Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.

A thymosin beta 4 ELISA using an antibody against the N terminal fragment thymosin beta 4 [1-14].

A thymosin beta 4 ELISA was developed in which thymosin beta 4, absorbed on microwells, competed with thymosin beta 4 in solution for the binding sites of an anti-thymosin beta 4 antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin beta 4 antibodies were raised in rabbits against intact thymosin beta 4 as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin beta 4[1-14] and thymosin beta 4[1-11]. The antibody against thymosin beta 4[1-14] was used in the thymosin beta 4 ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin beta 9 as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin beta 9, was fast, easy to perform and exhibited good assay characteristics.

A radioimmunoassay for parathymosin alpha using antibodies to synthetic N-terminal peptide 1-30.

Antibodies against the N-terminus of rat parathymosin alpha have been raised in rabbits by conjugating parathymosin alpha (1-30) to hemocyanin. A radioimmunoassay for parathymosin alpha was established by utilizing antibodies against the above polypeptide and parathymosin alpha(1-12)[Tyr] as tracer. The useful range was 5-450 pmol for parathymosin alpha. An epitope was located in the amino acid sequence 1-12. The antiserum failed to crossreact with the same molar concentrations of the partly homologous thymosin alpha 1 or prothymosin alpha. With this radioimmunoassay, parathymosin alpha was isolated from calf thymus after separation from prothymosin alpha by reversed phase HPLC. Endogenous proteases did not appear to generate N-terminal fragments of parathymosin alpha in rat liver extracts in a similar fashion to that observed for prothymosin alpha. Parathymosin alpha has a ubiquitous distribution in the human tissues examined, with levels ranging from 93 (brain) to 1043 (liver) ng of parathymosin alpha(1-30) equivalents/g (wet weight).

The identification of prothymosin alpha-like material in vertebrate lymphoid organs by a radioimmunoassay for the N-terminal decapeptide.

A radioimmunoassay (RIA) is described for the detection and quantitation of prothymosin alpha (ProT alpha), and its N-terminal fragments containing as a minimum the first ten amino acid residues. This range of peptides includes thymosins alpha 1 (T alpha 1) and alpha 11 (T alpha 11). Antibodies against T alpha 1 and the tracer T alpha 1(1-10)Tyr11(125I), an analogue of the major epitope, were utilized in this RIA. 50% displacement of the ligand was observed with 1.3 pmol of T alpha 1 and 6.4 pmol of ProT alpha. The partially homologous parathymosin alpha (ParaT alpha) showed less than 2% crossreactivity with ProT A. Sephacryl S-200 gel filtration separation of the peptides of calf thymus, chicken spleen and trout spleen extracts prepared by a method eliminating proteolysis, combined with the above RIA, showed the presence of a major immunoreactive peak. Its elution volume corresponded to that of rat ProT alpha (apparent mol. weight 36,000) for both calf (37,000) and chicken (35,000) tissues. In trout it corresponded to a significantly higher molecular weight (62,000). No detectable levels of shorter fragments, including T alpha 1, were observed in any of the above species. The levels of ProT alpha-like peptides in calf thymus, chicken spleen and trout spleen were found to be 246, 8.6 and 7.7 micrograms respectively, of rat ProT alpha equivalents per gram of fresh tissue. The significance of the presence of ProT alpha-like polypeptides in vertebrate species as distant as fish and mammals, the absence of short T alpha 1-like fragments, and the relative conservation of the N-terminus as suggested by the RIA is discussed.

Radioiodinated biotin derivatives for in vitro radioassays.

The synthesis of two radioiodinated biotin derivatives with the biotin-ureido group intact is described. This synthesis was performed by coupling (pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to tyramine, which was radioiodinated prior to this using a modified chloramine-T method. Two derivatives were produced, the N-[beta-(4-OH-3-125I-phenyl)ethyl] and the N-[beta-(4-OH-3,5-di 125I-phenyl)ethyl] biotin amides, depending on the amount of tyramine used in the radioiodination reaction. The final products were separated by thin layer chromatography (n-butanol: 2N NH4OH: ethanol, 3:1:1, v/v/v). The radioiodinated derivatives that were synthesized or their resulting mixture were found to complete with biotin for the avidin-binding sites; thus, they were capable of being used as tracers in biotin radioassays. The specific activity of their mixture was high-greater than 350 Ci/mmol-and they were stable for 2 mo at 4 degrees C.

