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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.

Summary Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin–PC (TPC), a novel helminth-based compound in collagen-induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 µg/0.1 ml) or orally (250 µg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme-linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (Treg) and regulatory B (Breg) cell phenotypes by fluorescence-activated cell sorter (FACS). TPC-treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P

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., 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.

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood–brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)–HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA–HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA–HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (~x3) prolonged survival.

To date, no truly efficacious drugs for Alzheimer's disease (AD) have been developed; moreover, all new anti-AD drugs developed since 2003 have failed. To succeed where previous ones have failed in drug development, new approaches for AD therapy are needed. Here we discuss the potential application of network medicine as a new approach to AD treatment. Unlike traditional approaches focused on a single target/pathway, network medicine targets and restores disease-disrupted networks through simultaneous modulation of numerous proteins (targets)/pathways involved in AD pathogenesis. We consider several drug candidates under development for AD therapy, including Keap1–Nrf2 regulators, endogenous neurogenic agents, and hypoxia-inducible factor 1 (HIF-1) activators. These drug candidates are multi-target ligands with the potential to further develop as network medicines, since they act as master regulators to initiate a broad range of cellular defense mechanisms/cytoprotective genes that exert their efficacy in a holistic way. We also explore their diverse mechanisms of action and potential disease-modifying effects, which may have profound implications for drug discovery.

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.

Background In areas where helminthes infections are common, autoimmune diseases are rare. Treatment with helminthes or their ova, improved clinical findings of several autoimmune diseases. Based on the helminthes biological activity, a novel chimeric be-specific molecule was synthetized of tuftsin-phosphoryl-choline (PC)-TPC. Objectives To study TPC treatment in established collagen induced arthritis (CIA) mice, and the mechanism of activity. Methods Arthritis was induced in DBA male mice by immunization with collagen emulsified in TB-mycobacteria at the tail base. Boost was given 3 weeks later. Treatment with TPC started when the clinical score was 2. Cytokines were measure in culture-fluid of splenocytes in-vitro. T regulatory cells and B regulatory cells were measured by FACS. TPC effect on TLR4 expression was studied using HEKTM-mTLR4 cells system and its inhibitor. M1 shift to M2 was performed in RAW macrophages differentiated to M1 by PMA followed by LPS. TPC was added and IL-6, TNFalpha, IL-10 were tested by ELISA. Results Starting TPC treatment of CIA mice after disease establishment, had a significant lower arthritis score in comparison with control vehicle subjected mice (i.e. TPC-6.8±0.8 vs vehicle-13.8±0.45; p via the phosphorylcholine end. Our data indicated that TPC significantly ameliorated established CIA by anti-inflammatory immunomodulatory activity. Conclusions Our data may lead to a novel bi-specific self small molecule for therapy of patients with advanced RA. Disclosure of Interest None declared

The finding that acetylcholinesterase (AChE) colocalizes with β-amyloid (Aβ) and promotes and accelerates Aβ aggregation has renewed an intense interest in developing new multifunctional AChE inhibitors as potential disease-modifying drugs for Alzheimer's therapy. To this end, we have developed a new class of selective AChE inhibitors with site-activated chelating activity. The identified lead, HLA20A, exhibits little affinity for metal (Fe, Cu, and Zn) ions but can be activated following inhibition of AChE to liberate an active chelator, HLA20. HLA20 has been shown to possess neuroprotective and neurorescuing activities in vitro and in vivo with the ability to lower amyloid precursor holoprotein (APP) expression and Aβ generation and inhibit Aβ aggregation induced by metal (Fe, Cu, and Zn) ion. HLA20A inhibited AChE in a time and concentration dependent manner with an HLA20A-AChE complex constant (K(i)) of 9.66 × 10(-6) M, a carbamylation rate (k(+2)) of 0.14 min(-1), and a second-order rate (k(i)) of 1.45 × 10 (4) M(-1) min(-1), comparable to those of rivastigmine. HLA20A showed little iron-binding capacity and activity against iron-induced lipid peroxidation (LPO) at concentrations of 1-50 μM, while HLA20 exhibited high potency in iron-binding and in inhibiting iron-induced LPO. At a concentration of 10 μM, HLA20A showed some activity against monoamine oxidase (MAO)-A and -B when tested in rat brain homogenates. Defined restrictively by Lipinski's rules, both HLA20A and HLA20 satisfied drug-like criteria and possible oral and brain permeability, but HLA20A was more lipophilic and considerably less toxic in human SHSY5Y neuroblastoma cells at high concentrations (25 or 50 μM). Together our data suggest that HLA20A may represent a promising lead for further development for Alzheimer's disease therapy.

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.

