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The common use of dental and orthopedic implants calls for special attention to the immune response leading to peri-prosthetic bone loss and implant failure. In addition to the well-established microbial etiology for oral implant failure, wear debris and in particular titanium (Ti) particles (TiP) in the implant vicinity are an important trigger of inflammation and activation of bone resorption around oral and orthopedic implants, presenting an unmet medical need. Here, we employed bacterial-derived lipopolysaccharides (LPS) to model infection and TiP to model aseptic inflammation and osteolysis. We assessed inflammation in vitro by measuring IL1β, IL6 and TNFα mRNA expression in primary macrophages, osteoclastogenesis in RANKL-induced bone marrow derived pre-osteoclasts and osteolysis in vivo in a mouse calvarial model. We also assessed the trans-epithelial penetrability and safety of the tested compound in rats. Our results show that a lipophilic super-active derivative of vasoactive intestinal peptide (VIP), namely stearyl-norleucine-VIP (SNV) presented superior anti-inflammatory and anti-osteoclastogenic effects compared to VIP in vitro. In the bacterial infection model (LPS), SNV significantly reduced IL1β expression, while VIP increased IL6 expression. In the aseptic models of osteolysis, SNV showed greater suppression of in vitro osteoclastogenesis than VIP, and significantly inhibited inflammation-induced osteolysis in vivo. We also observed that expression levels of the VIP receptor VPAC-2, but not that of VPAC-1, dramatically decreased during osteoclast differentiation. Importantly, SNV previously shown to have an increased stability compared to VIP, showed here significant trans-epithelial penetration and a clean toxicological profile, presenting a novel drug candidate that could be applied topically to counter both aseptic and infection-related bone destruction.

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH2)2-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

ABSTRACT The hygiene hypothesis claims that the lack of exposure to microorganisms in developed countries correlates with a rise in the incidence of autoimmune diseases. It was also found that helminths are able to modulate the immune response in hosts in order to survive. Consequently, several successful trials using helminths as a treatment for autoimmune patients have been reported. The helminth derivative, phosphorylcholine (PC), was discovered as an immunomodulatory molecule. We have recently shown in a murine model that when a conjugate of tuftsin and PC, termed TPC, is prophylactically administered before the onset of glomerulonephritis, it attenuates the development of systemic lupus erythematosus (SLE). The current study aimed to examine the TPC effect on the gut microbiome in a mouse model of lupus. TPC treatment altered the gut composition in the mice with active lupus, in correlation with a significant decrease in glomerulonephritis, followed by an increased level of anti-inflammatory interleukin 10 (IL-10), decreased levels of proinflammatory mediators, and expansion of the T regulatory cell population. Importantly, we found that TPC treatment altered the mouse gut microbiome composition, in correlation with a significant decrease in protein secretion and improved disease parameters. The major effects of TPC treatment on the gut microbiome included decreased abundances of Akkermansia and increased abundance of several genera, including Turicibacter, Bifidobacterium, unclassified Mogibacteriaceae, unclassified Clostridiaceae, Adlercreutzia, Allobaculum, and Anaeroplasma. Overall, our results associate microbial changes with the immunomodulation of glomerulonephritis in mice with lupus. IMPORTANCE Recently, several papers referred to the association of different bacteria with lupus in mice and humans. This is the first report to demonstrate the effect of a compound derived from helminths on the induction of remission in mice with lupus and its association with a bacterial change. We show that several genera, including Akkermansia, are associated with clinical and serological parameters of lupus, while other genera, including butyrate-producing bacteria, are associated with amelioration of disease following tuftsin and phosphorylcholine treatment.

A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-β, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.

Rheumatoid arthritis (RA) is characterized by chronic autoinflammation of the joints, with a prevalence of about 1% in Western populations. Evidence in recent years has linked RA to changes in the gut microbiota (dysbiosis). Interestingly, helminths have been shown to have therapeutic activity in RA. Specifically, a glycoprotein containing phosphorylcholine (PC) extracted from helminths was found to have immunomodulatory activity. We have previously developed a novel chimeric compound composed of tuftsin-PC (TPC) that attenuates the joint destruction in mice with collagen-induced arthritis (CIA). Here, we address the interrelationship between TPC immunomodulatory activity and the gut microbiota in CIA mice. Preventive therapy with TPC in mice with arthritis maintained a physiological arthritis score as well as a steady gut microbial environment, similar to that of healthy controls, in contrast to CIA mice with severe disease. The microbial composition differed significantly between healthy and phosphate-buffered saline-treated CIA mice, enabling classifying test samples by machine learning based on levels of a small number of bacterial species. Using these bacterial biomarkers, all TPC-treated CIA mice were classified as healthy. Thus, we describe a clear correlation between TPC treatment, healthy gut microbial communities, and prevention of arthritis. This is the first study to demonstrate the immunomodulatory effect of helminth derivatives in autoimmune diseases and the link to gut microbiota.

