An ELISA avoiding interference by heterophilic antibodies in the measurement of components of the plasminogen activation system in blood.

Endogenous heterophilic antibodies in blood are known to interfere with two-site enzyme-linked immunosorbent assays (ELISAs) evoking false positive signals. In the present study, we describe an assay for the assessment of components of the plasminogen activation system (uPA, tPA and PAI-1, and their complexes) in blood which is not susceptible to interference by heterophilic antibodies. In the ELISA format, two avian (duck, chicken) antibodies are employed in the pre-analyte and two mammalian (rabbit, goat) antibodies in the post-analyte stage. The assay is compared to our earlier reported ELISA for measuring uPA, tPA and PAI-1 components in tumor tissue extracts. Applying the so-called "nonsense formats", designed against non-existent components, to the NIBSC reference preparation of rheumatoid factor (RF), no response was found with the new assay, whereas a clear RF dose-dependent interfering signal was observed with the original assay designed for tumor tissue extracts. Analysis of tumor-tissue based international reference preparations (RBG EORTC 101094 and 040297), human anti-mouse antibodies (HAMA) containing sera, and sera from patients with rheumatoid arthritis (RA), also displayed no false positive signals. In conclusion, we have developed an ELISA that permits the determination of blood levels of components in the urokinase system, free from disturbance by endogenous heterophilic antibodies.

A class of asymmetrical nitrido 99mTc heterocomplexes as heart imaging agents with improved biological properties.

Asymmetrical heterocomplexes containing a terminal technetium-nitrogen multiple bond coordinated to one diphosphine ligand (PNP) and one dithiocarbamate ligand (DBODC), were obtained through a simple two-step procedure under controlled conditions. The resulting complexes [99mTc(N)(PNP)(DBODC)]+ are monocationic, and possess a distorted square-pyramidal geometry where the Tc triple bond N multiple bond occupies an apical position and the diphosphine and dithiocarbamate ligands span the residual four coordination positions on the basal plane through the two phosphorus atoms and the two sulfur atoms, respectively. Biodistribution data in rats demonstrated that these complexes were rapidly extracted by the myocardium, and retained in this region for a prolonged time. After a few minutes post-injection, lung uptake became negligible, and liver washout was extremely rapid and quantitative. Analysis of heart/liver uptake ratios for these complexes revealed that their values increased exponentially in time, and after 60 min post-injection liver activity was almost completely eliminated into the intestine. Comparison with heart/liver ratios determined for 99mTc sestamibi and 99mTc tetrfosmin showed that values for these latter compounds were approximately 10 times lower than those measured for [99mTc(N)(PNP)(DBODC)]+ complexes at 60 min post-injection. In conclusion, the monocationic tracers [99mTc(N)(PNP)(DBODC)]+ exhibit high myocardial uptake in rats and dramatically high heart/lung and heart/liver ratios, suggesting that this novel class of perfusion agents could be conveniently employed to obtain heart images with superior imaging quality.

Cytokine imbalance in pregnancies with fetal chromosomal abnormalities.

BACKGROUND: The aim of the present study is to investigate the levels of some of the cytokines which may be involved in the mechanisms leading to the impairment of placental perfusion and to the onset of uterine contractions in pregnancies with fetal genetic abnormalities compared with controls. METHODS: The amniotic fluid and maternal plasma levels of interleukin-6, interleukin-8 and tumour necrosis factor-beta in patients with fetal chromosomal abnormalities were measured, as well as in euploid pregnancies in the seventh week of gestation. RESULTS: An increase of interleukin-6 (P = 0.034) and a decrease of interleukin-8 (P < or =0.0001) in amniotic fluid, and a decrease of interleukin-6 in the maternal plasma (P = 0.026) was shown in pregnancies with fetal chromosomal abnormalities. A positive correlation was observed between amniotic interleukin-8 and serum interleukin-6 in the presence of fetal aneuploidy (P < 0.006). CONCLUSION: Further investigations of cytokine imbalance in pregnancies with poor outcome as a consequence of genetic disorders rather than infection is warranted.

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.

A multicentre observational study of radionuclide therapy in patients with painful bone metastases of prostate cancer.

