Antibody fragments labeled with fluorine-18 and gallium-68: In vivo comparison with indium-111 and iodine-125-labeled fragments

Although monoclonal antibodies have been radiolabeled with many different radionuclides, the application of positron emission tomography (PET) to the imaging of radiolabeled antibodies has been limited to the investigation of a small number of long-lived radionuclides. In this study, we labeled F(ab′)₂ fragments of a mouse monoclonal antibody (BB5-G1) specific for a human parathyroid surface antigen with the positron emitting radionuclides, gallium-68 and fluorine-18. The biodistribution of the fragments was evaluated in a nude mice model and the results were compared to those obtained with fragments labeled with iodine-125 and indium-111 using conventional labeling techniques. All labeled fragments bound to human parathyroid tissue implanted in nude mice, with parathyroid-to-muscle ratios reaching as high as 10:1, 4 h after administration. A major difference was observed in the uptake and clearance of the various labeled fragments through the kidney. The halogen activity cleared, but the metal radioactivity was retained in the kidney. The results indicate that the fluorine-18 or gallium-68 labeled fragment may be useful for parathyroid imaging with positron emission tomography.     

A mouse model for calculating the absorbed beta-particle dose from (131)I- and (90)Y-labeled immunoconjugates, including a method for dealing with heterogeneity in kidney and tumor.

Flynn, A. A., Green, A. J., Pedley, R. B., Boxer, G. M., Boden, R. and Begent, R. H. J. A Mouse Model for Calculating the Absorbed Beta-Particle Dose from (131)I- and (90)Y-Labeled Immunoconjugates, Including a Method for Dealing with Heterogeneity in Kidney and Tumor. Radiat. Res. 156, 28-35 (2001).Conventional internal radiation dosimetry methods assume that the beta-particle energy is absorbed uniformly and completely in the source organ and that the radioactivity is distributed uniformly in the source. However, in mice, a considerable proportion of the beta-particle energy can escape the source organ, resulting in large cross-organ doses. Furthermore, the distribution of radioactivity is generally heterogeneous in kidney and tumor. Therefore, a model was developed to account for cross-organ doses and for the effects of heterogeneity in kidney and tumor in mice for two of the most important radionuclides used in therapy, (131)I and (90)Y. Most mouse organs were modeled as single-compartment ellipsoids or cylinders, while heterogeneity in kidney and in tumor was addressed by using two compartments to represent the cortex and the medulla and viable and necrotic cells, respectively. The dimensions of these models were taken from previous studies, with the exception of kidney and tumor, which were defined using radioluminography and mosaics of high-power microscopy images. The absorbed fractions in each compartment were calculated using beta-particle point dose kernels. The self-organ dose was significantly higher for (131)I compared to (90)Y in all compartments, but a considerable amount of beta-particle energy was shown to escape the source organ for both radionuclides, with as much as 85% and 36% escaping the marrow for (90)Y and (131)I, respectively. The cortex was found to occupy a greater proportion of the total kidney volume than the medulla, and consequently the self-dose was higher in the cortex. In addition, the thickness of the viable shell in the tumor increased with tumor size, as did the self-dose fractions in both necrotic and viable areas. This dosimetry model improves dose estimates in mice and gives a conceptual basis for considering dosimetry in humans.     

The geterogenity of 137Cs and 90Sr distribution and dose loading on critical tissues of main seedling root

It is shown, that the roots of plants concentrate 137Cs and 90Sr from water solutions in different zones: 137Cs--mainly in a meristem zone, 90Sr--in a stretching zone. The similar character of radionuclide distribution was established regarding water and soil cultures. The real dose loading on critical tissues of main root have appeared to be much higher than it was expected from the assumption of uniform distribution of radionuclides in tissues.     

Engineered antibody fragments and the rise of single domains

With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragments retain the targeting specificity of whole mAbs but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Antibody fragments have been forged into multivalent and multi-specific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents. Single-domain antibodies are anticipated to significantly expand the repertoire of antibody-based reagents against the vast range of novel biomarkers being discovered through proteomics. As this review aims to show, there is tremendous potential for all antibody fragments either as robust diagnostic reagents (for example in biosensors), or as nonimmunogenic in vivo biopharmaceuticals with superior biodistribution and blood clearance properties.     

