Technetium labeling of monoclonal antibodies with functionalized BATOs: 2. TcCl(DMG)3CPITC labeling of B72.3 and NP-4 whole antibodies and NP-4 F(ab')2.

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive 2-carboxy-4-phenyl isothiocyanate (CPITC). The 99Tc complexes, where the dioxime was either dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO), were prepared and characterized. The 99mTc complex TcCl(DMG)3CPITC was prepared from a freeze-dried kit and used to label B72.3 (anti-TAG.72) and NP-4 (anti-CEA) whole antibodies, and the NP-4 F(ab')2 fragment. SDS-PAGE electrophoresis indicated that the labeling reagent was strongly bound to antibody. The labeled antibodies displayed high binding to affinity columns and good tumor uptake in GW39 tumor-bearing mice.     

An unexpected by-product obtained during the preparation of technetium(III) boronic acid adducts of dioximes. The single crystal structure of TcCl(DMG)2(BDI)BEt (DMG=dimethylglyoxime, BDI=butane-2, 3-dione imine-oxime)

An unusual Tc(III) boron-capped imine-oxime complex has been isolated from the reaction of ⁹⁹TcCl₃(CH₃CN)(PPh₃)₂, dimethyl glyoxime (DMG) and ethyl boronic acid (EtB(OH)₂). A single crystal X-ray structure analysis of this molecule ⁹⁹TcCl(DMG)₂(BDI)BEt (BDI=butane-2, 3-dione imine-oxime) shows it to be seven coordinate: TcClC₁₄H₂₅N₆O₅B, a=9.073(2), b=23.686(5), c=19.539(6) Å; β=93.77(2)°, P2₁/n, Z=8. Its structure is very similar to that of previously reported Tc(III) complexes ⁹⁹TcCl(dioxime)₃BR, except that one dioxime ligand on the molecule has been reduced to an imineoxime.     

Comparison of neutral and cationic myocardial perfusion agents: Characteristics of accumulation in cultured cells

Uptake and washout kinetics of two new neutral lipophilic technetium-99m-labeled boronic acid adducts of technetium tris(dioxime) (BATO complexes) were studied in monolayers of contractile chick heart cells and compared to the cationic myocardial perfusion agents, ^99mTc(CNCH₂C(CH₃)₂OCH₃)⁺₆ (Tc-MIBI) and ²⁰¹Tl⁺. ^99mTcCl(CDOH)₂(CDO)(BCH₃), where CDO = cyclohexanedione dioxime (CDO-MeB), had a 7-fold greater net accumulation than Tc-MIBI and the most rapid unidirectional washout with a fast initial phase and a slower secondary component. Incubation with cationic membrane transport inhibitors or metabolic inhibitors had little or modest influence, respectively, on uptake of these BATO complexes. Studies with NIH 3T3 fibroblasts indicated that the neutral complexes did not show myocyte specific accumulation.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Comparative biodistribution and antibody-dependent cellular cytotoxicity of native and heavy chain chimeric antibody.

We have recently chimerized the heavy chain of the pan-carcinoma monoclonal antibody (mAb) B72.3. Studies were undertaken to compare the IgG1 chimeric antibody, B72.3-1-3 with native murine B72.3 (nB72.3). Using fluorescence-activated cell sorting analysis, B72.3-1-3 demonstrated specific binding to fresh LS174T tumor cells. Biodistribution of 131I B72.3-1-3 was similar to 131I nB72.3 in nude mice bearing LS174T xenografts. Peak radiolocalization indices were noted on day 6 for B72.3-1-3 and day 8 for nB72.3. Both antibodies were capable of imaging LS174T tumors by radioimmunoscintigraphy. Antibody-dependent cellular cytotoxicity of LS174T by human peripheral blood lymphocytes was tested in 8h 51Cr release assays. With either no antibody or nB72.3, lymphocytes were not capable of killing LS174T cells. However, B72.3-1-3 at a concentration of 5 and 50 micrograms/ml mediated significant lysis of tumor cells by human lymphocytes. These results suggest that chimeric antibodies retain their binding properties to tumor cells and display biodistribution patterns similar to their unmodified counterparts. Such modifications may reduce the deleterious human antimouse antibody response to murine mAbs as well as augment antibody-dependent cellular cytotoxicity of tumor cells by human effectors.     