The steroidogenic enzyme 21-hydroxylase (21OH) is the main autoantigen in autoimmune primary adrenal failure (Addison’s disease). Autoantibodies against 21OH are immunological markers of an ongoing autoimmune process but are not directly involved in the tissue destruction. Autoreactive T cells are thought to mediate tissue damage, but the T cell antigen(s) has not been identified. To find out whether 21OH contains important immunodominant epitopes for T cells, we first immunized BALB/c and SJL inbred mouse strains with recombinant 21OH and showed that lymph node cells proliferated effectively following in vitro stimulation with recombinant 21OH (stimulation indices (SI) 20–40). We further synthesized a series of peptides based on 21OH with amino acid sequences with propensity to bind to major histocompatibility complex class II molecules. Only a few peptides could trigger lymphocytes of 21OH-primed mice to proliferate. One of these, 21OH (342–361), stimulated effectively 21OH-primed lymph node cells of SJL mice (SI = 4–8) and also, although to a lesser extent, of BALB/c mice (SI = 2.5). When SJL mice were immunized with 21OH (342–361), the immunizing peptide as well as peptide 21OH (346–361) triggered a significant proliferative response (SI = 24). A peptide from another part of 21OH, namely 21OH (191–202), did not stimulate the 21OH (342–361)-primed cells. Moreover, stimulation of lymph node cells of mice immunized with 21OH (342–361) with 21OH resulted in a significant proliferative response. We conclude that 21OH (342–361) is an immunodominant determinant for T cells in SJL and probably BALB/c mice. 21OH (342–361) corresponds to the substrate binding site of the enzyme. The p342–361 region may be involved in the pathogenesis of autoimmune adrenal failure in humans.

Blood infections caused by gram-negative bacteria are one of the major challenges facing modern medicine, despite treatment with the available conventional antibiotics (22). Mortality rates in the range of 20 to 80% for septicemia caused by gram-negative bacteria have been reported. Antibiotic treatment is often administered when the disease reaches an advanced stage, usually when symptoms appear, by which point insufficient time remains for the antibiotic to kill the pathogen before the onset of irreversible tissue damage. Moreover, in many cases the antibiotic is given before sensitivity tests have been performed to identify an effective treatment. The emergence of bacterial strains resistant to conventional antibiotics, the lack of a rapid means of diagnosing the infection, and the generally unknown antibiotic sensitivity pattern of the infecting bacteria are probably among the major causes of inefficient therapy and high mortality rates (26). Most of these blood infections are caused by opportunistic pathogens that are not usually capable of initiating bacteremia in otherwise healthy individuals. Host defense mechanisms of the innate, nonclonal immune system serve as the principal pathway for effective elimination of pathogens (11). Potent components of innate immunity are the macrophages and polymorphonuclear leukocytes (PMNs) that mediate the early clearance of bacteria by the phagocytic process (1, 23). Agents that enhance phagocytosis may promote clearance of the pathogen and termination of the infectious process. In some cases the phagocytotic process is mediated by adhesins expressed on the bacterial surface, in a process termed nonopsonic phagocytosis (18). However, the interaction of many bacteria with phagocytic cells is greatly facilitated by opsonins, which act as a bridge between the surfaces of these two types of cells (5, 8). The development of agents that can function as opsonins may provide a useful new approach to terminating the infectious process by enhancing bacterial attachment to phagocytic cells, followed by ingestion and digestion of the pathogen. For an agent to function as an opsonin, it must contain a moiety that recognizes a specific target molecule on the bacterial surface and another that recognizes specific receptors on phagocytic cells. Moreover, its toxicity must be relatively low, and most importantly, there should be little likelihood for the emergence of strains resistant to its action. In the present study, we describe the synthesis of antimicrobial peptide conjugates that act both as opsonins to enhance destruction by phagocytic cells and as agents that permeabilize the bacterial membrane to enhance eradication by hydrophobic antibiotics and other antimicrobial agents. The peptides are derived from polymyxin B (PMB) or polymyxin E (PME) covalently linked to a short chemotactic peptide. Early studies have established that although polymyxin-based antibiotics are relatively toxic, they can be rendered 10 to 15 times less toxic by cleaving the lipid moiety from the molecule (7, 10). These polymyxin-based peptides lack direct bactericidal activity but retain their ability to bind to lipopolysaccharides (LPSs) on the bacterial surface and to permeabilize the outer membrane (OM) to hydrophobic antibiotics that cannot otherwise penetrate the bacteria, as well as to other bactericidal agents, such as the complement (33). Since resistance to the parent polymyxins is rare (25), it is expected that resistance to these polymyxin-derived peptides will be rare as well. Indeed, we did not find strains resistant to the permeabilizing activity of a polymyxin B (PMB) nonapeptide (PMBN) among 59 PMB-sensitive strains tested (21). Moreover, administration of the nonapeptide in combination with erythromycin protected mice against a lethal dose of an erythromycin-resistant strain of Klebsiella pneumoniae (19). More recently, we showed that the polymyxin B nonapeptide binds to LPS, which is consistent with previous studies showing that the parent PMB and PME molecules bind to lipid A of LPS on bacterial surfaces (16, 25, 31). Encouraged by these results, we reasoned that conjugation of PMBN or PMEN to a moiety that recognizes specific receptors on phagocytic cells may confer opsonic activity on the molecule. We chose to use formyl methionine-leucine-phenylalanine (fMLF), a chemotactic peptide, as our conjugant because it has been studied extensively for its property of binding to specific receptors on phagocytic cells (17). It will be shown that conjugates consisting of polymyxin peptides covalently linked to fMLF enhance the bactericidal activities of conventional antibiotics, act as opsonins in promoting the phagocytic destruction of bacteria in vitro, and protect mice from lethal infections and bacteremia in vivo.

Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK-28, possessing the monoamine oxidase (MAO) and neuroprotective N-propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO-A and -B inhibitor in vitro (IC50, MAO-A, 0.037 +/- 0.02; MAO-B, 0.057 +/- 0.01). Acute (1-5 mg/kg) and chronic [5-10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 days]in vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO-A and -B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N-desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5-HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5-HIAA (5-hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N-methy-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5-HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO-A and -B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO-B inhibitors, for which it is being developed, as MAO-B inhibitors do not alter brain dopamine.

Iron-dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)-B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinson's disease, Alzheimer's disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO-B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO-B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO-B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron-dependent lipid peroxidation in rat brain homogenates with IC50 values (12-16 microM), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 microM were able to attenuate cell death induced by serum deprivation and by 6-hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti-Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non-selective MAO-A and MAO-B inhibitor (IC50 < 0.1 microM). However, HLA20 was more selective for MAO-B but had poor MAO inhibition, with an IC50 value of 64.2 microM. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.

Two hypothalamic peptide hormones, luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH), have been isolated from human milk and bovine colostrum. Acidified methanolic extracts, prepared from human milk, bovine colostrum and rat hypothalami, as well as synthetic LHRH and TRH markers were subjected to high-pressure liquid chromatography (HPLC). The eluates were tested for the presence of LHRH and TRH by specific radioimmunoassays. It was found that milk extracts contain significant amounts of LHRH (3.9 - 11.8 ng/ml) and TRH (0.16 - 0.34 ng/ml), which comigrate with the corresponding marker hormones and with those of hypothalamic origin. The HPLC-purified LHRH from both human and bovine milk was bioactive in a dose-response manner similar to synthetic LHRH.

Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing d-instead of l-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane.

Background and Purpose — We sought to determine the cerebroprotective potential of NAP, a synthetic octapeptide related to vasoactive intestinal peptide. Activity-dependent neuroprotective protein mediates some of the protective effects of vasoactive intestinal peptide. The neuroprotective NAP sequence is derived from activity-dependent neuroprotective protein. Methods — Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion by craniotomy and electrocoagulation. After dose-response and time-course experiments, the animals were injected with NAP (3 μg/kg) or vehicle intravenously 1 hour after stroke onset. Another group of rats was injected with the d -amino acid isomer of NAP (D-NAP) and served as a negative control. Rats were examined for motor and behavioral deficits 24 hours to 30 days later, and infarct volumes were determined. The effect of NAP administration on apoptotic death was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase-3 stainings. Results — NAP significantly reduced motor disability and infarct volumes compared with vehicle or D-NAP when tested at 24 hours after stroke onset (9.67±1.4% versus 17.04±1.18% and 19.19±1.9% of hemispheric volume, respectively; P P Conclusions — Our results indicate that the durable cerebroprotection by NAP involves antiapoptotic mechanisms.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcεRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5′-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcεRI stimulus.

Background: α-Bungarotoxin (α-BTX) is a highly toxic snake venom α-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind α-BTX with high affinity, and inhibit its interaction with AChR with an IC 50 of 2 nM. The design of these peptides was based on a lead peptide with an IC 50 of 3×10 −7 M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. Results: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of α-BTX with the lead peptide, as well as structure–function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC 50 values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against α-BTX lethality. Conclusions: Synthetic peptides with high affinity to α-BTX may be used as potential lead compounds for developing effective antidotes against α-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a modified HLA-A2.1/Db-beta2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts.