A multicentre observational study was conducted by the Italian Association of Nuclear Medicine between 1996 and 1998. Twenty-nine Nuclear Medicine Departments participated. The aims of the study were to systematically evaluate the efficacy, toxicity and repeatability of radionuclide therapy of painful bone metastases (RTBM) in a large number of patients and to assess its incidence in patients with prostate cancer. Out of 818 treatments performed with a single i.v. dose of 148 MBq of strontium-89 chloride or 1,295 MBq of rhenium-186 hydroxyethylidene diphosphonate (HEDP), 610 could be evaluated (527 with 89Sr and 83 with 186Re-HEDP). Eighty-one patients received multiple (up to five) RTBM. The total number of retreatments was 100. Patients were followed up for a period of 3-24 months. Results, assessed according to pain relief and consumption of analgesic drugs, were expressed at four levels: 1, no response; 2, mild response; 3, good response; 4, excellent response. Responses were: level 1 in 19%, level 2 in 21.3%, level 3 in 33.3% and level 4 in 26.4% of cases. Retreatments showed significantly (P<0.01) worse responses (48% levels 3+4), in comparison to first RTBM. Duration of palliation was 5.0+/-3.5 months, and was longer in cases of excellent response, in first RTBM, in patients with limited metastases and when 89Sr was used. Better responses were found in cases of limited skeletal disease, under good clinical conditions, when life expectancy exceeded 3 months, and in radiologically osteoblastic or mixed bone lesions. The only statistically significant predictive factor was life expectancy (P<0.001). Flare phenomenon (14.1% of cases) did not correlate with the response. Haematological toxicity (mild to moderate in most cases) mainly affected platelets, and was observed in 25.5% of cases overall and in 38.9% of retreatments. RTBM did not seem to prolong life, though in some cases scintigraphic regression of bone metastases was observed. The two radiopharmaceuticals did not show any statistically significant differences in palliative efficacy and toxicity, either in first RTBM or in retreatments.

Radionuclide therapy for painful bone metastases. An Italian multicentre observational study. Writing Committee of an Ad Hoc Study Group.

BACKGROUND: It has been affirmed that observational studies give analogous results to randomised controlled ones. METHODS: A multicentre observational trial was conducted between 1996-1998 in order to evaluate the efficacy of palliative radionuclide therapy for bone metastases in a large number of patients. An evaluation was made on 510 patients with prostate cancer and painful bone metastases, treated with a single iv. dose of 89Sr-chloride (527 treatments) or 186Re-HEDP (83 treatments), in 29 Italian Nuclear Medicine Departments. Eighty-one patients received up to five injections, totalling 100 retreatments. Patients were followed up for a period of 3 months-2 years. Results were expressed at four levels of response: excellent, good, mild, and nil. RESULTS: Responses were excellent in 26.4%, good in 33.3%, mild in 21.3% and nil in 19% of all treatments, while good and excellent responses were obtained in 48% of retreatments. No statistically significant correlations were found between response and age of patients, skeletal extension of tumour, pretherapeutic PSA levels, evidence of non-bony metastases, previous chemotherapy and/or external-beam radiotherapy; osteolytic lesions responded worse than osteoblastic or mixed ones. Hematological toxicity (mild to moderate), mainly affecting platelets, was observed in 25.5% of all treatments and in 38.9% of retreatments. No clear differences were found between the two radiopharmaceuticals employed. CONCLUSIONS: Bearing in mind that observational studies can provide just as accurate results as randomised controlled trials, this study confirms the main findings of various limited monocentre trials.

Plasminogen activator system in serum and amniotic fluid of euploid and aneuploid pregnancies.

OBJECTIVE: To compare euploid and aneuploid pregnancies with respect to maternal serum and amniotic fluid (AF) levels of the components of the plasminogen system. METHODS: The study population consisted of 123 single pregnancies at the 17th gestational week, 16 with minor chromosomal abnormalities, 15 aneuploid, and 92 euploid. RESULTS: Both groups with chromosomal abnormalities had significantly higher serum levels of urokinase plasminogen activator and its complexed form with its type-1 inhibitor compared with euploid pregnancies. In AF, tissue plasminogen activator was significantly lower in the aneuploid than the euploid group, whereas type-1 inhibitor of plasminogen activator was significantly higher in the cases with minor chromosomal abnormalities compared with euploid. At cutoff levels set at 100% sensitivity, the complexed form of urokinase plasminogen activator with its type-1 inhibitor had the strongest specificity (66.3%); after logarithmic transformation, its serum level was 7.53 times higher in aneuploidies than euploidies. CONCLUSION: Aneuploid pregnancies appear to be accompanied by abnormalities of the plasminogen activation system, which could lead to impaired placental perfusion and thus to abortion, fetal death, and fetal growth restriction.

Synthesis of a novel class of nitrido Tc-99m radiopharmaceuticals with phosphino-thiol ligands showing transient heart uptake.