Structure—distribution relationship studies of 99mTc-2,3-dioxime complexes

The structure-distribution relationship studies of a homologous series of anionic ^99mTc-2,3-dioximino-alkanes in mice, showed very low specific organ uptakes due to a combination of rapid clearance and excessive blood binding factors as found for the cationic ^99mTc-diamine analogues (Salako and Theobald, Nucl. Med. Biol.17, 437–441; 1990. The dioxime complexes showed a preference for hepatobiliary clearance unlike the diamine complexes which were cleared through the renal tract. The relationship between the amount cleared through the biliary tract and molecular weight of the dioxime complexes is binomial, with the optimum about the C-8 chain homologue. The hepatobiliary clearance model which was discovered for these complexes follows a multiple correlation equation which has a combination of four physico-chemical properties in the order: log P, charge, protein binding and molecular weight. It was observed particularly that the log P and charge have broadly similar magnitudes (to those in the urinary model of the diamine complexes) but reversed signs. The sign on the charge coefficient is negative, indicating that an overall negative charge is required for efficient hepatobiliary extraction and excretion of these Tc-complexes.     

Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab' fragments is described here. Chimeric mouse-human B72.3 Fab' and F(ab')2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab' bound weakly to the protein G-Sepharose it could be separated from F(ab')2 and eluted with a pH 7 wash whereas B72.3 F(ab')2 required elution at pH 2. Both Fab' and F(ab')2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab' or F(ab')2 fragments from CHO cell supernatant.     

Allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) inhibit tumour-specific angiogenesis by downregulating nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) production.

Angiogenesis, a crucial step in the growth and metastasis of cancers, is initiated with vasodilation mediated by nitric oxide (NO). The pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha), is a mediator of nitric oxide synthesis. We analyzed the effect of allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) on serum NO as well as TNF-alpha level during angiogenesis. In vivo antiangiogenic activity was studied using B16F-10 melanoma cell-induced capillary formation in C57BL/6 mice. Intraperitoneal administration of AITC and PITC at a concentration of 25 microg/dose/animal significantly inhibited tumour-directed capillary formation. Treatment of AITC and PITC significantly downregulated serum NO as well as TNF-alpha level in angiogenesis-induced animals compared to untreated control animals. The in vitro antiangiogenic study, using rat aortic ring assay, showed that both AITC and PITC at non-toxic concentrations inhibited the production of proangiogenic factors from B16F-10 melanoma cells which was evident with the inhibition of microvessel outgrowth from aortic rings. Both AITC and PITC significantly inhibited sodium nitroprusside as well as TNF-alpha-induced microvessel outgrowth from rat aortic ring. Administration of AITC and PITC also significantly reduced NO and TNF-alpha production by LPS-stimulated macrophages both in vivo as well as in vitro. Bio-assay using serum of angiogenesis-induced animals and supernatant from LPS-stimulated macrophages clearly confirmed the downregulatory action of AITC and PITC on TNF-alpha production. These results clearly demonstrated that AITC and PITC inhibited tumour-specific angiogenesis by downregulating NO and TNF-alpha production.     

Location of Chemically Modified Lysine 41 in the Structure of Bacteriorhodopsin by Neutron Diffraction

Purple membranes were prepared in which bacteriorhodopsin was labeled at lysine 41 with phenylisothiocyanate (PITC) and with perdeuterated PITC. The in-plane position of this small label containing only five deuterons was determined from the differences between the neutron diffraction intensities of the two samples. At 8.7-Å resolution the Fourier difference map revealed a well-defined site between helices 3 and 4. This position was confirmed by a refinement procedure in reciprocal space. Model calculations showed that the observed difference density had the right amplitude for the label. Thus it is possible to locate a small group in a large protein structure by replacing as few as five hydrogens by deuterium. The observed location of PITC restricts the number of possibilities for the assignment of helix B in the sequence (to which lysine 41 is attached) to one of the seven helices of the structure. Taking into account the size of the label and the length of the lysine side chain our result excludes helices 1, 2, and 7 as candidates for B.     