Cyclooxygenase-1 and cyclooxygenase-2 expression in rat kidney and adrenal gland after stimulation with systemic lipopolysaccharide

Cyclooxygenase-2 (COX-2) is a recently discovered isoform of cyclooxygenase that is inducible by various types of inflammatory stimuli. Although this enzyme is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins, the precise location, distribution, and regulation of prostaglandin synthesis remains unclear in several tissues. Using in situ hybridization histochemistry, we investigated the induction of COX-1 and COX-2 mRNA expression after systemic administration of a pyrogen, lipopolysaccharide (LPS), in kidney and adrenal gland in the rat. The COX-2 mRNA signals dramatically increased 1 h after LPS treatment in the kidney outer medulla and adrenal cortex, where almost no or little expression was observed in nontreated animals, and returned to control levels within 24 h. COX-2 mRNA levels increased in the kidney inner medulla 6 h after treatment. There was also a significant increase in mRNA levels in the kidney cortex and adrenal medulla. On the other hand, COX-1 mRNA levels did not show any detectable changes except in the kidney inner medulla, where a significant downregulation of mRNA expression was observed after LPS treatment. Light and electron immunocytochemistry using COX-2 antibodies showed that strong COX-2 immunoreactivity was localized to certain cortical cells of the thick ascending limb of Henle. In addition, based on double-staining with antiserum to nitric oxide synthase (NOS) four further cell populations could be identified in kidney cortex, including weakly COX-2-positive, NOS-positive macula densa cells. After LPS treatment, changes in COX-2 immunoreactivity could be observed in interstitial cells in the kidney medulla and in inner cortical cells in the adrenal gland. These results show that COX-2 is a highly induced enzyme that can be up-regulated in specific cell populations in kidney and adrenal gland in response to inflammation, leading to the elevated levels of prostaglandins seen during fever. In contrast COX-1 mRNA levels remained unchanged in this experimental situation, except for a decrease in kidney inner medulla.     

人胸腺内交错突细胞的免疫组织化学研究

本文用免疫组织化学方法检测胎儿期,儿童以及成人胸腺内交错突细胞的分布和ＨＬＡＤＲ抗原的表达．结果表明：Ｓ－１００抗体能够清楚显示胸腺内交错突细胞,这些细胞主要分布在皮,髓质交界处和髓质内,在皮质内的交错突细胞大都单个存在,偶而看到交错突细胞成团存在．交错突细胞的周围,常见有淋巴细胞形成玫瑰花结．出生后随年龄的增长,交错突细胞逐渐减少。ＨＬＡ－ＤＲ抗体能与胸腺内多种细胞反应,如皮,髓质内上皮细胞,巨噬细胞和交错突细胞,但它们染色强度不等．交错突细胞ＨＬＡ－ＤＲ抗体的阳性反应位于质膜和突起部,染色强阳性,巨噬细胞反应不尽一致一般多为阳性,胞质内未见有吞噬淋巴细胞碎片．上皮细胞染色一般由弱阳性到阳性,核阴性,仅有质膜呈阳性反应,有关ＨＬＡ－ＤＲ抗原表达的可能意义进行了讨论．     

基因工程小分子抗体的研究进展

随着分子生物学的发展,基因工程小分子抗体越来越受到人们关注,该抗体具有分子量小、结构简单、穿透性强、免疫源性低、低成本生产的优点,并且可与毒素、放射性核素、酶、细胞因子等结合用于肿瘤的诊断和治疗,临床应用前景十分广阔。对基因工程小分子抗体近几年的发展与应用作一综述。     

Distribution of amino acids and amino-acid enzymes in whole kidney and renal cortex. Effect of 24-h starvation

The levels of activity of amino-acid enzymes (alanine-, aspartate- and tyrosine transaminases, serine dehydratase, glutamate dehydrogenase, glutamine synthetase, arginase and adenylate deaminase) in the kidney cortex and whole kidney of control and 24-h starved adult rats have been determined from crude homogenates. The individual amino-acid content of the two kidney fractions indicated has also been studied. Serine dehydratase was found mainly in the cortex, whilst glutamine synthetase presence was mainly limited to the medulla. The distribution of adenylate deaminase and glutamate dehydrogenase is remarkably uniform along the kidney. Starvation induced decreases in arginase and adenylate deaminase activities as well as a decrease in cortical serine dehydratase. Analysis of the variance of the enzyme data showed no significant overall changes with respect to distribution and starvation. The same analysis applied to aminograms indicated only significant changes when the distribution was studied on starved samples as a whole, with changes mainly in the urea cycle and some essential amino acids. The results suggested a remarkable degree of uniformity in kidney composition both with respect to amino acid and enzyme distribution. This uniformity is not markedly affected by starvation despite the important r?le of kidney in the overall amino-acid economy of the Mammal.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Properties and differential regulation of two fatty acid binding proteins in the rat kidney