Clustering of the mast cell function-associated antigen by its specific monoclonal antibody (G63) inhibits the FcepsilonRI-mediated secretory response. The cytosolic tail of the mast cell function-associated antigen contains a SIYSTL stretch, a potential immunoreceptor tyrosine-based inhibition motif. To investigate the possible functional role of this sequence, as well as identify potential intracellular proteins that interact with it, peptides corresponding to residues 4-12 of the mast cell function-associated antigen's N-terminal cytoplasmic domain, containing the above motif, were synthesized and used in affinity chromatography of mast cell lysates. Both tyrosyl phosphorylated and thiophosphorylated mast cell function-associated antigen peptides bound the src homology domain 2 (SH2)-containing tyrosine phosphatases-1 (SHP-1), -2 (SHP-2) and inositol 5'-phosphatase (SHIP), though with different efficiencies. Neither the nonphosphorylated peptide nor its tyrosyl phosphorylated reversed sequence peptide bound any of these phosphatases. Point mutation analysis of mast cell function-associated antigen pITIM binding requirements demonstrated that for SHP-2 association the amino acid residue at position Y-2 is not restricted to the hydrophobic isoleucine or valine. Glycine and other amino acids with hydrophilic residues, such as serine and threonine, at this position also maintain this binding capacity, whereas alanine and acidic residues abolish it. In contrast, SHP-1 binding was maintained only when serine was substituted by valine, suggesting that the Y-2 position provides selectivity for peptide binding to SH2 domains of SHP-1 and SHP-2. These results were corroborated by surface plasmon resonance measurements of the interaction between tyrosyl phosphorylated mast cell function-associated antigen peptide and recombinant soluble SH2 domains of SHP-1, SHP-2 and SHIP, suggesting that the associations observed in the cell lysates may be direct. Taken together these results clearly indicate that the SIYSTL motif present in mast cell function-associated antigen's cytosolic tail exhibits characteristic features of an immunoreceptor tyrosine-based inhibition motif, suggesting it is a new member of the growing diverse family of immunoreceptor tyrosine-based inhibition motif-containing receptors.

Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an evolving immune response. This phenomenon has been associated most notably with the progression of naturally occurring or experimentally induced chronic autoimmune diseases. We have investigated the potential occurrence of epitope spreading in the context of antitumor cytotoxic T cell (CTL) responses using chicken ovalbumin (OVA) as a model antigen. Our results indicate that following rejection of OVA-expressing EG.7 tumor cells effectuated by a CTL response which is induced against the MHC class I-restricted immunodominant epitope OVA257-264, there occurs intramolecular diversification of the CTL response to two additional OVA-derived epitopes, OVA176-183 and OVA55-62, as well as intermolecular spreading to other endogenous tumor-derived determinants. It seems that CTL-mediated tumor cell destruction in vivo favors cross-presentation of additional epitopes with the consequent activation of additional tumor-reactive lymphocytes. The process of epitope spreading in that context has obvious important implications for the design of antigen-specific antitumor immunotherapies.

We report that the vanadium ligandl-Glu(γ)HXM potentiates the capacity of free vanadium ions to activate glucose uptake and glucose metabolism in rat adipocytes in vitro (by 4–5-fold) and to lower blood glucose levels in hyperglycemic rats in vivo (by 5–7-fold). A molar ratio of two l-Glu(γ)HXM molecules to one vanadium ion was most effective. Unlike other vanadium ligands that potentiate the insulinomimetic actions of vanadium,l-Glu(γ)HXM partially activated lipogenesis in rat adipocytes in the absence of exogenous vanadium. This effect was not manifested by d-Glu(γ)HXM. At 10–20 μm l-Glu(γ)HXM, lipogenesis was activated 9–21%. This effect was approximately 9-fold higher (140 ± 15% of maximal insulin response) in adipocytes derived from rats that had been treated with vanadium for several days. Titration of vanadium(IV) withl-Glu(γ)HXM led to a rapid decrease in the absorbance of vanadium(IV) at 765 nm, and 51V NMR spectroscopy revealed that the chemical shift of vanadium(IV) at −490 ppm disappeared with the appearance of a signal characteristic to vanadium(V) (−530 ppm) upon adding one equivalent of l-Glu(γ)HXM. In summary,l-Glu(γ)HXM is highly active in potentiating vanadium-activated glucose metabolism in vitro and in vivo and facilitating glucose metabolism in rat adipocytes in the absence of exogenous vanadium probably through conversion of trace intracellular vanadium into an active insulinomimetic compound. We propose that the active species is either a 1:1 or 2:1l-Glu(γ)HXM vanadium complex in which the endogenous vanadium(IV) has been altered to vanadium(V). Finally we demonstrate that l-Glu(γ)HXM- andl-Glu(γ)HXM·vanadium-evoked lipogenesis is arrested by wortmannin and that activation of glucose uptake in rat adipocytes is because of enhanced translocation of GLUT4 from low density microsomes to the plasma membrane.