Immunohistochemical studies with monoclonal antibodies B72.3 and MA5 on histologic and cytologic specimens from benign and malignant breast lesions

Monoclonal antibodies B72.3 and MA5 were tested by the avidin-biotin immunoperoxidase method in histologic sections of 38 benign, 22 precancerous and 22 cancerous breast lesions, as well as in fine needle aspiration (FNA) smears and cell blocks of 25 breast carcinomas. Neither B72.3 nor MA5 was specific for breast cancer cells: both also reacted with cells from benign and precancerous conditions. B72.3 as a "detector" of malignant cells or their precursors was superior to MA5, however: it was not reactive to cells in most benign breast lesions (mammary duct ectasia, fibroadenoma and ductal hyperplasia, with and without atypia). Cancerous cells had heterogeneous immunostaining with B72.3, which may lead to false-negative results in relatively hypocellular FNA samples. FNA samples prepared as both smears and cell blocks provided the most abundant cellular samples and the lowest false-negative immunostaining reaction of cancerous cells with B72.3.     

Provider-Initiated HIV Testing and Counselling in Rwanda: Acceptability among Clinic Attendees and Workers,Reasons for Testing and Predictors of Testing

Introduction

Routine provider-initiated HIV testing and counselling (PITC) may increase HIV testing rates, but whether PITC is acceptable to health facility (HF) attendees is unclear. In the course of a PITC intervention study in Rwanda, we assessed the acceptability of PITC, reasons for being or not being tested and factors associated with HIV testing.

Methods

Attendees were systematically interviewed in March 2009 as they left the HF, regarding knowledge and acceptability of PITC, history of testing and reasons for being tested or not. Subsequently, PITC was introduced in 6 of the 8 HFs and a second round of interviews was conducted. Independent factors associated with testing were analysed using logistic regression. Randomly selected health care workers (HCWs) were also interviewed.

Results

1772 attendees were interviewed. Over 95% agreed with the PITC policy, both prior to and after implementation of PITC policy. The most common reasons for testing were the desire to know one’s HIV status and having been offered an HIV test by an HCW. The most frequent reasons for not being tested were known HIV status and test not being offered. In multivariable analysis, PITC, age ≥15 years, and not having been previously tested were factors significantly associated with testing. Although workload was increased by PITC, HIV testing rates increased and HCWs overwhelmingly supported the policy.

Conclusion

Among attendees and HCWs in Rwandan clinics, the acceptability of PITC was very high. PITC appeared to increase testing rates and may be helpful in prevention and early access to treatment.     

Radiolabeling of immunoglobulin without loss of antibody activity--use of 14C-phenylisothiocyanate with human cytotoxic antibody.

Radioiodination of immunoglobulins, particularly human cytotoxic IgG, induced a marked loss of antibody activity. Phenylisothiocyanate (PITC) readily reacts with alpha-amino groups and the epsilon-amino groups of lysines to form phenylthiocarbamyl derivatives. Since PITC is commercially available with 14C, 3H, and 35S substitutions, it provided an alternative means for labeling immunoglobulin which preserved antibody activity. There were approximately 80 PITC binding sites per IgG molecule, and 14C-PITC was bound with an efficiency in excess of 80%. With as many as 40 PITC molecules bound per IgG molecule, full cytotoxic activity was retained by both anti-HLA isoantisera and human anti-melanoma autoantisera. Even with 70 to 80 molecules of PITC bound per IgG molecule, over 80% of the antibody activity remained. Radiolabeling of antibody with 3H, 14C, or 35S-substituted PITC will greatly facilitate studies of antibody binding, particularly to cell surface antigens.     

