Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer

Purpose Our objective was to study the cellular and nuclear uptake of ¹²³I-mouse IgG (¹²³I-mIgG) linked to peptides [GRKKRRQRRRPPQGYGC] harbouring the membrane-translocating and nuclear import sequences of HIV-1 tat protein.Methods Carbohydrates on mIgG were oxidized by NaIO₄, then reacted with a 40-fold excess of peptides. Displacement of binding of anti-mouse IgG (Fab specific; -mFab) to ¹²³I-mIgG by tat-mIgG or mIgG was compared. Internalization and nuclear translocation of ¹²³I-tat-mIgG in MDA-MB-468, MDA-MB-231 or MCF-7 breast cancer cells were measured. The immunoreactivity of imported tat-mIgG was evaluated by measuring binding of ¹²³I--mFab to cell lysate and by displacement of binding of ¹²³I-mIgG to -mFab by cell lysate. Biodistribution and nuclear uptake of ¹²³I-tat-mIgG, ¹²³I-mIgG and ¹²³I-tat were compared in mice bearing s.c. MDA-MB-468 tumours.Results There was a 15-fold decrease in affinity of -mFab for tat-mIgG compared with mIgG. Internalized radioactivity imported into the nucleus for ¹²³I-tat-mIgG in MDA-MB-468, MDA-MB-231 and MCF-7 cells was 61.5±0.6%, 60.3±3.6% and 64.7±1.0%, respectively. The binding of ¹²³I--mFab to lysate from MDA-MB-468 cells importing tat-mIgG was 17-fold higher than that for cells not exposed to tat-mIgG. Imported tat-mIgG competed with tat-mIgG for displacement of binding of ¹²³I-mIgG to -mFab. Conjugation of mIgG to tat peptides did not change tissue distribution. Nuclear localization for ¹²³I-tat-mIgG in MDA-MB-468 tumours was 28.1±5.6%, and for liver, spleen and kidneys it was 41.7±2.7%, 13.8±0.8% and 36.9±3.3%, respectively.Conclusion ¹²³I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes.     

Effect of adrenergic receptor ligands on metaiodobenzylguanidine uptake and storage in neuroblastoma cells

The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [¹²⁵I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were incubated for 15 min with varying concentrations of -agonist (clonidine, methoxamine, and xylazine), -antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), -antagonist (proranolol, atenolol), -agonist (isoprenaline and salbutamol), mixed / antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [¹²⁵I]MIBG (0.1 M). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [¹²⁵I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [¹²⁵I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. However, incubation with the other -agonists or antagonists failed to elicit significant reductions in uptake. In contrast, all of the -agonists significantly inhibited uptake (P<0.05); guanethidine >xylazine >clonidine=methoxamine. The -antagonists demonstrated a broad range of inhibition (phenoxybenzamine phentolamine prazosin yohimbine=tolazoline)(P<0.05). The mixed ligand, labetalol, inhibited MIBG uptake in a dose-dependent manner with an apparent IC₅₀ of 0.65 M. The retention studies demonstrated that unlabeled MIBG caused profound self-inhibition (P<0.01). Clonidine produced a modest inhibition of retention and yohimbine had no effect. Labetalol, phenoxybenzamine, guanethidine, and propranolol reduced uptake of [¹²⁵I]MIBG by neuroblastoma cells in culture. Although only labetalol has been reported to cause false-negative MIBG scans, our results suggest that these other drugs have the potential to interfere with MIBG imaging and therapy, particularly at high doses. Adrenergic drugs did not alter cytoplasmic retention of [¹²⁵I]MIBG in neuroblastoma cells but may have potential in tumors such as phenochromocytoma, where granular storage of MIBG has been observed. Inhibition of [¹²⁵I]MIBG retention by unlabeled MIBG supports the use of high specific activity radioiodinated MIBG for both diagnosis and therapy.     

Unexpected differences in early thyroidal trapping of iodide and pertechnetate

Compartmental analysis was applied to simultaneously acquired ¹³²I-iodide and ^99mTc-pertechnetate thyroid uptake data. The method allowed for arterio/venous differences in plasma tracer level and for an instantaneous phase of thyroid uptake.Observations in four thyrotoxic patients, before and during antithyroid drug therapy, revealed greater instantaneous uptake of TcO ₄ ^- . The results also revealed that instantaneous uptake of both I^– and TcO ₄ ^– may increase over the first six months of drug therapy. These findings could not be explained by the estimates of unidirectional clearance which were greater for I^- and varied little during the early stages of drug therapy.     