Colorimetric determination of reactive solid-supported primary and secondary amino groups.

A simple and sensitive method for the quantitative determination of solid-supported primary and/or secondary amino groups using commercially available reagents is described. The solid supports are treated in an aqueous environment with either 2-iminothiolane (ITL) or sulpho-succinimidyl-3-(4-hydroxyphenyl)propionate (sulpho-SHPP), which introduce one sulphydryl or one hydroxyphenyl group per amino group reacted, respectively. These groups are capable of reducing Cu2+ to Cu+ in alkaline medium. Thus, after removal of the excess reagents through washing, subsequent incubation of the solids with 2,2'-bicinchoninic acid (BCA) copper protein reagent results in production of Cu+ in the solution, which forms a chelate complex with BCA absorbing at 562 nm. The quantitation of the groups introduced on the surfaces, and therefore of the reacted amino groups, is carried out through standard curves of cysteine solutions for ITL, or tyrosine solutions for sulpho-SHPP-treated solids. Using ITL, only the primary amino groups are determined, whereas sulpho-SHPP provided the primary and secondary reactive amino groups. The method is versatile and can be used for the estimation of amino groups onto several biomedical solid matrices, and should provide useful information for the covalent immobilization of ligands (e.g. drugs, antibodies).

Development of specific anti-thymosin beta 10 antipeptide antibodies for application in immunochemical techniques.

We present theoretical and experimental data necessary for raising specific antibodies for thymosin beta 10, a 43-amino acid residues peptide occurring in human tissues. We postulate that thymosin beta 10 contains three major antigenic determinants (residues 2-8, 17-25, and 35-41). For antibody development, we synthesized the N-terminal fragment thymosin beta 10(1-16) as well as the C-terminal fragments thymosin beta 10(31-43) and thymosin beta 10(38-43), due to their putative antigenic properties and minimal structural similarity with the homologous peptide thymosin beta 4, which also occurs in humans. The putative antigenic determinant 17-25 is present in all beta-thymosins and was therefore not synthesized. All antisera raised against the above peptide fragments or the intact molecule of thymosin beta 10 were found capable of recognizing and binding synthetic or natural thymosin beta 10 with high specificity, showing minimal cross-reactivity with thymosin beta 4 isolated from bovine tissues or synthetic thymosin alpha 1. Due to its easy preparation and the highly specific affinity of the antibody raised against it for the intact peptide, the fragment thymosin beta 10(38-43) may be considered the antigen of choice for developing anti-thymosin beta 10 antibodies, which can eventually be applied to immunochemical studies.

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.

Differential expression of thymosins beta(4) and beta(10) during rat cerebellum postnatal development.

The beta-thymosins are a family of actin monomer-sequestering proteins widely distributed among vertebrate classes. The most abundant beta-thymosins in mammalian species are thymosin beta(4) (Tbeta(4)) and thymosin beta(10) (Tbeta(10)), two small peptides (43 amino acids) sharing a high degree of sequence homology. In the present work, we have analyzed the distribution of Tbeta(4) and Tbeta(10) in the developing and adult rat cerebellum using in situ hybridization and immunohistochemistry techniques. Our results show that the temporal and cellular patterns of expression of both beta-thymosins are different. In the young (7 and 18 postnatal days) and adult (1 and 4 months old) rat cerebellum, Tbeta(4) was mainly expressed in the glia (microglia, Golgi epithelial cells and oligodendrocytes), neurons (granule cells and Purkinje cells), and in the capillaries. In 14-month-old rats, the Tbeta(4) immunoreactivity was only detected in some microglia cells. In young and adult animals, most of the Tbeta(10) immunoreactivity was localized in several types of neuronal cells including granule cells, Golgi neurons and Purkinje cells. In old animals, a faint Tbeta(10) signal could be detected in a few Purkinje cells. Our results suggest that each beta-thymosin could play a different function in the control of actin dynamics.

