminished hepatic insulin signaling. Drops in blood glucose were observed even after destruction of ß-cells with streptozotocin. In the liver, we observed a rapid and persistent decrease in the expression of glucose-6-phosphatase (G6Pase) RNA and protein levels, accompanied by a drop in glucose-6-phosphate and increase in glycogen. TNF-α has previously been reported to suppress G6Pase expression. In humans, a genetic deficiency of G6Pase results in glycogen storage disease, type-I (GSD-1), a hallmark of which is potentially fatal hypoglycemia.

18ß-Glycyrrhetinic Acid Induces Metabolic Changes and Reduces Staphylococcus aureus Bacterial Cell-to-Cell Interactions.

The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18ß-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D 1H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell-cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Bispecific Anti-HIV Immunoadhesins That Bind Gp120 and Gp41 Have Broad and Potent HIV-Neutralizing Activity.

We have constructed bispecific immunoglobulin-like immunoadhesins that bind to both the HIV-envelope glycoproteins: gp120 and gp41. These immunoadhesins have N terminal domains of human CD4 engrafted onto the N-terminus of the heavy chain of human anti-gp41 mAb 7B2. Binding of these constructs to recombinant Env and their antiviral activities were compared to that of the parental mAbs and CD4, as well as to control mAbs. The CD4/7B2 constructs bind to both gp41 and gp140, as well as to native Env expressed on the surface of infected cells. These constructs deliver cytotoxic immunoconjugates to HIV-infected cells, but not as well as a mixture of 7B2 and sCD4, and opsonize for antibody-mediated phagocytosis. Most surprisingly, given that 7B2 neutralizes weakly, if at all, is that the chimeric CD4/7B2 immunoadhesins exhibit broad and potent neutralization of HIV, comparable to that of well-known neutralizing mAbs. These data add to the growing evidence that enhanced neutralizing activity can be obtained with bifunctional mAbs/immunoadhesins. The enhanced neutralization activity of the CD4/7B2 chimeras may result from cross-linking of the two Env subunits with subsequent inhibition of the pre-fusion conformational events that are necessary for entry.

Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype.

This study investigated resistance against trishexylaminomelamine trisphenylguanide (THAM-3ΦG), a novel antibacterial compound with selective microbicidal activity against Staphylococcus aureus. Resistance development was examined by culturing methicillin resistant S. aureus (MRSA) with sub-lethal doses of THAM-3ΦG. This quickly resulted in the formation of normal (WT) and small colonies (SC) of S. aureus exhibiting minimal inhibitory concentrations (MICs) 2× and 4× greater than the original MIC. Continuous cell passaging with increasing concentrations of THAM-3ΦG resulted in an exclusively SC phenotype with MIC >64 mg/L. Nuclear magnetic resonance (NMR)-based metabolomics and multivariate statistical analysis revealed three distinct metabolic profiles for THAM-3ΦG treated WT, untreated WT, and SC (both treated and untreated). The metabolome patterns of the SC sample groups match those reported for other small colony variants (SCV) of S. aureus. Supplementation of the SCV with menadione resulted in almost complete recovery of growth rate. This auxotrophism was corroborated by NMR analysis revealing the absence of menaquinone production in the SCV. In conclusion, MRSA rapidly acquires resistance to THAM-3ΦG through selection of a slow-growing menaquinone auxotroph. This study highlights the importance of evaluating and monitoring resistance to novel antibacterials during development.