Genetic polymorphism of Alpha-2-HS glycoprotein in Lombardy (Italy)

Summary 2HS-subtypes have been analysed in samples from 700 unrelated individuals from the Brescia area (Italy) by the isoelectric focusing technique and immunofixation. The observed allele frequencies were: 2HS*1= 0.7472; 2HS*2 = 0.2507; 2HS*V = 0.0021.     

Detection of α S1-casein in vomit from bottle-fed babies by enzyme-linked immunosorbent assay

Summary This study describes a sensitive enzyme-linked immunosorbent assay (ELISA) using rabbit anti-bovine S1-casein antibody for the detection of commercial milk and milk-containing vomit. The antibody does not react with other human body fluids such as breast milk. The stability of S1-casein antigenic activity was examined after storage at different temperatures and enzyme digestion. There was no decrease after storage for one year at room temperature but 40% of the activity was lost after 6 months at 37°C. Enzyme digestion (6 hours, 37°C) resulted in 65 70% loss of activity but the antibody reacted with the peptide fragments of S1-casein. Vomit samples from 3 normal infants were tested by ELISA, and a S 1-casein could be detected in 1 cm² stain.     

Application of immunoblotting to serum protein phenotyping with reference to α2HS-glycoprotein (AHS) typing of bloodstains

Summary Sera and bloodstain extracts were subjected to isoelectric focusing in polyacrylamide gels. The focused proteins were transferred to nitrocellulose membranes by diffusion or electrophoretically, then allowed to react with specific antiserum and, after washing, with peroxidase-labeled anti-rabbit IgG. The immune complexes formed on the membranes were detected with 4-chloro-1-naphthol and hydrogen peroxide. Serum group-specific component, ₂HS-glycoprotein, the sixth and the seventh component of complement, factor 13B, and plasminogen could be phenotyped with high sensitivity. Bloodstains as old as 6 months could be correctly typed for ₂HS-glycoprotein by the blotting technique.     

Stability of α-[11C]methyl-l-tryptophan brain trapping in healthy male volunteers

Purpose The purpose of this study was to assess the reproducibility in healthy volunteers of -[¹¹C]methyl-l-tryptophan ([¹¹C]MT) brain trapping imaging with positron emission tomography (PET), using volumes of interest (VOIs) and voxel-based image analysis.Methods Six right-handed healthy male volunteers (34.3±10.9 years) with a negative family history for psychiatric disorders were scanned twice in the resting condition, 22±17 days apart. An unbiased semiautomatic segmentation of the brain was used to define VOIs. The trapping constant K* (ml g^–1 min^–1) for [¹¹C]MT was calculated for the whole brain and seven brain regions using the graphical method for irreversible tracers. In addition, parametric maps of K* were obtained from dynamic scans using the same method. Comparison of test and retest K* functional images was performed using SPM99. Students paired t statistic was applied for comparisons of [¹¹C]MT brain trapping in a priori selected VOIs.Results [¹¹C]MT brain trapping in VOIs showed a mean variability 2.6±1.8% (0.3–5%) for absolute and 1.5±2.1% (1.4–4.1%) for normalized K*. Intraclass correlations between test and retest conditions were 0.61±0.34 for absolute K* values and 0.73±0.20 for K* values normalized by global mean. SPM99 analysis using a height threshold of p=0.05 (two tailed) and an extent threshold of 100 voxels showed no significant differences between scans.Conclusion Rest measurements in healthy male volunteers of the trapping constant for [¹¹C]MT, using PET, appeared to be stable during an average interval of 3 weeks.     

