Potential role for ESAT6 in dissemination of M. tuberculosis via human lung epithelial cells.

ESAT6 has recently been demonstrated to cause haemolysis and macrophage lysis. Our studies demonstrate that ESAT6 causes cytolysis of type 1 and type 2 pneumocytes. Both types of pneumocytes express membrane laminin, and ESAT6 exhibits dose-dependent binding to both cell types and to purified human laminin. While minimal ESAT6 was detected on the surface of Mycobacterium tuberculosis grown in vitro, exogenously provided ESAT6 specifically associated with the bacterial cell surface, and the bacterium-associated ESAT6 retained its cytolytic ability. esat6 transcripts were upregulated approximately 4- to approximately 13-fold in bacteria replicating in type 1 cells, and approximately 3- to approximately 5 fold in type 2 cells. In vivo, laminin is primarily concentrated at the basolateral surface of pneumocytes where they rest on the basement membrane, which is composed primarily of laminin and collagen. The upregulation of esat6 transcripts in bacteria replicating in pneumocytes, the specific association of ESAT6 with the bacterial surface, the binding of ESAT6 to laminin and the lysis of pneumocytes by free and bacterium-associated ESAT6 together suggest a scenario wherein Mycobacterium tuberculosis replicating in pneumocytes may utilize surface ESAT6 to anchor onto the basolateral laminin-expressing surface of the pneumocytes, and damage the cells and the basement membrane to directly disseminate through the alveolar wall.

Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 µM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a ß phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h ß half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Combined use of immunoassay and two-dimensional liquid chromatography mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples.

Peptides and monoclonal antibodies have both emerged as important therapeutic modalities, but each has challenges which limit their use. Non-recombinant chemical conjugation of peptides onto antibodies has the potential to minimize or eliminate altogether many of these limitations. Once such approach, pioneered by CovX has created the possibility for rapid stoichiometric fusion of pharmacophores to a single antibody platform. These molecules, called CovX-Bodies, maintain both the pharmacologic properties of a given peptide and the pharmacokinetic properties of a monoclonal antibody. The result is a new class of molecules wherein each component contributes desirable traits. In this paper, we demonstrate the use of immunoassay and two-dimensional liquid chromatography mass spectrometry (2DLC/MS) in combination to investigate the antibody conjugates of Glucagon-like peptide-1 (GLP-1) and analogs for intact protein metabolite identification directly from mouse serum. The information gained from combining these approaches has helped guide and expedite the optimization of our drug product development efforts.

Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin.

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.

Production and characterization of monoclonal antibodies against asexual stages of Plasmodium yoelii nigeriensis.

Swiss mice vaccinated with Plasmodium yoelii nigeriensis-soluble antigen and saponin, following a homologous 100% lethal challenge, showed 60% protection (6 out of 10 mice survived). Monoclonal antibodies (MAbs), generated by hybridizing the Sp2/0 myeloma cells with the splenocytes of each of these ten mice, separately, were screened using enzyme-linked immunosorbent assay (ELISA), and were characterized by using merozoite (Mz) invasion inhibition assay in vitro, immunofluorescence assay (IFA), passive transfer of protection and ELISA-based isotyping. Curiously, purified MAbs from each of the six protected mice showed a distinct dichotomy: only two or three of them inhibited >86% Mz invasion, whereas the remaining six to nine showed <58% Mz invasion inhibition. However, none of the purified MAbs from the nonprotected mice could inhibit >58% Mz invasion. Furthermore, the ability of the MAbs to inhibit Mz invasion appeared to correlate with their IFA-reactivity with the free-Mz, suggesting that these MAbs were directed against the Mz surface antigens involved in invasion. In passive transfer of protection experiments, pooled purified MAbs from protected mice, that inhibited >86% Mz invasion, transferred 60% protection from challenge; the remaining pooled purified MAbs from protected mice, and those from nonprotected mice, when transferred separately, imparted only 30 and 10% protection, respectively. Isotypically, the MAbs belonged to IgG(1), IgG(2a), IgG(2b), and IgG(3) subclasses. Our results indicate that purified MAbs against P. yoelii nigeriensis, produced from the hybrids generated using the splenocytes of vaccinated and protected mice, belonged to two distinct groups: a small group that inhibited >86% Mz invasion, strongly cross-reacted with free-Mz, transferred up to 60% passive protection, and belonged to IgG(2a) and IgG(3) subclasses, whereas the other relatively larger group inhibited <58% Mz invasion, weakly cross-reacted with free-Mz, and transferred only 30% passive protection.

Neuroimmunomodulatory effects of morphine in Leishmania donovani-infected hamsters.

OBJECTIVE: The effect of morphine on host defense during Leishmania donovani infection in golden hamsters was studied. METHODS: Hamsters were intracardially infected with L. donovani amastigotes and then monitored by spleen touch print microscopic examination. Morphine and naloxone were administered subcutaneously and intraperitoneally, respectively. Leukocytes were counted by a hemocytometer, and ex vivo phagocytosis was determined by the examination of stained adherent macrophages. RESULTS: Low doses of morphine, 1.75 and 2.5 mg/kg x 2, administered subcutaneously on day 0 and day 15 significantly (p < 0.05) suppressed the infection, whereas high doses (20.0 and 50.0 mg/kg x 2) exacerbated the infection. On day 30, hamsters treated with low doses of morphine showed a significant (p < 0.05) increase in the number of circulating leukocytes and the pool size and phagocytic activity of peritoneal macrophages ex vivo; in hamsters treated with high doses, all these parameters appeared to be diminished. The bone marrow of morphine-treated hamsters showed a fall in total cellularity and no change in the number of monocytes; however, in those treated with low doses, the infection was completely eliminated by day 30, and paradoxically, a significant (p < 0.05) potentiation of infection was observed in hamsters treated with high doses. The spleens of hamsters treated with both low and high doses of morphine showed a significant (p < 0.05) decrease and increase in weight, respectively; treatment with low doses also caused an almost 2-fold increase in the percentage of monocytes. Morphine apparently exerted its protective effects via naloxone-sensitive opioid receptors; naloxone pretreatment did not affect the potentiation of infection. CONCLUSION: Conditional doses of morphine apparently biphasically modulated the course of L. donovani infection in hamsters, at least in part through macrophage-mediated mechanisms.