Effect of N,N-dimethylglycine supplementation in parturition feed for sows on metabolism, nutrient digestibility and reproductive performance

The current pilot study assessed the influence of N,N-dimethylglycine (DMG) on insulin sensitivity, glucose and fat metabolism, nutrient digestibility and reproductive performance of sows in the peripartal period. At day 105 of gestation, 25 sows were randomly assigned to the control (n = 13) or the DMG group (n = 12). Sows from the DMG group were supplemented with 1 g DMG/kg feed until day 3 of lactation. After an overnight fast 1 day after farrowing, a blood sample of each sow was drawn. The plasma was analyzed for insulin, glucose, fructosamine, leptin, thiobarbituric acid reactive substances (TBARS), ferric reducing ability of plasma (FRAP), non-esterified fatty acids (NEFA) and triglycerides (TG) and an oral glucose tolerance test was performed. A rectal feces sample was collected and the apparent fecal digestibility (AFD) of crude fat (CFAT), crude protein (CP) and nitrogen-free extract (NFE) was calculated after proximate analyses. Finally, a colostrum sample was collected from each sow and analyzed for the presence of DMG. Reproductive performance parameters were recorded. The results showed an improvement in the AFD of CFAT, CP and NFE when DMG was supplemented. This beneficial effect confirms the hypothesis that DMG acts as an emulsifying agent. The improvement in digestibility in the DMG group was accompanied by a numerical increase in plasma TG (P = 0.067). Plasma NEFA concentrations were not different between treatment groups. DMG supplementation neither affected glucose clearance nor influenced plasma insulin, glucose, fructosamine or leptin levels. TBARS and FRAP also remained unaffected, despite previously reported anti-oxidative properties of DMG. Furthermore, no significant impact on reproductive performance could be recorded. In conclusion, DMG supplementation significantly improved nutrient digestibility. Possible beneficial effects on energy metabolism and reproductive performance of sows should be tested when DMG is supplemented for a longer period of time or at a higher dose.     

Immunocytochemical characterization of large-cell carcinomas of the lung. Role, limitations and technical considerations.

To clarify the use of cytologic preparations, particularly those previously stained by the Papanicolaou method, for the immunocytochemical evaluation of large-cell carcinomas (LCCs), 37 cytologic preparations from cases diagnosed as LCC were examined using a battery of immunocytochemical stains for keratin, chromogranin, common leukocyte antigen (CLA) and B72.3. Thirty-two specimens were from the thoracopulmonary region (12 fine needle aspirates of the lung, 7 bronchial brushings, 5 bronchial washings, 2 sputa and 6 pleural fluids); the remaining specimens were fine needle aspirates of 3 lymph nodes, 1 vertebral body and 1 liver. Of the specimens analyzed, 30 of 37 were positive for keratin and 7 of 35 were positive for B72.3 (6 were positive for both). Only 1 of 37 was positive for CLA while none of 37 was positive for chromogranin. Six specimens showed no reaction with either keratin, B72.3 or chromogranin. These results confirm that the majority of LCCs consist of epithelial cells of either a squamous or an adenocarcinomatous type. They also show that immunocytochemistry is a useful diagnostic adjunct that can be applied to cytologic preparations previously stained by the Papanicolaou method; this is important since immunostaining is often considered after undifferentiated malignant cells are encountered in a previously stained preparation. However, a thorough understanding of some technical limitations is critical in the evaluation of the results of this technique when it is applied to cytologic specimens.     

Anti-CEA and other antibodies in the study of gastrointestinal tumors.

Localization of gastrointestinal tumors by means of labeled monoclonal antibodies is a new, sensitive and suitable technique currently used in several centers. Encouraging results have been documented with several monoclonal antibodies by different authors. This article reviews our experience with radioimmunoscintigraphy in 59 patients with colorectal cancer in follow-up, using 131I and 111In labeled B72.3, and in 16 patients with primary gastrointestinal tumors using 99mTc anti-CEA monoclonal antibody (type F023C5). The sensitivity of both B72.3 and anti-CEA was greater than 70% either for primary tumors and abdominal recurrences or distant metastases except hepatic ones. A significant gradient in antibody uptake was measured on surgical biopsies between tumors and normal tissues allowing a good in vivo contrast for gamma detection. We have defined the impact of some factors affecting in vivo tumor targeting. In fact, pharmacodynamics of MAbs, percentage of injected dose bound to tissues were measured, and in particular antigenic content in tumor nodules was quantified. Furthermore, the results of RIS were compared to those obtained by CT and other imaging modalities.     