Cancer radioimmunotherapy with alpha-emitting nuclides

In lymphoid malignancies and in certain solid cancers such as medullary thyroid carcinoma, somewhat mixed success has been achieved when applying radioimmunotherapy (RIT) with -emitters for the treatment of refractory cases. The development of novel RIT with -emitters has created new opportunities and theoretical advantages due to the high linear energy transfer (LET) and the short path length in biological tissue of -particles. These physical properties offer the prospect of achieving selective tumoural cell killing. Thus, RIT with -emitters appears particularly suited for the elimination of circulating single cells or cell clusters or for the treatment of micrometastases at an early stage. However, to avoid non-specific irradiation of healthy tissues, it is necessary to identify accessible tumoural targets easily and rapidly. For this purpose, a small number of -emitters have been investigated, among which only a few have been used for in vivo preclinical studies. Another problem is the availability and cost of these radionuclides; for instance, the low cost and the development of a reliable actinium-225/bismuth-213 generator were probably determining elements in the choice of bismuth-213 in the only human trial of RIT with an -emitter. This article reviews the literature concerning monoclonal antibodies radiolabelled with -emitters that have been developed for possible RIT in cancer patients. The principal radio-immunoconjugates are considered, starting with physical and chemical properties of -emitters, their mode of production, the possibilities and difficulties of labelling, in vitro studies and finally, when available, in vivo preclinical and clinical studies.     

Immunochemical studies on blood group H and B substances from human hair

Summary Blood group H and B substances were extracted from urea-treated human hair of group O and B individuals, respectively, with methanol-ethyl ether (1:1,v/v) and chloroform-methanol (1:1,v/v). The blood group activities of H and B substances were destroyed by H-decomposing enzyme (-l-fucosidase) fromBacillus fulminans and B-decomposing enzyme (-d-galactosidase) fromClostridium sporogenes Maebashi, respectively. It is concluded therefore that the extract from the hair of group O contained blood group H-active glycolipid with -l-fucose as the non-reducing sugar and the one from group B contained blood group B-active glycolipid with -d-galactose as the non-reducing sugar.     

The distribution of the HLA-DQα alleles and genotypes in the Finnish population as determined by the use of DNA amplification and allele specific oligonucleotides

Summary Allele and genotype frequencies for the HLA-130 locus were determined for use in forensic analyses and paternity tests in Finland. The polymerase chain reaction (PCR) and the reverse dot blot format were employed to detect 6 different HLA-DQ alleles. All 6 HLA-DQ alleles were detected among the 112 unrelated individuals with allele frequencies ranging from 5.8% to 32.6%. The distribution of the observed genotypes is in Hardy-Weinberg equilibrium. Additionally, this Finnish population sample is statistically similar to 2 other Caucasian sample populations. The power of discrimination of this system in the Finnish population sample is 0.92, suggesting this method may prove suitable for identification purposes.     

Investigations into the use of 77Br labelled 5α-dihydrotestosterone for scanning the prostate

The potential use of a potent androgen labelled with a gamma emitting radionuclide for scanning the prostate was investigated. 2bromo 5-dihydrotestosterone was synthesized and subsequently labelled with ⁷⁷Br. The distribution of this compound was studied in rat and human tissues. The maximum concentration of radioactivity in the rat prostate at 1–4 h following the injection was found to be between 0.5–0.8% of the injected dose per gram of the tissue. In the human however, that value was 0.002–0.003%. Considering the overall results it was concluded that this compound is not appropriate for scanning the prostate.     