A novel class of technetium-99m radiopharmaceuticals showing high heart uptake is described. These complexes were prepared through a simple and efficient procedure, and their molecular structure fully characterized. They are formed by a terminal Tc(triple bond)N multiple bond and two bidentate phosphine-thiol ligands [R(2)P-(CH(2))(n)SH, n=2,3] coordinated to the metal ion through the neutral phosphorus atom and the deprotonated thiol sulfur atom. The resulting geometry was trigonal bipyramidal. Biodistribution studies were carried out in rats. The complexes exhibited high initial heart uptake and elimination through liver and kidneys. The washout kinetic from heart was dependent on the nature of the lateral R groups on the phosphine-thiol ligands. When R=phenyl, heart activity was rapidly eliminated within 10-20 min. Instead, when R=tolyl, cyclohexyl, persistent heart uptake was observed. Extraction of activity from myocardium tissue showed that no change of the chemical identity of the tracer occurred after heart uptake. On the contrary, metabolization to more hydrophilic species occurred in liver and kidneys.

Synthesis of hybrid distamycin-cysteine labeled with 99mTc: a model for a novel class of cancer imaging agents.

The synthesis of a hybrid constituted by distamycin A and cysteine labeled with the gamma-emitting radionuclide 99mTc to afford the conjugate complex 5 is reported. This new radiopharmaceutical is of potential interest as tumor imaging agent in diagnostic nuclear medicine. The preparation of the hybrid distamycin A-cysteine 4 has been achieved by coupling deformyldistamycin A and Boc-Dmt-OH. Compound 4 was then successfully labeled with 99mTc by reaction with the novel, high-electrophilic, metal-containing fragment [99mTc(N)(PP)]2+ (PP = diphosphine ligand) yielding the 1:1 complex 5.

An alternative approach to the preparation of (188)Re radiopharmaceuticals from generator-produced [(188)ReO(4)](-): efficient synthesis of (188)Re(V)-meso-2,3-dimercaptosuccinic acid.

A new efficient approach for the preparation of (188)Re radiopharmaceuticals starting from [(188)ReO(4)](-), produced at a carrier-free level through the (188)W/(188)Re generator system, is described. The reaction procedure was based on the combined action of different reagents and has been applied in detail to the preparation of the therapeutic agent (188)Re(V)-DMSA (H(2)DMSA [meso-2,3-dimercaptosuccinic acid]). The most efficient combination required the use of SnCl(2), oxalate ions, and gamma-cyclodextrin. These were reacted with [(188)ReO(4)](-) and H(2)DMSA to afford the final radiopharmaceutical in high radiochemical purity, at room temperature, and in weakly acidic solution. The role played by the various reagents in the reaction was investigated. It was found that SnCl(2) behaved as the actual reducing agent, whereas oxalate and gamma-cyclodextrin greatly enhanced the ease of reduction of [(188)ReO(4)](-) through the action of two hypothetical mechanisms. In the first step of the reaction, oxalate ions gave rise to the formation of Re(VII) complexes with the concomitant expansion of the coordination sphere of the metal. This process strongly favored the electron transfer between Sn(2+) and Re(+7) centers, giving rise to intermediate reduced rhenium complexes. These species were further stabilized by the formation of transient host-guest aggregates with gamma-cyclodextrin and finally converted into (188)Re(V)-DMSA through simple replacement of the coordinated ligands by H(2)DMSA.

EORTC receptor and biomarker study group report analytical and technical evaluation of a thymidine kinase radio-enzymatic assay in breast cancer cytosols.

BACKGROUND: High thymidine kinase (TK) activity in cancer cells could counteract adjuvant chemotherapy directed at the inhibition of de novo DNA synthesis. TK is an independent prognostic factor in breast cancer patients receiving adjuvant chemotherapy. MATERIAL AND METHODS: In this paper, we describe the effects of extraction and dilution buffer composition on TK enzymatic activity values obtained in breast cancer cytosols with the Prolifigen serum TK-REA kit (Sangtec Medical, Sweden). RESULTS: The addition of MgCl2 and ATP early in the assay, preferably during the extraction of tumor tissue, seems critical to stabilise the enzyme. Furthermore, the use of normal calf serum to dilute both standards and samples is necessary to obtain satisfactory parallelism between TK values in serial dilutions of breast cancer cytosols. CONCLUSION: Based on the data reported here, the manufacturer has changed the cytosol diluent composition and is adding a specific cytosol assay insert to the Prolifigen TK-REA kit. As evidenced by the laboratory reproducibility, these modifications to the serum assay led to an adequate, standardized protocol for analyzing TK activity in breast tumor cytosols.

Underestimation of regional myocardial perfusion with Tc-99m sestamibi single-day rest-stress SPECT: a "drug washout" pitfall?

