Labeled leukocyte imaging: current status and future directions.

The ability to radiolabel inflammatory cells that migrate to foci of infection was a significant milestone in the evolution of infection imaging. More than 20 years after being approved for clinical use in the United States, labeled leukocyte imaging using cells labeled with [(99m)Tc]exametazime or [(111)In]oxine remains the radionuclide procedure of choice for diagnosing most infections in the immunocompetent population. In the central nervous system, labeled leukocyte imaging is useful for differentiating infection from tumor; in the postoperative setting, this test facilitates the differentiation of infection from normal postoperative changes. Labeled leukocyte imaging accurately diagnoses mycotic aneurysms and infected prosthetic vascular grafts. In patients with fever of unknown origin, a negative study excludes, with a high degree of certainty, infection as the source of fever. Labeled leukocyte imaging accurately diagnoses pedal osteomyelitis and is useful for distinguishing infection from the neuropathic joint in this population. Together with bone marrow imaging, the labeled leukocyte study is the imaging procedure of choice for diagnosing prosthetic joint infection. There are limitations to the test. Most of the leukocytes labeled are neutrophils, and the procedure is most useful for detecting neutrophil-mediated inflammatory processes, i.e., bacterial infections. It is less useful for illnesses in which the predominant cellular response is other than neutrophilic, such as most opportunistic infections and spinal osteomyelitis. The in vitro labeling procedure is time consuming and is not routinely available. Results of in vivo leukocyte labeling methods have been variable; none are available in the United States. Labeled leukocyte imaging suffers from inherently poor quality images. Single photon emission compute tomography/computed tomography improves lesion localization, and will undoubtedly improve the accuracy of the test. Efforts to develop methods of labeling white cells with positron emitting compounds are underway and, if successful, should further strengthen the role of nuclear medicine in infection imaging.

Analysis of hepatocyte distribution and survival in vascular beds with cells marked by 99mTC or endogenous dipeptidyl peptidase IV activity.

Knowledge of the kinetics of cell distribution in vascular beds will help optimize engraftment of transplanted hepatocytes. To noninvasively localize transplanted cells in vivo, we developed conditions for labeling rat hepatocytes with 99mTc-pertechnetate. The incorporated o9mTc was bound to intracellular proteins and did not impair cell viability. When 99mTc hepatocytes were intrasplenically injected into normal rats, cells entered liver sinusoids with time-activity curves demonstrating instantaneous cell translocations. 99mTc activity in removed organs was in liver or spleen, and lungs showed little activity. However, when cells were intrasplenically transplanted into rats with portasystemic collaterals, 99mTc appeared in both liver sinusoids and pulmonary alveolar capillaries. To further localize cells, we transplanted DPPIV+ F344 rat hepatocytes into syngeneic DPPIV-recipients. Histochemical staining for DPPIV activity demonstrated engraftment of intrasplenically transplanted cells in liver parenchyma. In contrast, when 99mTc hepatocytes were injected into a peripheral vein, cells were entrapped in pulmonary capillaries but were subsequently broken down with redistribution of 99mTc activity elsewhere. Intact DPPIV+ hepatocytes were identified in lungs, whereas only cell fragments were present in liver, spleen, or kidneys. These findings indicate that although the pulmonary vascular bed offers advantages of easy accessibility and a relatively large capacity, significant early cell destruction is an important limitation.

Studies of liver repopulation using the dipeptidyl peptidase IV-deficient rat and other rodent recipients: cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule.

The feasibility of liver repopulation with hepatocytes has been shown, although clinical applications demand significant hepatic replacement. To show whether portal vascular bed in large animals could accomodate a greater cell number, we analyzed liver repopulation in syngeneic Fischer 344 rats deficient in dipeptidyl peptidase IV. This system allowed localization of transplanted normal hepatocytes in liver or various ectopic sites, as well as dual studies for analysis of gene expression. Interestingly, the product of a dipeptidyl peptidase IV substrate inactivated bile canalicular adenosine triphosphatase (ATPase) activity in normal but not in dipeptidyl peptidase IV-deficient rats, which allowed localization of dipeptidyl peptidase IV-deficient hepatocytes in normal rat liver for additional reversed transplantation systems. Further studies with genetically marked cells showed that because of the size difference between hepatocytes and portal vein radicles, intrasplenically transplanted cells were distributed in periportal areas (zone 1) in mice, whereas in larger animals (rats or rabbits) cells were also distributed downstream to midlobular (zone 2) or perivenous (zone 3) areas. Transplantation of an escalating number of hepatocytes showed that adult rats tolerated intrasplenic injection of a large cell number in single sessions (up to 1 X 10(8), approximately 10% to 15% of the host hepatocyte mass). Morphometric analysis of recipient livers showed survival of a significantly greater cell number with incorporation in host liver plates. At 4 weeks, transplantation of 2 x 10(7) hepatocytes into adult rats led to a survival of 1.4 +/- 1.0 x 10(6) transplanted cells/cm3 liver, whereas after transplantation of 5 x 10(7) cells or 7.5 x 10(7) cells, the number of surviving transplanted cells in the liver significantly increased to 4.1 +/- 1.4 x 10(6) transplanted cells/cm3 liver (mean, 2.9-fold; P<.003) and 5.5 +/- 1.3 x 10(6) transplanted cells/cm3 liver (mean, 3.9-fold; P<.003), respectively. When cells were injected in greater numbers, transplanted hepatocytes retained normal function and produced more serum albumin or hepatitis B surface antigen in deficient hosts. These data indicate the feasibility in larger animals of significant liver repopulation with hepatocyte transplantation. Use of dipeptidyl peptidase IV-deficient rats should help further analysis of mechanisms in liver repopulation.