Immunoelektrophoretische Differenzierung der Proteine faulen Leichenblutes

Zusammenfassung Leichenblute verschiedener Fäulnisgrade wurden immunoelektrophoretisch untersucht. Für die Differenzierung der einzelnen Blutplasmafraktionen wurden Anti-Humanserum vom Kaninchen und die spezifischen Antiseren der Behringwerke gegen die nachstehend aufgeführten Proteine benutzt: Präalbumin, Albumin, ₁-Lipoprotein, ₂-Makroglobulin, ₂-Lipoprotein, ₁-Globulin (Transferrin),-Lipoprotein, Fibrinogen, ₂-A-Globulin, ₂-M-Globulin,- Globulin.Infolge der mangelnden Proportionalität zwischen Fortschritt der Fäulnis und Leichenalter gestattet die Immunoelektrophorese des Leichenblutes keine Todeszeitbestimmung.Albumin und- Globulin widerstehen der Fäulnis am längsten. Es folgen ₂-Makroglobulin, Transferrin und Fibrinogen. Die Proteine, die am reichlichsten im Menschenblut vorhanden sind, lassen sich im faulen Leichenblut auch am längsten nachweisen. Die Proteolyse folgt jedoch nicht nur quantitativen Gesetzen. Die Lipoproteine bilden insofern eine Ausnahme, als sie früher durch die Fäulnis eliminiert werden, als es ihrem Anteil am Gesamtprotein entspricht.Die Eiweißkörper des faulen Leichenblutes weisen im Vergleich zu ihren Homologen frischer Blutproben z. T. Veränderungen der Wanderungsgeschwindigkeit auf. Bei erhaltener Antigenstruktur treten Bruchstücke in Erscheinung, die mit dem Antiserum in ungleichmäßig geschwungenen oder aufgesplitterten Linien präcipitieren.Im Doppeldiffusionstest nachOuchterlony ließen sich im Vergleich zu den frischen Homologen keine Veränderungen der Antigenkomponenten der Proteine des faulen Leichenblutes nachweisen.Mit Unterstützung der Deutschen Forschungsgemeinschaft, der hiermit unser herzlicher Dank ausgesprochen wird.In Anlehnung an einen Vortrag auf der Tagung der Deutschen Gesellschaft für Gerichtliche und Soziale Medizin vom 30. 9. bis 3. 10. 62 in Münster (Westf.).Herrn Professor G.Weyrich zum 65. Geburtstag gewidmet.     

Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins

Summary Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), ₂-macroglobulin (₂-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human ₂-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next.Supported in part by the Cooperation Research Program of the Primate Research Institute     

Positive 131Cesium scanning in parathyroid adenoma

In a case of primary hyperparathyroidism, a palpable nodule, at the base of the right lobe of the thyroid, proved cool during ^99mTc scanning, but hot when scanned with radiocesium. The uptake of this tracer was higher than the uptake of ⁷⁵Se-selenomethionine after suppression with T₃.The authors discuss the possibility of cases of false-positive radiocesium uptake in extrathyroid nodules, and in particular, the use of this tracer for the detection of parathyroid adenoma by scanning.     

Radiolabelling of monoclonal antibodies: Optimization of conjugation of DTPA to F(ab′) 2-fragments and a novel measurement of the degree of conjugation using Eu(III)-labelling

F(ab) ₂ fragments (at concentrations of 5–30 mg/ml) derived from monoclonal antibodies raised against human prostate specific acid phosphatase were derivatized with a bicyclic anhydride of diethylenetriaminepentaacetic acid (cDTPAA) in the molar ratios of cDTPAA/F(ab) ₂ of 1:1, 5:1, 10:1 or 50:1. The most optimal product, aimed at radioimaging of prostatic cancer was obtained when the antibody fragment concentration was at least 10 mg/ml and the molar ratio of cDTPAA to F(ab) ₂ was 5:1 cDTPAA was added dissolved in dimethylsulfoxide (DMSO). Under these conditions, 1.8–2.2 DTPA molecules/F(ab) ₂ molecule were bound, giving a coupling efficiency of 37%–44%, and the labelling efficiency with ¹¹¹In (3 mCi/1 mg protein) was 95%±3% (n=7). The antibody fragment completely retained its immunoreactivity measured by radioimmunoassay and showed no aggregation when studied using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).For evaluation of the degree of conjugation of DTPA to the antibody fragment, a novel technique was developed relying on the use of EuCl₃, and the measurement of europium fluorescence employing time resolved fluorometry. Results by EuCl₃ labelling were identical to those obtained by the conventional ¹¹¹InCl₃ labelling method.     

γ-Glutamyltransferase test as a new tool for identification of seminal stains

Summary A simple qualitative method for identification of seminal stains based on a high activity of -glutamyltransferase (-GTP) in human semen is described. It employs the release of -naphthylamine from N--glutamyl--naphthylamide by the -GTP action; -naphthylamine couples with Fast Garnet GBC salt to produce a strong brownish-red color. The data on its simplicity, specificity, and stability show that the present method is suitable for medicolegal examination of seminal stains as a preliminary test.     