Myocardial perfusion scintigraphy with a Tc-99m sestamibi single-day SPECT protocol is a widely used technique to examine patients with possible or known coronary artery disease. A 76-year-old man with a clinical history suggestive of ischemic heart disease underwent Tc-99m sestamibi myocardial SPECT imaging with a same-day rest and stress protocol after temporary discontinuation of his current therapy, which included calcium channel and beta blockers and nitrates. The scintigraphic pattern was consistent with an asymptomatic infarction of the posterolateral myocardial wall and periinfarct ischemia. One week later, the patient had a Tc-99m sestamibi myocardial SPECT study at rest without discontinuing therapy, and scintigraphic images showed normalization of the posterolateral wall perfusion defect. The angiographic study showed a 90% stenosis of the circumflex artery. This case suggests that, during a 1-day cardiac SPECT protocol, washout of therapeutic pharmaceuticals may be responsible for underestimation of myocardial rest perfusion in territory supplied by a coronary artery with a critical stenosis.

Endocrine-disrupting agents on healthy human tissues.

A vast number of substances have been suggested as possibly contributing to perturbation of the endocrine system. Several have been tested with different approaches ranging from yeast expression system of human oestrogenic receptors to human breast cancer cells assays. Surprisingly, no inhibition-binding experiments to steroid receptors on healthy human tissue have been performed so far. Our study provides inhibition binding experiments to oestrogens, progesterone, testosterone and retinoic acid receptors in prostate and uterine human tissue of organochlorine pesticides, phthalate esters, oestrogenic constituents derived from plants and phenol derivates. Affinities of significant extent of phthalates on oestrogenic, progestinic and androgenic receptors have not been detected. As for retinoic acid receptors, mono(2-ethylexyl)phthalate provokes a notable reduction of the binding of the tritiated retinoic acid, phtalic acid ethyl-n-butyl ester and 4-octylphenol show an affinity comparable to that of isoflavonoid genistein, whereas 4-nonylphenol reduces the binding of retinoic acid in prostate.

A CD(4)/T(4) receptor peptide ligand labeled with technetium-99m: synthesis and biological activity.

The octapeptide D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH(2) ([D-Ala(1)]TNH(2)), an analog of peptide T (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH) associated with CD(4)/T(4) receptors involved in human immunodeficiency virus infection, was combined with the chelating polyazamacrocycle 1,4,8,11-tetraazacyclotetradecane (cyclam) to afford the bifunctional ligand cyc-[D-Ala(1)]TNH(2). This was then reacted with [(99m)TcO(4)](-) and Sn(2+) to yield the monocationic complex [(99m)Tc(O)(2)(cyc-[D-Ala(1)]TNH(2))](+). Biological activity of both the cyclam-peptide conjugate and the resulting Tc-99m complex were evaluated by measuring their chemotactic indexes. Results showed that N-cyclam acylation and subsequent labeling with Tc-99m of [D-Ala(1)]TNH(2) were tolerated, and both cyc-[D-Ala(1)]TNH(2) and [(99m)Tc(O)(2)(cyc-[D-Ala(1)]TNH(2))](+) retained the high chemotactic capacity of the original octapeptide. Biodistribution of the Tc-99m complex was carried out in rats. Fast blood clearance and no accumulation in organs of interest were observed.

Quality control of 99Mo/99Tcm generators: results of a survey of the Radiopharmacy Working Group of the Italian Association of Nuclear Medicine (AIMN).

A multicentre survey of the quality control of 99Tcm generators has been completed: 245 generators from seven different commercial sources were tested over a period of 2 years. The results indicate that the mean pH of the eluates was 5.8 +/- 0.6; the aluminium contents were typically < 10 ppm; the radiochemical purity was 99.8 +/- 0.4% and the median 99Mo content was 3.8 x 10(-4) percent. The elution profiles gave a volume of 1.9 ml to obtain 50% of the total eluted activity and of 4.9 ml to obtain 95%. Other radionuclide impurities and heavy metal breakthrough were evaluated by graphite furnace absorption spectrometry and inductively coupled plasma mass spectrometry. National guidelines for the standardization of radiopharmacy procedures are currently being compiled.

Immunometric assays of ras and c-erbB-2/neu overexpression in breast cancer: a pilot study.