The understanding of the molecular mechanisms leading to peptide action entails the identification of a core active site. The major 28-aa neuropeptide, vasoactive intestinal peptide (VIP), provides neuroprotection. A lipophilic derivative with a stearyl moiety at the N-terminal and norleucine residue replacing the Met-17 was 100-fold more potent than VIP in promoting neuronal survival, acting at femtomolar–picomolar concentration. To identify the active site in VIP, over 50 related fragments containing an N-terminal stearic acid attachment and an amidated C terminus were designed, synthesized, and tested for neuroprotective properties. Stearyl-Lys-Lys-Tyr-Leu-NH 2 (derived from the C terminus of VIP and the related peptide, pituitary adenylate cyclase activating peptide) captured the neurotrophic effects offered by the entire 28-aa parent lipophilic derivative and protected against β-amyloid toxicity in vitro . Furthermore, the 4-aa lipophilic peptide recognized VIP-binding sites and enhanced choline acetyltransferase activity as well as cognitive functions in Alzheimer’s disease-related in vivo models. Biodistribution studies following intranasal administration of radiolabeled peptide demonstrated intact peptide in the brain 30 min after administration. Thus, lipophilic peptide fragments offer bioavailability and stability, providing lead compounds for drug design against neurodegenerative diseases.

Healthy persons manifest a high frequency of T cells reactive to epitopes of the self 60-kDa heat-shock protein (hsp60) molecule. It was reasoned that a self hsp60 peptide, p458m, might provide T cell help for a response to the T independent capsular polysaccharide of Streptococcus pneumoniae type 4 (PS4). The conjugate vaccine (PS4-p458m) induced resistance to challenge with >300,000 times the minimal lethal dose of pneumococci. PS4 conjugated to other immunogenic carriers (tetanus toxoid, a pneumolysin peptide, and others) or a commercial pneumococcal vaccine were far less effective. The effectiveness of the PS4-p458m conjugate was associated with an increased IgG1 antibody response to PS4, with long-term memory, and with T cell responses to the p458m peptide. Thus, T cell reactivity to a self epitope in a conjugate vaccine can be mobilized to induce help for resistance to a lethal infection.

Tumor necrosis factor alpha (TNFalpha), an early cytokine produced by activated macrophages, plays an essential role in normal and pathological inflammatory reactions. The excessive production of TNFalpha is prevented by the so-called "macrophage-deactivating factors." This study examines the role of two structurally related neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating peptide (PACAP), as inhibitors of TNFalpha. Both VIP and PACAP inhibit TNFalpha production from lipopolysaccharide-stimulated RAW 246.7 cells in a dose- and time-dependent manner. Although the activated cells express mRNA for all three VIP/PACAP receptors, agonist and antagonist studies indicate that the major receptor involved is VIP1R. VIP/PACAP inhibit TNFalpha gene expression by affecting both NF-kB binding and the composition of the cAMP responsive element binding complex (CREB/c-Jun). Two transduction pathways, a cAMP-dependent and a cAMP-independent pathway, are involved in the inhibition of TNFalpha gene expression and appear to differentially regulate the transcriptional factors involved. Because TNFalpha plays a central role in various inflammatory diseases such as endotoxic shock, multiple sclerosis, cerebral malaria, and various autoimmune conditions, the down-regulatory effect of VIP/PACAP may have a significant therapeutic potential.

Excitotoxic damage may be a critical factor in the formation of brain lesions associated with cerebral palsy. When injected at birth, the glutamatergic analog ibotenate induces mouse brain lesions that strikingly mimic human microgyria. When ibotenate is injected at postnatal day 5, it produces transcortical necrosis and white matter cysts that mimic human perinatal hypoxic-like lesions. Vasoactive intestinal peptide (VIP) has potent growth-related actions and neuroprotective properties that influence mitosis and neuronal survival in culture. The goal of this study was to assess the protective role of VIP against excitotoxic lesions induced by ibotenate in developing mouse brain. VIP cotreatment reduced ibotenate-induced microgyric-like cortical lesions and white matter cysts by up to 77 and 85%, respectively. VIP protective effects were reproduced by a peptide derived from activity-dependent neurotrophic factor (ADNF), a trophic factor released by VIP-stimulated astrocytes, and by stearyl norleucine VIP, a specific VIP agonist that does not activate adenylate cyclase. Neither forskolin, an adenylate cyclase activator, nor pituitary adenylate cyclase-activating peptide, provided VIP-like protection. VIP and neurotrophic analogs, acting through a cAMP-independent mechanism and inducing ADNF release, could represent new avenues in the understanding and prevention of human cerebral palsy.