A pragmatic approach to metal artifact reduction in CT: merging of metal artifact reduced images

The purpose of this study was to improve metal artifact reduction (MAR) in X-ray computed tomography (CT) by the combination of two artifact reduction methods. The presented method constitutes an image-based weighted superposition of images processed with two known methods for MAR: linear interpolation of reprojected metal traces (LI) and multi-dimensional adaptive filtering of the raw data (MAF). Two weighting concepts were realized that take into account mean distances of image points from metal objects or additional directional components. Artifact reduction on patient data from the jaw and the hip region shows that although the application of only one of the MAR algorithms can already improve image quality, these methods have specific drawbacks. While MAF does not correct corrupted CT values, LI often introduces secondary artifacts. The corrective impact of the merging algorithm is almost always superior to the application of only one of the methods. The results obtained with directional weighting are equal to or in many cases better than those of the distance weighting scheme. Merging combines the advantages of two fundamentally different approaches to artifact reduction and can improve the quality of images that are affected by metal artifacts.Symbols LI linear interpolation algorithm - MAF multi-dimensional adaptive filtering algorithm - MAR metal artifact reduction - f(x, y) object function at (x, y) - F(), F() normalized filter function in the direction of the coordinates and - I primary X-ray intensity - I₀ transmitted X-ray intensity - N_D number of detectors per detector row - p attenuation - p(,), p(,) projection data (fan and parallel geometry) - p_th lower threshold of the attenuation of adaptive filtering - p_max maximum value of the attenuation of adaptive filtering - (x, y) image point - projection angle in fan geometry - angle within the fan relative to the central ray - , , z, , minimum functions for adaptive filtering - projection angle in parallel geometry - orthogonal distance of a ray to the center of rotation in parallel geometry     

Control of the RIA method as viewed from the standpoint of the investigation on the kinetics of the insulin-125I-antibody reaction

In this paper attempts were made towards the optimalization and the control of some parameters of the RIA reaction.Basing on the law mass action, as well as on the Scatchard's and Sips's equations, the equilibrium constants for the reversible reaction: insulin-¹²⁵I-antibody for different incubation temperatures were calculated. Moreover a characteristic of the antiinsulin antibody by means of the heterogeneity coefficient was done, as well as the values of the thermodynamic function increments were calculated, which made possible to point out explicitly the optimal shape of the standard curve.     

Biodistribution of iodine-125 tyramine transforming growth factor α antisense oligonucleotide in athymic mice with a human mammary tumour xenograft following intratumoral injection

The Watson-Crick base pairing rule provides the underlying principle for the antisense (AS) approach to inhibiting gene expression. Transforming growth factor (TGF) was the first growth factor to be associated with tumorigenesis, thus making the TGF (mRNA) a potential target for AS therapy and offering the potential for monitoring of the progression of malignancy by non-invasive imaging with radiolabelled AS phosphodiester. Probe labelling and biodistribution were studied in the present report. A 23-mer oligonucleotide sequence was synthesized and grafted in 5 with a tyramine group which was further radioiodinated. The radiolabelled AS was injected intratumorally in mammary tumour-bearing BALB/c mice (3 weeks after inoculation of 7·10⁶ NS2T2A mammary cells). Biodistribution was monitored by sequential scintigraphy and organ radioactivity after autopsy. The 5 tyramine group allowed specific and stable radiolabelling of the AS with¹²⁵I. The¹²⁵I AS oligonucleotide was rapidly cleared from the tumour by intestine and kidneys. Four hours after intratumoral injection, 6.5%±1.5% of the dose was retained in the tumour as non-degraded¹²⁵I AS. It is concluded that 5 tyraminylated AS provides information on the biodistribution of AS oligonucleotide following intratumoral injection. These data will contribute to the pharmacology of AS oligonucleotides which can be used for therapy.     

Frequency of D1S80 and HLA DQα alleles in a Chinese population

Allele frequency distributions for the D1S80 (MCT118) and HLA DQ loci were determined in a Chinese population sample using the polymerase chain reaction (PCR). A total of 25 alleles and 100 phenotypes were observed for D 1 S80. The frequency of allele 18 was higher than allele 24 only in this Chinese population when compared to other reported populations. A total of 6 alleles and 21 possible phenotypes were observed for HLA DQ. The power of discrimination was 0.97 and 0.93 for D1S80 and HLA DQ, respectively.