A 125I-radioimmunoassay for diethylstilbestrol in serum of patients with prostatic cancer treated with stilphostrol.

A new radioimmunoassay for determining diethylstilbestrol in serum using N-(4'-OH-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)-hex anamide as a radiotracer and a double antibody as a separation reagent is described. The radiotracer is prepared by synthesizing 6-(4-O-diethylstilbestryl)-hexanoic acid and coupling its succinimidyl ester with mono-[125I]tyramine in tetrahydrofuran (16 h, 20-22 degrees C). The standard curve is linear (semi-log transformation) and the assay is sensitive (< 0.022 pmol/tube), reproducible (intra- and interassay coefficient of variation values, 5.3 and 8.1%, respectively), and accurate (recovery values, 95-101%), with a non-specific binding less than 3.2%. Diethylstilbestrol concentrations measured in sera of nine patients with prostatic cancer by the proposed assay ranged from 0.170 to 2.517 mumol/l, which corresponded to an only three-fold dosage variation. In all cases tested, dosing was adequate to retain markers of prostatic cancer in serum within accepted limits; nevertheless, individualization of dosing may be necessary to minimize toxicity.

Analytical techniques for determining biotin.

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Preparation of the N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)hexanam ide for diethylstilbestrol radioimmunoassays.

The synthesis of a radioiodinated diethylstilbestrol (DES) derivative is described. This derivative was prepared by coupling the previously synthesized active ester of 6-(4-O-diethylstilbestryl)hexanoic acid with mono-[125I]iodotyramine in dry tetrahydrofuran (20 to 22 C, 16 hours). The mono-[125I]iodotyramine was prepared using a chloramine-T method and purified by paper electrophoresis. The final product, N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O- diethylstilbestryl)hexanamide, was separated by thin-layer chromatography (cyclohexane/ethanol/NH4OH 2.5 N/acetone; 40:50:5:20, v/v/v/v); it was stable for 2 months in ethanol at 4 C and had a specific activity higher than 540 Ci/mmol. The [125I]DES amide synthesized was found to retain the immunoreactivity of DES, since it competed with [3H]DES or DES in an in vitro radioimmunoassay system for the binding sites of a rabbit anti-DES antibody; thus, it seems to be capable of replacing the tritiated tracer used so far in DES radioimmunoassays.

Direct radioimmunoassay for diethylstilbestrol in serum.

A radioimmunoassay method for the measurement of diethylstilbestrol directly on 50 microliters of human serum was established using a tritium-labeled radioligand and a double antibody as a separation reagent. The assay was sensitive (less than 0.17 micrograms/L), reproducible (intra-assay and interassay coefficient of variation values, 8.14% and 8.25%, respectively), and accurate (recovery up to 95%). Factors influencing the assay are identified and discussed.

Determination of serum biotinidase activity with biotinyl derivatives of lodotyramines as substrates.

We synthesized biotinylated mono- and di-iodotyramine and their radioactive counterparts and used these substances as substrates to estimate serum biotinidase activity in a radioassay system. The Km values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 microM, respectively, whereas, the maximum velocities of the enzymatic reaction were 27.0 and 8.7 nmol.min-1.mL-1, respectively. Both substrates competed with biocytin for the same active site of the enzyme and the Ki values were 7.30 and 9.56 microM for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [125I]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the well-established colorimetric method (r = 0.9377, n = 31). However, for routine use, the assay may be accomplished by diluting the radiotracer with biocytin instead of its "cold" counterpart, because it is a commercially available reagent. The values obtained in this case were very well correlated with those determined by the colorimetric assay as well (r = 0.9289, n = 31).

Technetium labeled bombesin-like peptide: preliminary report on breast cancer uptake in patients.