Brain network dysfunction in Alzheimer’s disease (AD) involves many proteins (enzymes), processes and pathways, which overlap and influence one another in AD pathogenesis. This complexity challenges the dominant paradigm in drug discovery or a single-target drug for a single mechanism. Although this paradigm has achieved considerable success in some particular diseases, it has failed to provide effective approaches to AD therapy. Network medicines may offer alternative hope for effective treatment of AD and other complex diseases. In contrast to the single-target drug approach, network medicines employ a holistic approach to restore network dysfunction by simultaneously targeting key components in disease networks. In this paper, we explore several drugs either in the clinic or under development for AD therapy in term of their design strategies, diverse mechanisms of action and disease-modifying potential. These drugs act as multi-target ligands and may serve as leads for further development as network medicines.

In this study, we examined the possibility of introducing methotrexate (MTX) to the carboxylate rather than to the ε-amino side chains of proteins. We found that MTX-amino compounds covalently linked to the carboxylate moieties of macromolecules, undergo unusual peptide-bond cleavage, with the release of the MTX amino derivatives from the conjugates. This event takes place at an accelerated rate under acidic conditions, and at a slower rate at physiological pH values. The glutamate portion of MTX is responsible for this behavior, with little or no contribution of the p-aminobenzoate-pteridine ring that is linked to the α-amino side chain of the glutamate. Carboxylate-linked Fmoc-Glu-γ-CONH-(CH2)6-NH2 undergoes hydrolysis in a nearly indistinguishable fashion. A free α carboxylate moiety is essential for this effect. Carboxylate linked Fmoc-glutamic-amide-γ-CONH-(CH2)6-NH2 undergoes no hydrolysis under acidic conditions. Based on these findings, we engineered a cysteine specific MTX containing reagent. Its linkage to bovine serum albumin (BSA) yielded a conjugate with profound antiproliferative efficacy in a MTX-sensitive glioma cell line. In conclusion, carboxylate linked MTX-amino derivatives in particular, and carboxylate linked R-α-GLU-γ amino compounds in general are equipped with'built-in chemical machinery' that releases them under mild acidic conditions.

Affecting an estimated 5% of adults over 65 years of age, Parkinson’s disease and Alzheimer’s disease are the most common neurodegenerative disorders. Accumulating evidence suggests that oxidative stress induced by the breakdown of iron homeostasis is a major contributor to the neuronal loss observed in neurodegeneration. Thus, brain-permeable iron chelators may present potential therapeutic benefits. In the present study, iron-chelating hydroxamate groups were introduced into the NAP (NAPVSIPQ) peptide, whose neuroprotective qualities have been widely demonstrated. Our experiments revealed that the novel dihydroxamate peptide 3is capable of inhibiting iron-catalyzed hydroxyl radical formation and lipid peroxidation, abilities that are not part of the repertoire of its parent peptide. In addition, peptide 3was superior to native NAP in protecting human neuroblastoma cell cultures against the toxicity of hydrogen peroxide. These results suggest that NAP-based iron chelators deserve further investigation in the search for drug candidates for neurodegeneration. [ABSTRACT FROM AUTHOR]

Objectives: Administration of intravenous Ig (IVIG) is a recognized, safe and efficient mode of immunomodulatory therapy for many autoimmune diseases. Anti-idiotypic antibody binding to pathogenic autoantibodies and hence inhibition of binding to the corresponding antigen is one postulated mechanism of the beneficial effect of IVIG. The aim of this study was to fractionate the anti-beta-2-glycoprotein-I (β2GPI) anti-idiotypic antibodies from a commercial IVIG preparation and use it as a treatment in an experimental antiphospholipid syndrome (APS) mouse model. Methods: Anti-β2GPI polyclonal antibodies were purified on a β2GPI column. The purified antibodies were bound to CN–Br-activated sepharose and employed for purification of IVIG-anti-anti-β2GPI (anti-idiotypic antibodies), defined as specific intravenous Ig (sIVIG). The idiotype specificities were confirmed by competition assays. The effect of sIVIG in vitro was tested in a trophoblast and choriocarcinoma matrigel/invasion assay (i.e. proliferation and metalloproteinase (MMP)2/MMP9 expression) and in vivo in a fetal loss model of APS. Results: Anti-β2GPI antibodies inhibited human trophoblast cell invasion in vitro. The function was attributed to the Fab portion of the anti-β2GPI Igs, since β2GPI-related synthetic peptides specific for the Fab part of the anti-β2GPI antibodies neutralized its activity. APS sIVIG fraction reduce human trophoblast invasion in vitro by 560 times more than the whole IVIG compound and improved the MMP2 and MMP9 production by trophoblast cells. sIVIG improved significantly (200 times more) the pregnancy outcome in BALB/c mice passively infused with anti-β2GPI antibodies, in comparison to treatment with IVIG (P < 0.02). Conclusions: Based on the current results, we propose that APS sIVIG may be considered as potential specific therapeutic safe compound for developing a treatment for APS patients early fetal loss. [ABSTRACT FROM AUTHOR]