The expression of the ras and c-erbB2 oncoproteins (p21 and p185, respectively), together with estrogen receptor (ER) and progesterone receptor (PgR) determination, has been retrospectively analyzed in 68 primary breast carcinomas and in 19 normal breast tissue samples. The aims of this study were: a) to explore the association between ras and c-erbB2 expression; b) to evaluate the relationship between ras and c-erbB2 expression and both steroid receptor status and the classical clinical and pathological parameters; and c) to compare two different methods for p185 determination. p185 and p21 were measured by enzyme immunoassay (EIA); p185 was also determined by Western blotting (WB); ER and PgR were assayed by radioligand binding assay. The highest value of p185 in benign breast lesions was used as the threshold to distinguish between positive and negative samples. With this threshold the c-erbB2 oncoprotein was overexpressed in 41.2% (with EIA) and in 50% (with WB) of cancer samples. The concordance rate between the two methods was 79.4. No significant association was found between p21 and p185 levels either in cancer or in normal breast tissue samples. Increasing levels of tumor p21 were associated with a shorter time to recurrence and overall survival. Increasing levels of p185 were associated with a significantly shorter time to recurrence (p185 EIA: p = 0.04, p185 WB: p = 0.029) and overall survival (p185 EIA: p = 0.04, p185 WB: p = 0.029).

New insights on flow-independent mechanisms of 99mTc-HMPAO retention in nervous tissue: in vitro study.

UNLABELLED: SPECT using 99mTc-hexamethyl propyleneamine oxime (HMPAO) mainly reflects regional cerebral blood flow, however metabolic abnormalities also affect the retention of 99mTc-HMPAO. METHODS: To rule out any flow factor, a test-tube model was used to evaluate the effects of metabolic alterations both on intracellular trapping of 99mTc-HMPAO and on extracellular glutamate and lactate dehydrogenase (LDH) outflow from rat brain slices. RESULTS: Under control conditions, slices took up 7.0%+/-1.4% of 99mTc-HMPAO contained in the medium, whereas prelabeled slices released 10.8%+/-2.6% of their radioactive content; glutamate and LDH outflow were 49.1+/-21.6 pmol/mg protein/ min and 4.8+/-0.9 U/L/mg protein/min, respectively. The control medium was altered by adding a metabolic poison (5 mmol/L azide), removing glucose and replacing O2 with N2 to mimic ischemia (in vitro ischemia) and replacing Krebs solution with hypotonic medium to evoke cell lysis. Both azide and in vitro ischemia induced a significant increase in 99mTc-HMPAO release (15.8%+/-3.3% and 18.3%+/-6.2%, respectively), without any modification in LDH efflux. However, only azide reduced the uptake of the tracer. Conversely, glutamate outflow was massive during in vitro ischemia and was far lower during azide treatment. Under hypotonic medium conditions, the release of 99mTc-HMPAO, glutamate and LDH were dramatically increased. Surprisingly, a two-fold increase of 99mTc-HMPAO uptake was also found. When 1 mmol/L glutathione was added to the medium, to convert native lipophilic 99mTc-HMPAO into hydrophilic derivatives, tracer uptake was inhibited both under control and hypotonic medium conditions. CONCLUSION: This study provides evidence that not only poisoning of the tissue but also in vitro ischemia induced a reduction of 99mTc-HMPAO retention. Moreover, we demonstrated that injuries causing cell membrane disruption led to hyperfixation of 99mTc-HMPAO.

Design and synthesis of a redox-active Tc-99m radiopharmaceutical with ferrocenedithiocarboxylate [FcCS = Fe(C5H4CS2)(C5H5)-].

The synthesis, at tracer level, of two Tc-99m complexes having the same chemical composition and structure, but differing by one electron in the total electron counting, is reported. These compounds have been prepared by reacting [99mTcO4]- with the piperidinium salt of the ligand ferrocenedithiocarboxylate {[Fe(II)(C5H4CS2)(C5H5)]- = FcCS}, in the presence of N-methyl S-methyldithiocarbazate as donor of N3-groups, and triphenylphosphine or SnCl2 as reducing agents. The formation of the neutral complex [99mTc(N)(FcCS)2] (compound A) and of the monocationic, mixed-valence complex [99mTc(N)(FcCS) (FcCS)]+ (compound B) {FcCS = [Fe(III)(C5H4CS2)(C5H5)]} was obtained in high yield. Both complexes comprise a terminal Tc triple bond N multiple bond and two FcCS ligands coordinated to the metal center through the two sulfur atoms of the -CS2 group, but they differ in the oxidation state of one of the two iron atoms of the coordinated FcCS ligands. In complex A, the two Fe atoms are both in the +2 oxidation state, while in B, one Fe atom is in the +2 and the other is in the +3 oxidation state. Thus, B is a mixed-valence Fe(II)-Fe(III) complex. B is easily converted into A by one-electron exchange with various reductants such as triphenylphosphine and excess SnCl2. Biodistribution studies in rats showed that complexes A and B are mostly retained in lungs and liver without any significant uptake in organs such as heart and brain.