Bombesin-like peptides are neurotransmitters and cancer growth factors. Several tumors, breast cancer among them, show one or more than one of the three known bombesin receptors. We have synthesized and labeled with technetium 99m a new pentadecapeptide, analogue to the leu13 amphibian bombesin (99mTc BN). Labeling yield was 83 +/- 4%. Prone Scintimammography was performed on five patients affected by breast cancers (T categorization: two T1b and three T1c), after injecting 0.7 mg, 185 to 296 MBq (5 to 8 mCi) of the peptide. Total body scan did not show free technetium biodistribution. No adverse reaction was observed. Prone Scintimammography with 99mTc Sestamibi (99mTc SM) was also performed few days later. 99mTc BN detected all 5 cancers, whereas 99mTc SM only four: all the T1c and one T1b cancer. Two of them showed axillary node invasion that was detected by both the radiotracers. A fibroadenoma present on contralateral breast to the one with cancer, was not detected neither by 99mTc SM nor by 99mTc BN. Tumor/breast normal tissue ratio (T/B) was constantly higher with 99mTc BN than with 99mTc SM. Maximal T/B was measured as 1.79 with 99mTc SM and 2.25 with 99mTc BN 5 min after fast i.v. administration. In conclusion our 99mTc BN is taken up by primary breast cancer showing higher T/B than 99mTc SM (p < 0.01). In our limited scale, 99mTc BN appears to be safe and, in our limited scale, even more accurate than 99mTc SM for detecting breast cancer.

Synthesis, chemical, radiochemical and radiobiological evaluation of a new 99mTc-labelled bombesin-like peptide.

A new pentadecapeptide bombesin analogue was prepared by Fmoc synthesis, purified by HPLC and identified by electron ionization mass spectrometry. The biological activity of the new peptide was tested on isolated human colonic muscle cells and compared to native bombesin. Labelling of the new biomolecule with Tc-99m yielded a single radioactive species which remained stable at room temperature for eight hours. In a binding assay, the radiolabelled peptide showed high affinity for oat-cell carcinoma (Kd = 9.8 nM) and colorectal adenocarcinoma (Kd = 27.2 nM). Biodistribution studies, performed in normal rodents, indicated uptake by organs that normally express bombesin receptors, such as liver, intestines and kidneys. Scintigraphic studies, performed in nude mice transplanted with small cell lung carcinoma and colon cancer cells, showed significant tumor uptake two hours p.i. The new synthetic pentadecapeptide appears to have promise for several malignancies, including oat-cell lung carcinoma, colorectal cancer and gastroenteropancreatic (GEP) tumors.

Effect of copolymer composition on the physicochemical characteristics, in vitro stability, and biodistribution of PLGA-mPEG nanoparticles.

The physicochemical properties, the colloidal stability in vitro and the biodistribution properties in mice of different PLGA-mPEG nanoparticle compositions were investigated. The nanoparticles were prepared by a precipitation-solvent evaporation technique. The physical characteristics and the colloidal stability of the PLGA-mPEG nanoparticles were significantly influenced by the composition of the PLGA-mPEG copolymer used to prepare the nanoparticles. PLGA-mPEG nanoparticles prepared from copolymers having relatively high mPEG/PLGA ratios were smaller and less stable than those prepared from copolymers having relatively low mPEG/PLGA ratios. All PLGA-mPEG nanoparticle compositions exhibited prolonged residence in blood, compared to the conventional PLGA nanoparticles. The composition of the PLGA-mPEG copolymer affected significantly the blood residence time and the biodistribution of the PLGA-mPEG nanoparticles in liver, spleen and bones. The in vivo behavior of the different PLGA-mPEG nanoparticle compositions did not appear to correlate with their in vitro stability. Optimum mPEG/PLGA ratios appeared to exist leading to long blood circulation times of the PLGA-mPEG nanoparticles. This may be associated with the effects of the mPEG/PLGA ratio on the density of PEG on the surface of the nanoparticles and on the size of the nanoparticles.

Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGA-mPEG nanoparticles.

The effect of nanoparticle dose on the biodistribution and pharmacokinetics of conventional PLGA and stealth poly(Lactide-co-glycolide)-monomethoxypoly(ethyleneglycol) (PLGA-mPEG) nanoparticles was investigated. The precipitation-solvent diffusion method was used to prepare PLGA and PLGA-mPEG nanoparticles labeled with 125I-cholesterylaniline. These were administered intravenously (i.v.) in mice and at predetermined time intervals the animals were sacrificed and their tissues were excised and assayed for radioactivity. Within the dose range applied in this study, blood clearance and mononuclear phagocyte system (MPS) uptake of the PLGA nanoparticles depended on dose whereas they were independent of dose in the case of the PLGA-mPEG nanoparticles. Increasing the dose, decreased the rates of blood clearance and MPS uptake of the PLGA nanoparticles, indicating a certain degree of MPS saturation at higher doses of PLGA nanoparticles. The dose affected the distribution of PLGA nanoparticles between blood and MPS (liver) but it did not affect the nanoparticle levels in the other tissues. Within the range of doses applied here, the PLGA nanoparticles followed non-linear and dose-dependent pharmacokinetics whereas the PLGA-mPEG nanoparticles followed linear and dose-independent pharmacokinetics. In addition to the prolonged blood residence, the dosage-independence of the pharmacokinetics of the PLGA-mPEG nanoparticles would provide further advantages for their application in controlled drug delivery and in drug targeting.

Breast cancer takes up 99mTc bombesin. A preliminary report.

BACKGROUND: Several tumors including lung, prostate, ovarian, colon, and exocrine pancreatic cancer show receptors for the amphibian neurotransmitter and growth factor bombesin (BN) and its mammalian counterparts gastrin-releasing peptide and neuromedin B. Also breast cancer has been reported to show such receptors: the presence of BN receptors in primary breast cancer has been demonstrated on cultured cells and by autoradiography on breast tissue samples. Authors who have studied BN receptors in breast cancer do not agree on their frequency in primary cancer, but indicate that 100% of metastatic breast cancers show such receptors. METHODS: We examined three primary breast cancer patients with 99mTc BN and 99mTc sestamibi one week before surgery. One of them showed axillary node invasion. The same acquisition technique was used for breast and chest imaging with both radiopharmaceuticals, whereas total body images were acquired only with 99mTc BN. Also the administered radioactivity was different: 20 mCi of 99mTc sestamibi and 5-8 mCi of 99mTc BN. Dynamic images were acquired for 20 mins after iv injection with the patient in ventral decubitus and the gamma camera positioned in a lateral view, as is generally done in Khakhali's prone scintimammography. Anterior chest images were acquired for 30 mins. Prone scintimammography was performed one hour after administration of both tracers. ROIs were drawn on tumors and surrounding breast with the same technique in order to calculate the tumor to breast ratio (T/B). In addition, total body scan was performed one hour and three hours after 99mTc BN administration. All three patients underwent breast conserving surgery with lymphadenectomy. Postoperative pathologic assessment showed the following T and N stages in the three patients: T1bN0, T1c-N0, and T1cN1. RESULTS: All three cancers were imaged with both tracers. The T/B of 99mTc BN was always higher than that of 99mTc sestamibi. Chest uptake was always much higher with 99mTc sestamibi than with 99mTc BN. Comparison between 99mTc BN and 99mTc sestamibi images gave other intriguing results: in the N1 patient both tracers clearly imaged the invaded node, but on the 99mTc BM image the primary tumor was larger than on the 99mTc sestamibi image and the node was smaller. It is known that 99mTc BN is not taken up by vessels and inflammatory tissue. The time activity curves of the two tracers were significantly different in all patients, with an increase in 99mTc BN uptake in the first three to five minutes, followed by a less sharp uprise of the curve, quite similar to a plateau. CONCLUSIONS: Our first impression is that 99mTc BN is a useful breast cancer seeking agent and very promising for lymph node staging.