A protein from rat kidney was characterized that had several properties common to a multigene family of fatty acid binding proteins identified in other tissues. The putative kidney fatty acid binding protein (FABP) was purified from the soluble fraction of kidney homogenates using gel filtration and ion exchange chromatography. It was relatively abundant, had an apparent molecular mass of 15.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, bound equimolar amounts of oleic acid, and could be distinguished from other FABPs on the basis of size, amino acid composition, and tissue distribution. Polyclonal antibodies to kidney FABP were obtained and used to show that only kidney contained the 15.5-kDa protein, although the antibodies also recognized a slightly larger and less abundant protein in kidney that also was present in bladder. Rat kidney also contained heart FABP, and the properties of both FABPs in rat kidney were compared. The distribution of both proteins within the kidney differed, with kidney FABP being localized almost exclusively within the cortex, whereas heart FABP was found both in cortex and medulla. Kidney FABP was expressed developmentally after the neonatal period, whereas heart FABP was present in both neonatal and adult kidney at comparable amounts. Hypertension induced by mineralocorticoids or infusion of angiotensin II caused a marked suppression of kidney FABP expression, whereas amounts of heart FABP in kidney were unchanged. The studies showed that rat kidney contains at least two FABPs, and that these proteins are differentially regulated, suggesting that functional differences between the proteins may exist.     

Postembedding immunogold histochemistry for the localization of laminin and its E4 and P1 fragments in mouse kidney embedded in LR-White and LR-Gold

Summary A method is presented which permits the ultrastructural localization of laminin and its E4 and P1 subunits in the renal cortex of the mouse embedded in LR-White or LR-Gold. It was performed with postembedding immunogold histochemistry using polyclonal antibodies against either the entire laminin molecular or the E4 fragment or with a monoclonal antibody against the P1 fragment. Localization of laminin was achieved in LR-White and in LR-Gold embedded kidney. Using polyclonal antibodies against the entire laminin molecule, laminin could be localized with direct as well as with indirect immunogold histochemistry with a gold labelled IgG as secondary antibody. In contrast, immunostaining for the E4 or the P1 fragments was possible only with antibodibodies directly labelled with gold.     

Angiotensin converting enzyme predominates in the inner cortex and medulla of the rat kidney

Regional distribution of angiotensin converting enzyme(ACE) in the rat kidney was studied. The ACE activities in the inner cortex and outer medulla were about 10 and 5 times those in the outer cortex, respectively. The activity in the inner medulla or papilla was much the same as that in the outer cortex. Immunofluorescence was greatest in the proximal tubules in the inner cortex, while the outer medulla and the inner medulla or papilla showed a weak fluorescence. The brush border membranes isolated from the inner cortex also possessed about 10 times the ACE activity seen in the outer cortex. The results indicate that the major source of renal ACE is not the proximal convoluted tubules in the outer cortex, but rather the brush border membranes of proximal tubules in the inner cortex. The contribution of ACE in the inner cortex would therefore be predominant.     

Immunocytochemical localization of gamma-glutamyl-transferase on isolated renal cortical tubular fragments

The localization of gamma-Glutamyltransferase (gamma-GT, E.C.2.3.2.2) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney gamma-GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments. The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral gamma-GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.     

Relative biological effectiveness of 103Pd and 125I photons for continuous low-dose-rate irradiation of Chinese hamster cells

Monolayers of Chinese hamster lung cells (CCL-16) in a polystyrene phantom were irradiated in vitro by 103Pd and 125I sources at dose rates of 6 to 72 cGy/h. Cell survival curves for acute high-dose-rate irradiation (over 30 Gy/h) were also measured using nearly monoenergetic X-ray beams which were designed to simulate the mean energies of photons emitted by 125I and 103Pd and also using a clinical 250 kVp X-ray beam. A profound dose-rate effect is observed over the dose-rate range of 6 to 20 cGy/h. An inverse dose-rate effect was observed for both radionuclides, with its onset occurring at a dose rate of about 20-30 cGy/h. The average RBE of 103Pd relative to 125I was determined to be 1.45 +/- 0.07, 1.41 +/- 0.07, 0.70 +/- 0.07 and 1.49 +/- 0.07 at dose rates of 6.9, 12.6, 19.0 and 26.7 cGy/h, respectively. Because 103Pd implants are generally prescribed at a higher initial dose rate (21 cGy/h) than the corresponding 125I implants (7 cGy/h), the effects of both dose rate and photon energy on biological response must be considered together. For the CCL-16 cells, the RBE of 103Pd at 19.0 cGy/h relative to that of 125I at 6.9 cGy/h was estimated to be 2.3 +/- 0.5.     