A panel of antibodies useful in the cytologic diagnosis of metastatic melanoma.

Five antibodies, 2D.1 (pan-leukocyte), AE-1,3 (anti-keratin), B72.3 (anti-carcinoma), ME 1-14 (alpha-chondroitin sulfate proteoglycan) and polyclonal S-100 protein (P-S100), were tested to determine if this panel could be used immunocytochemically to differentiate melanoma from nonmelanoma. A total of 161 cases were evaluated: 145 fine needle aspirates of various body sites and 16 effusions, consisting of 52 melanomas, 41 adenocarcinomas, 11 squamous cell carcinomas, 14 undifferentiated carcinomas, 8 small cell carcinomas, 8 miscellaneous carcinomas, 8 primary central nervous system (CNS) tumors, 7 lymphomas/leukemias, 4 sarcomas and 8 benign effusions. The 52 melanomas were stained by ME 1-14 (in 31 cases) and by P-S100 (in 39 cases), but not by B72.3, AE-1,3 or 2D.1. The 82 carcinomas reacted with P-S100 (in 25 cases), B72.3 (in 37 cases), AE-1,3 (in 68 cases) and 2D.1 (in 1 case), but not with ME 1-14. Lymphomas were stained only by 2D.1 (5 of 7 cases). The eight primary CNS tumors reacted solely with ME1-14 (in 3 cases) and P-S100 (in 3 cases). The eight benign effusions exhibited staining by ME 1-14 (in 1 case), P-S100 (in 1 case), AE-1,3 (in 3 cases) and 2D.1 (in 8 cases), but not by B72.3. Thirty-six cases (including 11 melanomas) failed to stain with any antibody. In summary, 41 of 52 melanomas and 4 of 8 CNS tumors stained with ME1-14, P-S100 or both and were negative for B72.3, AE-1,3 and 2D.1. Only 2 of 101 other nonmelanomas exhibited this pattern. Thus, this panel distinguishes melanoma from other neoplastic and nonneoplastic processes in the majority of cases.     

Synthesis and DNA binding properties of dioxime-peptide nucleic acids

Peptide nucleic acids (PNAs) C- or N-modified with dioxime ligands were prepared by solid-phase synthesis using iron(II)-clathrochelates as protected dioxime building blocks. These PNA bind complementary DNA sequence specifically, though with much reduced affinity in comparison with nonmodified PNA. The dioxime-PNA conjugates bind Cu2+ and Ni2+ at microM concentration.     

Synthesis, characterization, and biological evaluation of neutral nitrido technetium(V) mixed ligand complexes containing dithiolates and aminodiphosphines. A novel system for linking technetium to biomolecules

A new biomolecule labeling method that utilizes the [(99m)Tc(N)(PNP)](2+) metal fragment is presented. Thus, a series of nitrido mixed-ligand M(V) complexes (M = (99m)Tc, (99g)Tc, Re), [M(N)(Ln)(PNP)], where Ln is the dianionic form of a dithiolate or substituted-dithiolate ligand and PNP is an aminodiphosphine, is described. (99m)Tc complexes can be prepared using either a two-step or a three-step procedure starting from generator-eluted pertechnetate through a prereduced mixture of [(99m)Tc(N)]-containing species, followed by sequential or contemporary addition of the relevant dithiolate and aminodiphosphine. The reactions of 2,3-dimercaptopropionic acid (H(2)L1) with [Tc(N)(PNP)](2+) were investigated in detail. It was found that this bidentate ligand coordinated the metal fragment through the [S(-),S(-)] donor atom pair, to yield neutral mixed-ligand complexes [(99m)Tc(N)(L1)(PNP)] in high specific activity. The additional carboxylic functional group was not involved in metal coordination, thus remaining available for conjugation to target-specific molecules. Dithiolates incorporating pendant functional group(s) gave rise to a 1:1 diastereoisomeric mixture of syn-[M(N)(Ln)(PNP)] and anti-[M(N)(Ln)(PNP)] derivatives, depending on the relative orientation of the dithiolate substituent(s) with respect to the terminal nitrido group, and no isomeric conversion was detected. (99m)Tc species had been proven to be identical with the (99g)Tc complexes prepared at the macroscopic level by comparison of the corresponding radiometric and UV/vis HPLC profiles. Challenge experiments with cysteine or glutathione indicated that these physiological agents had no effect on the stability of this class of mixed-ligand (99m)Tc-complexes. Biodistribution studies in rats of selected (99m)Tc-complexes showed a rapid clearance from the blood and tissues after 60 min pi.     