Mast cells are known to accumulate in various inflammatory processes, some of which are known to be associated with increased local and systemic levels of acute-phase reactants such as serum amyloid A (SAA) or with amyloid deposition. The mechanism(s) by which mast cells are recruited to these sites, however, has not been fully elucidated. It has recently been shown that SAA interacts with extracellular matrix (ECM) components and thereby acts as a chemoattractant and regulator of immune cell migration. On the basis of these observations, we examined the effect of SAA on mast cell adhesion to ECM, an essential step in cellular transmigration. We could first demonstrate strong specific binding of recombinant human SAA (rSAA) to murine mast cells using flow cytometry. Moreover, radiolabeled rSAA was found to bind, in a saturable manner, to mast cells, reaching a binding affinity of 10(-8) M. When immobilized by preincubation with ECM, SAA or its proteolytically degraded amyloid A fragment (amino acid residues 2-82), which contains RGD-related adhesion motif but not the COOH-terminal portion of SAA (amino acid residues 77-104), induced the adhesion of resting mast cells to ECM or laminin. SAA and AA, in soluble or immobilized forms, did not activate mast cells to release mediators. Mast cell adhesion to the immobilized ECM-SAA complex appeared to occur through an integrin recognition, inasmuch as adhesion was calcium dependent and could be blocked by an RGD-containing peptide or by anti-CD29 monoclonal antibody. Genistein also inhibited adhesion, indicating that tyrosine kinase activity was involved. These data suggest that SAA bound to ECM may serve as an important inducer of mast cell adhesion, thus regulating mast cell recruitment and accumulation at these sites, which in turn could potentiate further pathology.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

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.

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.

Human serum amyloid P component (hSAP) and human C-reactive protein (hCRP) are normal serum constituents related to the pentraxin family of plasma proteins. hSAP has morphological and immunochemical identity and extensive sequence similarity to the amyloid P (AP) component found in normal tissues and particularly in amyloid deposits. hCRP and its proteolytic products have been previously shown to bind and to interact with various types of human leukocytes. Binding-displacement experiments with 125I-labeled hSAP and hCRP show that both proteins have specific high-affinity binding sites on normal human polymorphonuclear leukocytes (PMN) and each can compete efficiently with the binding of the other. Scatchard analysis of hSAP-displacement curves reveals a heterogeneous population of hSAP-binding sites existing on the PMN cells, among them about 300,000 low-affinity binding sites with Kdor = 5 x 10(-6) M and about 30,000 high-affinity binding sites with Kdor = 5 x 10(-8) M. hAP was found to be degraded by enzymes from human neutrophils to yield a mixture of low-molecular-mass peptides, similarly to the case of CRP reported previously. The binding of hSAP can be efficiently inhibited by this peptide mixture. The results suggest that both hCRP and hSAP, together with related peptides, may participate in vivo in an unknown mechanism of regulation of human neutrophils.

Synthetic peptides related to amino acid residues 29-42 of human serum amyloid A (SAA), Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-His-Ala-Arg-Gly-Asn-Tyr, were found to inhibit the adhesion of human T-lymphocytes and of mouse M4 melanoma cells to surfaces coated with the major cell adhesive glycoproteins of the extracellular matrix, laminin or fibronectin. Correspondingly inhibitory activity was manifested by the entire 14-residue peptide, by its YIGSD laminin-related domain, and by RGN, the fibronectin-related domain. Intact recombinant SAA (rSAA) and its 1-76 fragment, an amyloid A (AA) protein, also inhibited cell adhesion. The peptides did not inhibit collagen and ADP-induced aggregation of human platelets. Proteolysis of SAA by lysosomal enzymes originating from human neutrophils led to generation of specific peptide segments some of which pertain to the 29-42 domain. It is suggested that the acute-phase protein SAA might be involved, either directly or via its peptide fragments, in inhibition of inflammatory reactions or metastatic processes which depend on integrin and possibly other extracellular-matrix-specific receptors mediated specific recognition and interactions with immobilized components of blood-vessel walls.

Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) have been subdivided into type I receptors and II receptors. Using rat brain stem as a tissue containing type I receptors and rat lung as a tissue containing type II receptors, we investigated the binding of 125 I-labelled PACAP, vasoactive intestinal peptide (VIP) and the related peptide, helodermin to these and, by chemical cross-linking, the relative molecular weight of the receptors. Type I receptors showed an M r , of 58,000 and type II an M r , of 54,000. After deglycosylation type I receptors showed an M r , of 49,000 and type II receptors an M r , of 41,000 demonstrating a high degree of glycosylation of both subtypes

The most prevalent lung cancer, non-small cell lung cancer (NSCLC) has receptors for vasoactive intestinal peptide (VIP). Here the effects of a VIP antagonist (VIP-hyb) on NSCLC growth were investigated. In vivo, when VIPhyb (10 micrograms, s.c.) was daily injected into nude mice, xenograft formation was significantly inhibited by approximately 80%. In vitro, VIP (100 nM) stimulated colony formation approximately 2-fold, whereas 1 microM VIPhyb inhibited colony formation by approximately 50% when adenocarcinoma cell line NCI-H838 was used. The attenuation of tumor proliferation is receptor mediated, as VIPhyb inhibited specific 125I-labeled VIP binding to cell lines NCI-H157 and NCI-H838 with an IC50 of 0.7 microM. VIP (10 nM) increased the cAMP levels 5-fold when cell line NCI-H838 was used, and 10 microM VIPhyb inhibited the increase in cAMP caused by VIP. Northern blot analysis and radioimmunoassays have shown VIP mRNA and VIP-like immunoreactivity in NSCLC cells. These data suggest that VIP may be a regulatory peptide in NSCLC and that VIPhyb is a VIP receptor antagonist that inhibits proliferation.

We read the paper by Trebicz-Geffen published in the April issue of the International Journal of Experimental Pathology with interest (Trebicz-Geffen et al. 2008). In their study, the authors reported molecular analysis of the multiple osteochondroma (MO)-associated EXT1 gene [MO (Bovee & Hogendoorn 2002) was previously known as hereditary multiple exostoses (HME) (Solomon 1964)], and documented their findings with mRNA and protein measurements, and with DNA sequence analysis. As these results are difficult to reconcile with aspects of current understanding of EXT1 function, and with previous studies on EXT1 and EXT2 expression in hereditary MO and solitary osteochondromas (SO), we would like to comment on their results in perspective. Their observation that EXT1 mRNA and protein levels are elevated in patients with MO who carry an EXT1 mutation was surprising. EXT1 has been shown to act as a tumour suppressor gene (Bovee et al. 1999; Bernard et al. 2001). In a subset (but not all) of MO patients, loss of the remaining wild type allele can be shown in the osteochondromas (Bovee et al. 1999). As 70% of the mutations found in EXT1 are non-sense frame shift or splice site mutations, leading to a premature stopcodon (Wuyts & Van Hul 2000), one would expect down-regulation at both mRNA and protein levels in MO. We reported that EXT1 and/or EXT2 mRNA levels are down-regulated in MO as compared with normal human growth plate (Hameetman et al. 2007a), and decreased levels of EXT1 and/or EXT2 protein in the cartilage cap cells of MOs have been reported previously (Bernard et al. 2001). In addition, their observation that normal EXT1 levels are present in SOs is surprising. Somatic mutations of EXT1 and EXT2 are very rare or absent in these tumours (Hecht et al. 1997, Hecht et al. 2002; Bernard et al. 2001). Recently, however, we have shown that homozygous deletion of EXT1 occurs in the cartilaginous cap of SOs (Hameetman et al. 2007b), suggesting that (similar to MOs) EXT1 is inactivated in SOs. These findings suggest that EXT1 mRNA and protein levels are likely to be down-regulated in both conditions, and this has been confirmed at the mRNA level (Hameetman et al. 2007a). The authors do not discuss these discrepancies. They propose that up-regulation of EXT1 is a compensation mechanism for the defective EXT1. This is difficult to reconcile with the alternative, which is that inactivation of EXT1 is causative for osteochondromas. An explanation for these conflicting data may lie in the different tissues that were used in the two studies. Our study used fresh frozen material from the cartilage cap, with confirmed cartilaginous morphology, whereas their study used cultured cartilage cap cells, without clarifying which markers were used to confirm this. It is therefore impossible to exclude that the cultured cells had a fibroblast-like phenotype without evidence about morphology and biochemistry. Furthermore, as control material, we used normal age-matched growth plate, and they used normal cartilage from patients undergoing total joint replacement. EXT1 mRNA expression is high in both human (Hameetman et al. 2007a) and mouse (Stickens et al. 2000) growth plate. Endogenous EXT1 mRNA levels have not been determined previously in human articular cartilage, and have not been documented in elderly people with severe osteoarthritis, which is the most likely source of material for their study. However, high EXT expression would not be expected, because proliferating and prehypertrophic chondrocytes (i.e. the cells that express EXT in the growth plate) are infrequent in articular cartilage. Moreover, postpuberty, one would expect low or absence of expression of EXT in human articular cartilage, as the downstream signalling targets of EXT, such as Wnt, transforming growth factor, and bone morphogenetic protein, are also altered to favour chondrocyte catabolic activity (Hopwood et al. 2007). The immunohistochemical staining of EXT1 in articular cartilage is difficult to evaluate but does not suggest high expression in figures 5 and 6 of the paper. Comparison to low EXT expression in articular cartilage will mean that there appears to be an increase in osteochondromas, whereas comparison with high endogenous EXT1 expression in growth plate cartilage will suggest down regulation. As osteochondromas are presumed to arise from growth plate cartilage (Hameetman et al. 2007b; Clement et al. 2008), we believe that this is the more relevant control. Another explanation, which could account for the difference in findings, may lie in the approach used for the Western blot analysis. Pre-immune sera showed different patterns in normal, SO and hereditary MO samples. The use of a standard more stably expressed internal control, such as alpha tubulin, could overcome this problem and might lead to different conclusions. Thus we suggest that the findings are difficult to interpret based upon current understanding on the function of EXT1, and on comparative expression (Bernard et al. 2001; Hameetman et al. 2007a), and that there are technical issues, including choice of positive controls, which might account for the observations reported.

