Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents

Neurotransmitter Transporter-Like: A Male Germline-specific SLC6 Transporter Required for Drosophila Spermiogenesis

The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl), is expressed only in the male germline. Mobilization of a transposon inserted near the 3′ end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism

Up-regulation of IL-18 and predominance of a Th1 immune response is a hallmark of lupus nephritis

There is evidence that nephritis is dominated by a Th1 immune response in systemic lupus erythematosus. Since IL-18 promotes polarization of the immune response toward Th1, we investigated the role of this cytokine in lupus nephritis (LN). A total of 133 lupus patients and 44 healthy subjects were enrolled. Demographic and clinical characteristics with renal biopsy data were recorded. IL-18 along with IFN-γ and IL-4, two prototypical of Th1 and Th2 cytokines, were measured in serum by ELISA. Peripheral blood lymphocytes were analysed by flow cytometry for IFN-γ and IL-4. IL-18 expression was determined by immunohistochemistry in 13 renal biopsy specimens from patients with LN and 2 controls. Serum IL-18 was higher in lupus patients than in controls. Levels of IL-18 correlated with urinary microalbumin and were increased in patients with LN when compared to those without LN. IL-18 expression was also increased within the glomeruli of nephritic patients and was primarily detected within the mesangial matrix and in infiltrating mononuclear cells. Measurement of IFN-γ and IL-4 in either sera or peripheral blood lymphocytes showed high IFN-γ along with low IL-4 expression in LN patients compared to patients without nephritis. A positive correlation between serum IL-18 and IFN-γ levels was found. IL-18 may play a prominent role in the pathogenesis of LN by promoting a cytokine imbalance towards a Th1 immune response. Measurement of IL-18 may be helpful for the early identification of lupus patients with LN and may help gauge the response to treatment in patients with active LN undergoing treatment

Insight into the bronchodilator activity of Vitex negundo

CONTEXT: Vitex negundo Linn. (Verbenaceae) is traditionally used in hyperactive respiratory disorders.Objective: This study explored the mechanisms underlying the effectiveness of Vitex negundo in hyperactive respiratory disorders.MATERIALS AND Methods: Crude extract of V. negundo leaves was obtained. For in vivo bronchodilatory activity in anesthetized rats, different doses (1, 3, 10, 30, and 50 mg/kg) of the crude extract of V. negundo (Vn.Cr) were tested. The underlying mechanisms were studied in isolated guinea pig tracheal strips, suspended in organ baths at 37 °C.Results: Intravenous doses of the crude extract of Vn.Cr showed dose-dependent bronchodilatory effect (9-50%) against carbachol (CCh; 100 g/kg)-induced bronchoconstriction, similar to aminophylline. In isolated guinea-pig tracheal strips, Vn.Crrelaxed CCh (1 M) and high K(+) pre-contractions with respective EC50 values of 0.72 (0.48-1.10; n = 5) and 3.38 mg/mL (1.84-6.21; n = 4), similar to papaverine. Diltiazem also relaxed both contractions with more potency against high K(+) pre-contraction (p \u3c 0.05). Pre-incubation of the tracheal strips with Vn.Cr potentiated the isoprenaline inhibitory concentration response curves (CRCs), similar to papaverine.DISCUSSION: The inhibitory effect against CCh and high K(+) suggests involvement of phosphodiesterase (PDE) inhibitory pathway(s), in addition to an inhibitory effect on Ca(++) entry. This finding was further strengthened when pre-treatment of the tracheal strips potentiated the isoprenaline CRCs.CONCLUSION: RESULTS suggest Vn.Cr possesses a combination of papaverine-like PDE inhibitor and diltiazem-like Ca(++) entry blocking constituents, which partly explain its bronchodilatory effect, thus validating its medicinal importance in asthma