Targeted Delivery of Cyclotides via Conjugation to a Nanobody.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

Charting novel allergens from date palm pollen (Phoenix sylvestris) using homology driven proteomics.

Pollen grains from Phoenix sylvestris (date palm), a commonly cultivated tree in India has been found to cause severe allergic diseases in an increasing percentage of hypersensitive individuals. To unearth its allergenic components, pollen protein were profiled by two-dimensional gel electrophoresis followed by immunoblotting with date palm pollen sensitive patient sera. Allergens were identified by MALDI-TOF/TOF employing a layered proteomic approach combining conventional database dependent search and manual de novo sequencing followed by homology-based search as Phoenix sylvestris is unsequenced. Derivatization of tryptic peptides by acetylation has been demonstrated to differentiate the 'b' from the 'y' ions facilitating efficient de novo sequencing. Ten allergenic proteins were identified, out of which six showed homology with known allergens while others were reported for the first time. Amongst these, isoflavone reductase, beta-conglycinin, S-adenosyl methionine synthase, 1, 4 glucan synthase and beta-galactosidase were commonly reported as allergens from coconut pollen and presumably responsible for cross-reactivity. One of the allergens had IgE binding epitope recognized by its glycan moiety. The allergenic potency of date palm pollen has been demonstrated using in vitro tests. The identified allergens can be used to develop vaccines for immunotherapy against date palm pollen allergy. THE SIGNIFICANCE OF THE STUDY: Identification of allergenic proteins from sources harboring them is essential in developing therapeutic interventions. This is the first comprehensive study on the identification of allergens from Phoenix sylvestris (date palm) pollen, one of the major aeroallergens in India using a proteomic approach. Proteomic methods are being increasingly used to identify allergens. However, since many of these proteins arise from species which are un-sequenced, it becomes difficult to interpret those using conventional proteomics. Date palm being an unsequenced species, the IgE-reactive proteins have been identified using a stratified proteomic workflow incorporating manual de novo sequencing and homology-based proteomics. This study also gives an insight into the presence of glycan nature of the IgE binding epitopes. Five proteins have been found to be common with coconut pollen allergens and presumably responsible for cross-reactivity. These can be used in diagnostics to differentiate patient cohorts allergic to both coconut and date palm pollen from true date palm pollen allergic subjects. This would also determine better specific immunotherapy regimes between the two cohorts. The allergens identified herein have potential towards vaccine development in date palm pollen allergy as well as in enriching the existing catalogue of allergenic proteins.

Hepatoprotective effect of Arctium lappa root extract on cadmium toxicity in adult Wistar rats.

This study was performed to determine the effects of Arctium lappa (Al) to protect against cadmium damage in the rat liver. Male rats received a single i.p. dose of CdCl2 (1.2 mg/kg body weight (BW)) with or without Al extract administered daily by gavage (300 mg/kg BW) for 7 or 56 days. After 7 days, Al caused plasma transaminase activity to diminish in groups Al (glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)) and CdAl (GPT). After 56 days, GOT and GPT plasma activities were reduced in the Cd group. No alteration in plasma levels of creatinine, total bilirubin, and total protein were observed. GOT liver activity increased in the Cd group. No alteration was observed in superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and malondialdehyde (MDA) dosage. In the Cd group, hepatocyte proportion decreased and sinusoid capillary proportion increased. In the Al and CdAl groups, the nuclear proportion increased and the cytoplasmic proportion decreased. The hepatocyte nucleus density reduced in Cd and increased in the Al group. After 56 days, there was no alteration in the Cd group. In Al and CdAl groups, the nuclear proportion increased without cytoplasmic proportion variation, but the sinusoid capillary proportion was reduced. The hepatocyte nucleus density decreased in the Cd group and increased in the Al and CdAl groups. In conclusion, the liver function indicators showed that A. lappa protected the liver against cadmium toxicity damage.

Lunasin is prevalent in barley and is bioavailable and bioactive in in vivo and in vitro studies.