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.

Normal human monocytes and macrophages generate potent procoagulant activity (PCA) resembling tissue factor (TF) in response to various stimuli. In this study we show that tuftsin, a natural stimulator of many functions of monocytes and macrophages, also stimulates a potent PCA in mixed mononuclear cells and monocytes, and a mild PCA in lymphocytes and cell lines of monocytic origin (U937 and THP). No activity was generated by several lymphoid cell lines and HL-60 cells. The PCA resembled TF in that it accelerated clotting through the extrinsic coagulation pathway and was inhibited by concanavalin-A and by monoclonal anti-TF antibodies. The induction of TF-like activity by tuftsin was dose- and time-dependent. It was located in the cell membrane and did not require T cells for expression. Generation of TF- like activity was prevented by actinomycin D, while cytarabine had no effect on this process, suggesting that expression of the activity depends on protein synthesis. Studies with various tuftsin analogs suggest that tuftsin stimulates generation of TF-like activity, as well as other functions of monocytes via the same receptors. The results with the monocytic cell lines show that tuftsin affects mainly mature cells. The induction of TF-like activity in mononuclear cells by tuftsin constitutes an important link between mononuclear cells and the immune and coagulation systems. It may play a major role in the pathogenesis of thromboembolism and fibrin deposition in various inflammatory and immunologic disorders.

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 antibeta-2-glycoprotein-I (b2GPI) anti-idiotypic antibodies from a commercial IVIG preparation and use it as a treatment in an experimental antiphospholipid syndrome (APS) mouse model. Methods: Antib2GPI polyclonal antibodies were purified on a b2GPI column. The purified antibodies were bound to CN‐Br-activated sepharose and employed for purification of IVIG-anti-anti-b2GPI (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-b2GPI antibodies inhibited human trophoblast cell invasion in vitro. The function was attributed to the Fab portion of the anti-b2GPI Igs, since b2GPIrelated synthetic peptides specific for the Fab part of the anti-b2GPI 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 antib2GPI 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 patient’s early fetal loss.

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple CNS targets. We have synthesized a multifunctional non-toxic, brain permeable iron chelator drug, M-30, possessing propargyl monoamine oxidase (MAO) inhibitory neuroprotective and iron-chelating moieties, from our prototype iron chelator VK-28. In the present study M-30 was shown to possess a wide range of pharmacological activities, including pro-survival neurorescue effects, induction of neuronal differentiation and regulation of amyloid precursor protein (APP) and beta-amyloid (Abeta) levels. M-30 was found to decrease apoptosis of SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via reduction of the pro-apoptotic proteins Bad and Bax, and inhibition of the apoptosis-associated phosphorylated H2A.X protein (Ser 139) and caspase 3 activation. In addition, M-30 induced the outgrowth of neurites, triggered cell cycle arrest in G(0)/G(1) phase and enhanced the expression of growth associated protein-43. Furthermore, M-30 markedly reduced the levels of cellular APP and beta-C-terminal fragment (beta-CTF) and the levels of the amyloidogenic Abeta peptide in the medium of SH-SY5Y cells and Chinese hamster ovary cells stably transfected with the APP 'Swedish' mutation. Levels of the non-amyloidogenic soluble APPalpha and alpha-CTF in the medium and cell lysate respectively were coordinately increased. These properties, together with its brain selective MAO inhibitory and propargylamine- dependent neuroprotective effects, suggest that M-30 might serve as an ideal drug for neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, in which oxidative stress and iron dysregulation have been implicated.