Labeling of monoclonal antibodies with radionuclides.

Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as ethylenediaminepentaacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides.

Solid phase synthesis of thymosin beta 9.

Thymosin beta 9, a 41 residue thymic polypeptide, has been synthesized by a solid phase method. A modification of the low HF method was used to deprotect and cleave the peptide from the resin. Thymosin beta 9 was then obtained in analytically pure form by a one-step purification procedure in 32% yield. The activity of thymosin beta 9 in the terminal deoxynucleotidyl transferase assay was greater than calf thymus fraction 5, but comparable to thymosin beta 4. In contrast to thymosin alpha 1, neither beta 4 nor beta 9 was active in the rosette inhibition assay.

Effect of arsenical drugs on glutathione metabolism of Litomosoides carinii.

Despite centuries of therapeutic use, the mechanism of action of arsenicals against various diseases remains unknown. Because of the known inhibition of sulfhydryl-containing enzymes by arsenicals, we investigated the possibility that the anti-filarial effects of arsenical drugs might be exerted specifically through impairment of parasite thiol metabolism. We find: (1) arsenicals readily inhibit glutathione reductase of Litomosoides carinii but have little effect upon mammalian enzyme. (2) Administration of Melarsen B to filaria-infected gerbils causes decreases in filarial - but not host - glutathione reductase and reduced glutathione. (3) Such in vivo treatment does not, however, acutely affect parasite energy (ATP) metabolism. These results support the proposition that arsenicals may act through preferential interference with parasite thiol metabolism. The much greater susceptibility of parasite glutathione reductase to inhibition by arsenicals suggests that this enzyme may be a useful point of attack for new drugs.

Distribution of the inducer of differentiation bis-acetyl-diaminopentane in murine erythroleukemia cells.

Exposure of murine leukemia cells in culture to bis-acetyl-diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [3H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [3H]BADP was capable of binding to the plasma membrane-enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.

New compounds: N1,N8-bis(2,3-dihydroxybenzoyl)spermidine and analogs as potential iron-chelating drugs.

N1,N8-bis(2,3-dihydroxybenzoyl)spermidine was synthesized and evaluated as an iron-chelating drug. Homologs were prepared and evaluated together with a series of N,N'-bis(2,3-dihydroxybenzoyl)-alpha,omega-diaminoalkanes. Analogous 2-hydroxbenzoyl compounds also were synthesized and evaluated.

Rhodotorulic acid--investigation of its potential as an iron-chelating drug.

The use of rhodotorulic acid (RA) as an iron-chelating drug was suggested by experiments in hypertransfused rats in which urinary and fecal iron excretion were significantly enhanced in response to RA. The toxicity of the drug appears to be minimal at a parenteral dose less than 250 mg/kg. An increased excretion of zinc was the only notable side effect of the drug at the doses used. When administered i.v. to humans, RA was only 16% more effective than desferrioxamine (DF). Pharmacokinetic studies showed that RA persisted in the bloodstream of dogs 6 times longer than desferrioxamine after an intravenous injection. Accordingly RA was evaluated as a potential repository drug. While animal experiments were encouraging, human subjects experienced a painful local reaction to RA administered either i.m. or s.c. as a suspension in physiological saline. Accordingly it appears that RA is best looked at as a second line drug, unless a means can be found to obviate local inflammatory reactions.

Tetramisole analogues as inhibitors of alkaline phosphatase, an enzyme involved in the resistance of neoplastic cells to 6-thiopurines.

A series of tetramisole derivatives was synthesized and tested for inhibitory activity against alkaline phosphatase which was partially purified from a murine ascitic neoplasm resistant to 6-thiopurines (Sarcoma 180/TG). These agents included derivatives substituted with halogens, CH3, or NO2 groups at either the meta or para position of the phenyl ring of tetramisole and 2,3-dehydrotetramisole. The phenyl ring of tetramisole and 2,3-dehydrotetramisole was also replaced by a naphthyl ring, and the phenyl ring of 2,3-dehydrotetramisole was substituted by a thienyl ring system. The presence of both the thiazolidine and dihydroimidazole rings of tetramisole was found to be essential for enzyme inhibitory activity. Substitution of a naphthyl for the phenyl group and dehydrogenation at the 2,3 position of the thiazolidine ring were found to significantly enhance inhibitory activity for alkaline phosphatase. Tests employing (S)-(-)-6-(4-bromophenyl)-2,3,5,6-tetrahydroimidazo[2,1-b]thiazole oxalate in combination with 6-thioguanine demonstrated that the inhibitor of alkaline phosphatase was capable of increasing the toxicity of 6-thioguanine to Sarcoma 180/TG cells in tissue culture.