Lunasin, a unique 43-amino acid peptide found in a number of seeds, has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model. To elucidate the role of cereals in cancer prevention, we report here the prevalence, bioavailability, and bioactivity of lunasin from barley. Lunasin is present in all cultivars of barley analyzed. The liver and kidney of rats fed with lunasin-enriched barley (LEB) show the presence of lunasin in Western blot. Lunasin extracted from the kidney and liver inhibits the activities of HATs (histone acetyl transferases), yGCN5 by 20% and 18% at 100 nM, and PCAF activity by 25% and 24% at 100 nM, confirming that the peptide is intact and bioactive. Purified barley lunasin localizes in the nuclei of NIH 3T3 cells. Barley lunasin added to NIH 3T3 cells in the presence of the chemical carcinogen MCA activates the expression of tumor suppressors p21 and p15 by 45% and 47%, decreases cyclin D1 by 98%, and inhibits Rb hyperphosphorylation by 45% compared with the MCA treatment alone. We conclude that lunasin is prevalent in barley, bioavailable, and bioactive and that consumption of barley could play an important role of cancer prevention in barley-consuming populations.

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut.

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E(2) (PGE(2)) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE(2) increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.

Adaptation and performance of an immuno-PCR assay for the quantification of Aviscumine in patient plasma samples.

An immuno-polymerase chain reaction (IPCR) assay is used to evaluate the kinetic behaviour of the novel anti-cancer drug Aviscumine in plasma samples taken from 41 patients during a 3-year clinical trial. The ultrasensitive IPCR assay employed the amplification of a detection-antibody linked marker-DNA and an internal competitor DNA for standardization, thus enabling the detection of the antigen in concentrations far below the detection limit of conventional enzyme-linked immuno-sorbent assay (ELISA). The quantification of Aviscumine was carried out using external calibration curves obtained from individual patient plasma samples, collected previous to the administration of Aviscumine, which were spiked with known amounts of the reference substance Aviscumine. Additional controls were measured containing standardized human serum spiked with Aviscumine to assure the continuous general reproducibility of the assay as well as to estimate differences between individual patients. Average recovery was found to be 95+/-19% and the average deviation in precision of the assay was determined to be 9+/-5%. Data for the quantification of Aviscumine were obtained from all patient samples investigated with the exception of a single patient. The collected data provided the basis for the valid routine quantification of patient samples for the calculation of the pharmacokinetic behaviour of Aviscumine in patient plasma.

Allergen skin test reaction patterns in children (

BACKGROUND: Genetic studies of atopy rely upon evidence of abnormal IgE production, usually elevated total IgE or skin prick test (SPT) reactions. However, these measures may change with subject age. METHODS: We screened 1,099 members of atopic families (aged 6-87 years) by serum total IgE and SPT for 14 allergens. For those SPT negative, we screened for Amb a 1- and Der p 1-specific IgE. Der p 1 IgE-Der p 1 allergen binding affinities were done on randomly selected subjects. RESULTS: There were significantly fewer atopics 10 years old (75.8%) based upon any SPT-positive result. Children 10 years old = 82.3%). Among those SPT-positive for house dust mite extract, there was a positive correlation between Der p 1 binding affinity and the wheal area of the house dust mite extract. There was a positive correlation between the number of SPT-positive reactions and total IgE for both age groups. However, there was only a significant relationship between SPT-positive wheal area and total IgE for those >10 years old and no apparent relationship between wheal area and total IgE for those

Tumor-associated trypsin inhibitor.

Tumor-associated trypsin inhibitor (TATI) is a low-molecular-weight (6 kDa) trypsin inhibitor that has been used as a marker for ovarian cancer. It is also expressed together with tumor-associated trypsin by many other tumors, and increased serum concentrations of TATI occur in connection with these. TATI is a prognostic marker for ovarian, bladder, and kidney cancer, which may be associated with the participation of trypsin in protease cascades contributing to tumor invasiveness.

Pharmacokinetic features, immunogenicity, and toxicity of B43(anti-CD19)-pokeweed antiviral protein immunotoxin in cynomolgus monkeys.