Tissue distribution of smg p25A, a ras p21-like GTP-binding protein, studied by use of a specific monoclonal antibody

We made a monoclonal antibody specifically recognizing smg p25A among many ras p21-like GTP-binding proteins and investigated the tissue distribution of smg p25A by use of this antibody. By immunoblot analysis, smg p25A was detected in rat brain and bovine adrenal medulla but not in bovine adrenal cortex or other rat tissues including thymus, spleen, lung, heart, liver and kidney. However, by immunocytochemical studies, smg p25A was detected not only in the synaptic areas of rat brain and the chromaffin cells of bovine adrenal medulla but also in the endocrine cells of rat pancreatic islets, the acinar cells of rat exocrine pancreas and the exocrine cells of rat submaxillary gland. These results suggest that smg p25A is involved in the regulation of secretory processes not only in synapses but also in other endocrine and exocrine secretory cells.     

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.     

Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L ‐[³H]‐carnitine in a Na⁺‐dependent and ‐independent manner. OCTN2 mediates the Na⁺/L ‐carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na⁺/L ‐carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na⁺‐independent L ‐carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na⁺‐independent L ‐carnitine uptake was inhibited by both, anti‐OCTN2 and anti‐OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high‐ and a low‐affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na⁺‐independent L ‐carnitine uptake exhibited trans‐stimulation by intravesicular L ‐carnitine or betaine. This trans‐stimulation was inhibited by anti‐OCTN3 antibody, but not by anti‐OCTN2 antibody, indicating that OCTN3 can function as an L ‐carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla. J. Cell. Physiol. 223: 451–459, 2010. © 2010 Wiley‐Liss, Inc.     

Dimeric dihydrodiol dehydrogenase in monkey kidney. Substrate specificity, stereospecificity of hydrogen transfer, and distribution

Chemical cross-linking and NADPH binding studies suggested that the native dihydrodiol dehydrogenase from monkey kidney is a basic dimer having a molecular weight of 78,000 and one active site per the subunit. The enzyme oxidized specifically trans-dihydrodiols of benzene and naphthalene, whereas it catalyzed the reduction of dihydroxyacetone and dihydroxyacetone phosphate at a physiological pH, 7.4. The Km and kcat values for dihydroxyacetone phosphate were 5.0 mM and 4.3 s-1, respectively. The enzyme transferred the 4-pro-R hydrogen atom of NADPH to the carbonyl substrate. Immunochemical experiments using an antibody against the dimeric enzyme revealed the specific distribution of the enzyme in the kidney of this animal. By immunohistochemical staining with the specific antibody, the immunoreactivity was found in proximal and distal tubules of the cortex, and in the loop of Henle of the medulla.     

Long-term development of the radionuclide exposure of murine rodent populations in Belarus after the Chernobyl accident

As a determinant of the associated health risks, the behavior of radionuclides in natural ecosystems needs to be better understood. Therefore, the activity concentration of various long-lived radionuclides released due to the Chernobyl accident, and the corresponding contributions to the whole-body dose rate, was studied as a function of time in mammalian indicator species inhabiting the natural forest ecosystems of Belarus, the bank vole (Clethrionomys glareolus) and the yellow-necked mouse (Apodemus flavicollus). The activity concentrations of ¹³⁷Cs, ¹³⁴Cs, ⁹⁰Sr, ²³⁸Pu, ^239,240Pu, ²⁴¹Pu and ²⁴¹Am in soil and in animals were measured at five monitoring sites with different ground deposition of radionuclides at different distances from the destroyed reactor. The observed temporal pattern of the radionuclide activity concentration in the studied animal populations reflects the changes in biological availability of these isotopes for biota, mostly due to fuel particle destruction and appearance of dissolved and exchangeable forms of radionuclides. The time course of ^134+137Cs activity concentrations in animal populations appeared as a sequence of increase, peak and decrease. Maximal levels of radiocesium occurred 1–2 years after deposition, followed by an exponential decrease. Concentrations of incorporated ⁹⁰Sr increased up to the tenth year after deposition. The activity concentrations of transuranic elements (²³⁸Pu, ^239,240Pu, ²⁴¹Pu and ²⁴¹Am) were much lower than those of the other radionuclides, in the studied animals. A considerable activity of ²⁴¹Am in animals from areas with high levels of contamination was firstly detected 5 years after deposition, it increased up to the tenth year and is expected to increase further in the future. Maximal values of the whole-body absorbed dose rates occurred during the year of deposition, followed by a decrease in the subsequent period. Generally, this decrease was monotonic, mainly determined by the decrease of the external γ-ray dose rate, but there were exceptions due to the delayed maximum of internal exposure. The inter-individual distributions of radionuclide concentrations and lifetime whole-body absorbed doses were asymmetric and close to log-normal, including concentrations and doses considerably higher than the population mean values.     

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.