Applications of monoclonal antibodies in clinical cytology as exemplified by studies with monoclonal antibody B72.3. The George N. Papanicolaou award lecture

The monoclonal antibody (MAb) B72.3, reactive with a high-molecular-weight, glycoprotein, tumor-associated antigen, designated TAG-72, has been previously shown to be reactive with formalin-fixed, paraffin-embedded tissue sections of adenocarcinomas of the ovary, colon and breast, but not a variety of normal adult tissues. It has demonstrated utility as an immunocytochemical adjunct for the diagnosis of carcinoma in cell blocks and cytocentrifuge preparations of human serous effusions, with selective reactivity for tumor cells (particularly adenocarcinoma) over reactive mesothelium. Using the avidin-biotin complex (ABC) method of immunoperoxidase staining and formalin-fixed, paraffin-embedded cell suspensions, MAb B72.3 detected tumor cells in effusions from all of 21 patients with adenocarcinoma of the breast. No reactivity was demonstrated in any cell type in benign effusions from 41 patients. In contrast, MAb B72.3 showed no reactivity to leukemic or lymphomatous effusions, or to mesothelial cells from malignant effusions. MAb B72.3 also detected adenocarcinoma cells in effusion specimens from 12 of 12 patients with adenocarcinoma of the lung and 16 of 16 patients with adenocarcinoma of the ovary. MAb B72.3 has recently been used with fine needle aspiration (FNA) biopsy specimens and the corresponding surgically excised tumors to determine cellular reactivity. Using the ABC immunoperoxidase method, fine needle aspirates and corresponding surgically excised tumors were analyzed for TAG-72 expression. Positive staining with MAb B72.3 was observed in needle aspirates of 27 of 27 adenocarcinomas and adenosquamous carcinomas of the lung, 17 of 21 adenocarcinomas of the breast, 6 of 6 adenocarcinomas of the colon and in carcinomas from other body sites. In contrast, 21 small-cell carcinomas of the lung, 13 malignant melanomas, 2 lymphomas and 2 sarcomas did not stain with the antibody. Benign lesions from the breast, lung, pancreas, parotid and thyroid also showed no staining. In many patients, tumor-bearing tissue had also been resected and was available for comparative examination with MAb B72.3. In more than 90% of these patients, the staining patterns of the tumor cells in the aspirates were found to be predictive of the patterns of antibody reactivity in the comparable surgically resected tumors. From these studies, it is concluded that MAb B72.3 defines a tumor-associated antigen that is expressed in neoplastic cells versus benign cells, that is most selectively expressed in carcinomas and that may be used as a novel adjunct for the diagnosis of neoplasms in effusions and in fine needle aspiration biopsies.     

Preparation of no-carrier-added technetium-99m complexes via metal-assisted cleavage from a solid phase

A novel method for the preparation of no-carrier-added (nca) complexes [99mTc(CO)3L] (L = diethylenetriamine or picolylamine-N-acetic acid) is described. The ligands were covalently bound to a solid support of organic polymers via formation of a tertiary amine from the chelating unit. This C-N bond to the solid phase is selectively cleaved during the formation of the technetium complexes by intramolecular nucleophilic attack of a remaining hydroxy ligand to the alpha-carbon. The complex [99mTc(CO)3L] is released into solution while uncomplexed ligand and uncleaved complex remain solid-phase bound. High specific activity technetium complexes can then be isolated by simple filtration. Cleavage yield depends on temperature, pH, and ligand. Up to 50% release from the solid phase could be achieved under optimized conditions. Corresponding to the 99mTc concentration, free ligand is present in concentrations lower than 10(-7) M. If a targeting vector is conjugated to these ligands, no-carrier-added radiopharmaceuticals can be prepared in that way.