We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral protein (PAP) immunotoxin in 13 cynomolgus monkeys. The disposition of B43-PAP in two monkeys, when administered as a single i.v. bolus dose, was characterized by a slow clearance (1-2 ml/h/kg) with a very discrete peripheral distribution. B43-PAP was retained and distributed largely in the blood as the sole compartment with no significant equilibration with the extravascular compartment. The circulating B43-PAP immunotoxin detected in monkey plasma samples by ELISA and protein immunoblotting was both immunoreactive with, and active against, human leukemic cells in vitro. In systemic immunogenicity and toxicity studies, which involved 11 cynomolgus monkeys, each monkey received a total of seven i.v. doses of B43-PAP at a specific dose level of the dose escalation schedule. B43-PAP-treated monkeys mounted a dose-dependent humoral immune response against both the mouse IgG and PAP moieties of the immunotoxin. When administered i.v. either on an every-day or every-other-day schedule, B43-PAP was very well tolerated, with no significant clinical or laboratory signs of toxicity at total dose levels ranging from 0.007 to 0.7 mg/kg. A transient episode of a mild capillary leak with a grade 2 hypoalbuminemia and 2+ proteinuria was observed at total dose levels equal to or higher than 0.35 mg/kg. At total dose levels of 3.5 and 7.0 mg/kg, B43-PAP caused dose-limiting renal toxicity due to severe renal tubular necrosis. The present study completes the preclinical evaluation of B43-PAP and provides the basis for its clinical evaluation in children with therapy-refractory B-lineage acute lymphoblastic leukemia.

Fate of cadmium bound to phytochelatin in rats.

The fate cadmium(Cd) bound to phytochelatin [PC, (gamma-Glu-Cys)n-Gly)] was studied in rats using synthesized 109Cd-PC. Less Cd was absorbed through the digestive tracts than CdCl2, but the ratio of renal Cd to hepatic Cd was higher. After parenteral administration of Cd-PC, Cd was distributed mainly in the liver, kidney, small intestine and pancreas. More Cd was found in the kidney than the liver after Cd-PC (n = 5) administration. Most of the Cd was bound to the high molecular weight fraction in the hepatic cytosol 0.5 hr after administration and moved to the metallothionein fraction at 6 hr. The tissue distribution of Cd was not affected even when free PC (n = 5) was administered 3 hr after or before Cd injection. The distribution in the kidney increased only in the case of the simultaneous administration of Cd with PC. These findings show that the absorbance of Cd bound to PC from the alimentary tract is lower than that of CdCl2 although absorbed Cd is distributed to the kidney more than CdCl2, and Cd is liberated from PC soon after uptake by the cells.

A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator.

A full length cDNA of a barley leaf messenger, found to increase in amount during infection attempts by the powdery mildew fungus (Erysiphe graminis), is characterized. The messenger encodes a polypeptide of 261 amino acid residues with a calculated mass of 29.2 kDa and a pI of 4.6. Sequence comparisons as well as serological studies demonstrate that the encoded protein is closely related to a family of mammalian proteins believed to have functions associated with the multifunctional Ca(2+)-dependent protein kinases.

Blood clearance and organ distribution and tissue concentration of native, homopolymerized and IgG-conjugated ribosome-inactivating proteins.

1. The blood clearance, organ distribution and tissue concentration of several native, homopolymerized, and IgG-conjugated 125I-labelled ribosome-inactivating proteins (RIPs) were determined in mice. 2. Native RIPs were cleared rapidly from blood, with half-lives of 4-8 min, and were concentrated mainly in the kidneys. 3. After conjugation to IgG the three RIPs studied showed increased blood half-lives and decreased concentrations in the kidneys. 4. The two homopolymers studied had blood half-lives and kidney concentrations intermediate to those of free and conjugated RIPs. 5. These results indicate that after IgG-conjugation the increased half-lives of the RIPs studied were at least in part due to the larger molecular size of the conjugates and to their lower renal excretion.

The prolamin antibody reactivity against hordein polypeptides in sera from patients with coeliac disease.

The antibody reactivity against the barley prolamin, hordein, was investigated by an immunoblotting technique, in sera from patients with untreated coeliac disease, patients with other gastrointestinal diseases and healthy controls. No characteristic hordein polypeptide antibody pattern could be connected to coeliac disease, as observed in a similar study using different fractions of the wheat prolamin, gliadin. Gliadin- and hordein-immunoadsorbent column experiments demonstrated that the prolamin reactivity originates from the same population of antibodies. It is speculated that distinct antigenic epitopes characteristic for untreated coeliac disease, might reside within a N-terminal repeat region of gliadin.

Radioimmunoassays of abrin and ricin in blood.

Radioimmunoassays for abrin and ricin are described. There is little cross-reactivity between the two toxins. The procedures described are capable of determining blood concentrations down to 50-100 pg/ml, permitting identification of abrin and ricin poisoning and monitoring of the blood concentrations in cancer patients treated with these agents.